Antiseizure Activity of Mitragyna inermis in the Pentylenetetrazol-Induced Seizure Model in Mice: Involvement of Flavonoids and Alkaloids.

Objective: This study aimed to investigate whether Mitragyna inermis (Willd.) Otto Kuntze organic and aqueous extracts are able to control seizures induced by pentylenetetrazol (PTZ) in mice based on flavonoid fingerprints and alkaloidal contents. Materials and Methods: Ethanolic extract and decoction-derived fractions from roots, leaves, and stems were subjected to chromatographic fingerprinting using AlCl3 and screening for their antiseizure effects using PTZ-induced acute seizure model. From the fractions that showed potent bioactivities, plausible antiseizure alkaloids were isolated using thin layer chromatography, and their structures were elucidated using 1H NMR, 2D NMR, 13C NMR, and FAB-HR (+ve or -ve). Results: All fractions, with the exception of the dichloromethane and hexane fractions, revealed remarkable flavonoid fingerprints. An acute PTZ-induced seizure test revealed that ethanolic extract of stem bark [500 mg/kg body weight (bw)], ethyl acetate extract of stem bark (500 mg/kg bw), and aqueous extract of leaves (300 mg/kg bw) significantly delayed the occurrence of hind limb tonic extension (HLTE); however, a non-significant delay was observed in the onset of first myoclonic jerk compared with control animals. Isolation yielded four main alkaloids: that are, pteropodine (1), isopteropodine (2), mitraphylline (3) and corynoxeine (4). Corynoxeine is a new compound derived from M. inermis. Conclusion: This study suggests that flavonoid fingerprints are tracers of M. inermis anticonvulsant ingredients. The stem bark ethanolic and ethyl acetate extracts and leaf aqueous extracts contain anticonvulsant bioactive principles that delay notifying the HLTE occurring in male naval medical research institute mice. Furthermore, alkaloidal contents also remain plausible bioactive anticonvulsant principles. All observations support the traditional use of M. inermis to manage epilepsy. However, further studies are needed to understand the effects of alkaloid fractions, flavonoids, and the isolated compounds as promising antiseizure agents derived from M. inermis in experimental animals.

Rac2 regulates immune complex-mediated granule polarization and exocytosis in neutrophils.

A key molecule for neutrophil degranulation is Rac2 guanosine triphosphatase. Neutrophils from Rac2 knockout mice (Rac2-/-) exhibit impaired primary granule exocytosis in response to cytochalasin B/f-Met-Leu-Phe, while secondary and tertiary granule release is unaffected. Coronin 1A, a protein involved in actin remodeling, is diminished in Rac2-/- neutrophils. However, primary granule exocytosis from Rac2-/- neutrophils has not been determined using more immunologically relevant stimuli. We sought to determine the role of Rac2 in degranulation and actin cytoskeleton rearrangement in response to immobilized immune complexes and relate this to intracellular coronin 1A localization. We used bone marrow neutrophils from wild-type and Rac2-/- mice stimulated with immobilized immune complexes. Secretion of primary (myeloperoxidase), secondary (lactoferrin), and tertiary granule (MMP-2 and MMP-9) products was evaluated. Subcellular colocalization of coronin 1A with actin and the primary granule marker CD63 was determined by deconvolution microscopy. We found major differences in myeloperoxidase, MMP-2, and MMP-9 but not lactoferrin release, along with diminished filopodia formation, CD63 polarization, and colocalization of coronin 1A with CD63 in immune complex-stimulated Rac2-/- bone marrow neutrophils. Rac2 and coronin 1A were found associated with granules in cytochalasin B/f-Met-Leu-Phe-activated human neutrophils. This report confirms a role for Rac2 in immunologically relevant stimulation of neutrophil granule exocytosis. Rac2 appears to attach to neutrophil granules, polarize CD63+ granules to the cell surface in a manner dependent on coronin 1A, and induce filopodia formation. Our studies provide insight into mechanisms of Rac2-mediated regulation of granule exocytosis.

Inhibition of Advanced Glycation End-Products by Tamarindus indica and Mitragyna inermis Extracts and Effects on Human Hepatocyte and Fibroblast Viability.

Tamarindus indica and Mitragyna inermis are widely used by herbalists to cure diabetes mellitus. The aim of this study is to investigate the inhibitory potential of aqueous and various organic solvent fractions from both plants and some isolated compounds against advanced glycation end-products (AGEs). For this purpose, an in vitro BSA-fructose glycation model was used to evaluate the inhibition of AGE formation. Furthermore, the effects of the fractions on mouse fibroblast (NIH-3T3) and human hepatocyte (HepG2) survival were evaluated. The leaf, stem, and root fractions of both plants exhibited significant inhibition of AGEs formation. The IC50 values appeared to be less than 250 µg/mL; however, all fractions presented no adverse effects on NIH-3T3 up to 500 µg/mL. Otherwise, our phytochemical investigation afforded the isolation of a secoiridoid from the Mitragyna genus named secoiridoid glucoside sweroside (1), along with three known quinovic acid glycosides: quinovic acid-3ß-O-ß-d-glucopyranoside (2), quinovic acid-3-O-ß-d-6-deoxy-glucopyranoside, 28-O-ß-d-glucopyranosyl ester (3), and quinovic acid 3-O-α-l-rhamnopyranosyl-(4→1)-ß-d-glucopyranoside (4). In particular, 1-3 are compounds which have not previously been described in Mitragyna inermis roots. However, the isolated compounds did not exhibit AGE inhibitory activity. Further investigation on these potent antiglycation fractions may allow for the isolation of new antidiabetic drug candidates.

First report on the synthesis and structural studies of trans-Phakellistatin 18: a rotamer of marine natural product phakellistatin 18.

Phakellistatin peptides from marine organisms are the sources of proline-rich cyclic peptides with reported significant antitumor activities. Phakellistatin 18 (1), reported from marine sponge Phakellia fusca, contains three proline-peptide linkages in cis form. We attempted the total synthesis of natural product 1 through solution-phase macrocyclization approach, as a result, the synthetic cyclic peptide 2 was obtained as a rotamer of natural product having all three proline residues in trans-conformation. Here, we describe the synthesis, structural, and cytotoxicity studies of trans-Phakellistatin 18 (2), and its analog [Ala1,3,6]-Phakellistatin 18 (3). Detailed NMR studies were carried out to characterize the synthesized peptides, and anti-cancer screening was performed by using MTT assay. The synthetic trans-Phakellistatin 18 (2) (IC50=67.5 ± 2.938 µM) showed comparable cytotoxicity against HepG2 cancer cell line with standard drug doxorubicin (IC50=63.88 ± 6.48 µM). Here, the first synthetic and structural studies on trans-Phakellistatin 18 (2), and its anticancer screening against HepG2 cell line was reported.

Design, Synthesis and Characterization of [G10a]-Temporin SHa Dendrimers as Dual Inhibitors of Cancer and Pathogenic Microbes.

As the technologies for peptide synthesis and development continue to mature, antimicrobial peptides (AMPs) are being widely studied as significant contributors in medicinal chemistry research. Furthermore, the advancement in the synthesis of dendrimers' design makes dendrimers wonderful nanostructures with distinguishing properties. This study foregrounds a temporin SHa analog, [G10a]-SHa, and its dendrimers as globular macromolecules possessing anticancer and antibacterial activities. These architectures of temporin SHa, named as [G10a]-SHa, its dendrimeric analogs [G10a]2-SHa and [G10a]3-SHa, and [G10a]2-SHa conjugated with a polymer molecule, i.e., Jeff-[G10a]2-SHa, were synthesized, purified on RP-HPLC and UPLC and fully characterized by mass, NMR spectroscopic techniques, circular dichroism, ultraviolet, infrared, dynamic light scattering, and atomic force microscopic studies. In pH- and temperature-dependent studies, all of the peptide dendrimers were found to be stable in the temperature range up to 40-60 °C and pH values in the range of 6-12. Biological-activity studies showed these peptide dendrimers possessed improved antibacterial activity against different strains of both Gram-positive and Gram-negative strains. Together, these dendrimers also possessed potent selective antiproliferative activity against human cancer cells originating from different organs (breast, lung, prostate, pancreas, and liver). The high hemolytic activity of [G10a]2-SHa and [G10a]3-SHa dendrimers, however, limits their use for topical treatment, such as in the case of skin infection. On the contrary, the antibacterial and anticancer activities of Jeff-[G10a]2-SHa, associated with its low hemolytic action, make it potentially suitable for systemic treatment.

Discovery of stylissatin A analogs exhibiting potent nitric oxide inhibition.

The cyclic peptide stylissatin A(STA) was obtained from the Papua New Guinean marine spongeStylissamassaas a potent nitric oxide (NO) inhibitor.Among its reported analogs,cyclo-{Glu6, Ala2}-STA1potentlyinhibited theinterleukin-2 and proliferation of T-cells indicating position 2 of sequence playing important part in biological activities of this compound.In current studies, second generation analogs of STAwere synthesizedaround its most active analog1by screening position 2 of analog1with different amino acid. All analogs2-6were identified by mass, and NMR techniques.The synthesized analogswere also evaluated against NO generation by lipopolysaccharide (LPS)-stimulated murine J774.2macrophages, ROS inhibition from whole blood phagocytes, and T-cell proliferation from Jurkat cells.All analogswere found to be inactive towards interleukin-2, T-cells proliferation, and ROS inhibition. The analog2showed a potent suppression of NO (IC50 = 46.0 ± 2.2 µM) that was superior to the activityreported for natural product STA.Further attempts to optimizeanalog2afforded new nitric oxide inhibitors2a-2fwhich were found less active than2.The analog2also downregulated the transcription of pro-inflammatory molecules, tumor necrosis factor-α, interlukin-1ß, caspase-1 and ASC which further highlights its anti-inflammatory and possible therapeutic potential. Analog2was non-toxic to BJ and Vero cell lines of normal mammalian origin.

Antioxidant and Cytotoxic Activity of a New Ferruginan A from Olea ferruginea: In Vitro and In Silico Studies.

Studies of the ethyl acetate extract bark extract of Olea ferruginea led to the isolation of one new compound Ferruginan A (1) in addition to two known compounds, Ferruginan (2) and cycloolivil (3). Structures of the isolated compounds were confirmed by mass spectrometry (MS) and NMR spectral data. The ethyl acetate fraction and compounds (1-3) were evaluated against breast cancer cell line (MCF-7) and as antioxidants using the free radical scavenging assay. Results revealed that compound 2 exhibits significant antioxidant activity with an IC50 value of 21.74 µg/mL. In addition, the ethyl acetate fraction showed good cytotoxic activity (79.31% inhibition at 250 µg/mL), whereas compounds 1-3 exerted mild cytotoxic activity (IC50 = 8.03-12.01 µg/mL) as compared to the standard (IC50 = 4.41 µg/mL) against MCF-7. Docking studies suggested that antioxidant activity is due to the chelation of compounds with copper present in the active site of tyrosinase. These results suggest that the extract exhibits considerable antioxidant activity, and the isolated compounds exert moderate anticancer activity.

Synthesis of [1-8-NαC]-zanriorb A1, alanine-containing analogues, and their cytotoxic and anti-inflammatory activity.

The synthesis of the orbitide[1-8-NαC]-zanriorb A1, isolated from the medicinal plant Zanthoxylum riedelianum, was investigated by solution-phase macrocyclization of a linear peptide and on-resin solid-phase macrocyclization with an acylsulfonamide safety-catch linker. The solution-phase route produced a mixture of proline rotamers, and the main component was assigned as the trans, cis rotamer, identical to the natural product. The on-resin cyclization was less successful, producing mainly a linear peptide, and minor cyclic products as rotameric mixtures. Although the natural product was reported to be significantly cytotoxic against Jurkat leukemia T cells, our synthetic peptides were inactive, suggesting the presence of other rotamers or impurities in the naturally isolated material. Additional analogues of zanriorb A1 were synthesized in which proline and glycine residues were replaced with alanine. While these analogues were not cytotoxic, several of them inhibited the production of nitric oxide in lipopolysaccharide (LPS)-stimulated macrophages. The most active compound, cyclic[Ala5,6,8 ]-zanriorb A1 had an IC50 of 22 µM and was more potent compared with the standard NG-monomethyl-l-arginine acetate (L-NMMA) with an IC50 of 98 µM, indicating their strong anti-inflammatory potential.

Serum Stable and Low Hemolytic Temporin-SHa Peptide Analogs Disrupt Cell Membrane of Methicillin-Resistant Staphylococcus aureus (MRSA).

Anti-microbial peptides (AMPs) have attracted major attention due to their potential bio-activities against some multidrug resistant pathogens. The present study evaluated the mechanism of actions of highly potent AMP temporin-SHa analogs, i.e., [G4a]-SHa, [G7a]-SHa, and [G10a]-SHa, against methicillin-resistant Staphylococcus aureus (MRSA) NCTC (13277) with minimum inhibitory concentrations (MICs) of 14.35, 7.16, and 3.58 µM, respectively. These analogs exhibited significant anti-MRSA activity at physiological salt concentration, 30% fetal bovine serum, and 30% human serum. [G4a]-SHa and [G7a]-SHa were non-hemolytic and non-cytotoxic to normal mouse fibroblast 3T3 cell and human Caco-2 cell line. Atomic force microscopy revealed that these analogs have profound effect on the morphological changes in MRSA surface with significant leakage of cell cytoplasmic content. Propidium iodide uptake kinetic assay and (bis-(1,3-dibutylbarbituric acid) trimethine oxonol) DiBAC4(3) membrane depolarization assay demonstrated that these analogs display a membrane disrupting property, characterized by elevation of plasma membrane permeability and rapid transmembrane potential depolarization. [G10a]-SHa showed a significant anti-biofilm activity against biofilm forming S. aureus (ATCC 6538). Acute in vivo toxicity studies revealed that [G10a]-SHa possesses some toxic effect at 100-mg/kg dose. While [G4a]-SHa at 100 mg/kg, i.p. has no toxic effect even after 48 h, [G7a]-SHa also did not show any toxic effect at the dose of 100 mg/kg, i.p. during 24-h observation of animals. In conclusion, [G4a]-SHa, [G7a]-SHa, and [G10a]-SHa show improved activity against MRSA and stability compared to SHa peptide. Although highly potent, [G10a]-SHa, due to its hemolytic activity, might be more suitable for topical application, whereas [G4a]-SHa and [G7a]-SHa have potential to be used for systemic application.

Peptide conjugates of 18ß-glycyrrhetinic acid as potent inhibitors of α-glucosidase and AGEs-induced oxidation.

18ß-Glycyrrhetinic acid (18ß-GA) is known for several biological activities, and has been the focus of extensive research for the development of therapeutic agents. In the current study, 18ß-GA-peptide conjugates 2-11 were evaluated for their in vitro α-glucosidase inhibitory and antiglycation activities. Structure-activity relationship (SAR) established and molecular interactions of active bioconjugates with the enzyme's binding sites were predicted through molecular modeling approach. In tripeptide moiety of conjugates 2-11, peptide residue at position 1 was found to have a significant role on α-glucosidase inhibition. The most active 18ß-GA-peptide conjugates 5 (18ß-GA-Cys1-Tyr2-Gly3), and 8 (18ß-GA-Pro1-Tyr2-Gly3) exhibited several-fold potent α-glucosidase inhibition (IC50 values 20-28 µM), as compared to standard drug acarbose (IC50 = 875.8 ± 2.10 µM). Kinetic studies of potent compounds, 4-8 revealed that conjugate 5 exhibits competitive-type of inhibition, while conjugates 6-8 showed a non-competitive type of inhibition. The simulation studies also supported the kinetic results that conjugate 5 (18ß-GA-Cys1-Tyr2-Gly3) inhibits the α-glucosidase enzyme by blocking its substrate binding site. AGEs-induced NO• inhibitors play an important role in controlling the inflammation associated with diabetes mellitus. The peptide conjugates 2-11 were also evaluated in vitro for AGEs-induced NO• inhibition using RAW 264.7 macrophage cell line. Our data revealed that conjugates 7-10 were the more potent AGEs-induced NO• inhibitors, comparable to standards rutin, and PDTC. The peptide conjugate 5 (a competitive inhibitor of α-glucosidase) also exhibited a strong inhibitory activity against AGEs-induced NO• production. Furthermore, peptide conjugates 2-11 were found non-cytotoxic to mouse fibroblast NIH-3T3, and murine macrophages RAW 264.7 cell lines. In conclusion, our data demonstrates that besides possessing strong α-glucosidase inhibition, the newly synthesized peptide conjugates also alleviated the AGEs-induced NO• production in RAW macrophages. Dual inhibition of α-glucosidase enzyme, and AGEs-induced NO• production by 18ß-GA-peptide conjugates qualify them for further research in anti-diabetic drug discovery.

Isolation, characterization, and hepatoprotective properties of betulinic acid and ricinine from Tetracarpidium conophorum seeds (Euphorbiaceae).

The present study is to isolate and characterize betulinic acid and ricinine from T. conophorum seeds. Phytochemical investigation on hexane fraction of T. conophorum seeds led to the isolation of two compounds, Betulinic acid (1), and Ricinine (2). Betulinic acid and ricinine were screened against HepG2 cells and tested in vivo in CCl4 -induced experimental rats model. Results from this study showed that the compounds had hepatoprotective and cytotoxic activities. It was observed that betulinic acid inhibited HepG2 cell with percentage inhibition of 54% compared with standard doxorubicin (64%), while ricinine was inactive against HepG2 cell lines. Furthermore, molecular docking was carried out on betulinic acids and ricinine, with binding energies of -11.2 kcal/mol and -5.4 kcal/mol, respectively, indicating strong binding sites and interactions with Hepatitis B Virus DNA polymerase. Therefore, findings from this study suggest that betulinic acid possess cytotoxic and hepatoprotective properties, while ricinine exhibited hepatoprotection in CCl4 -induced liver damage. PRACTICAL APPLICATIONS: Medicinal plants contain unrestricted ability to make compounds that intrigue researchers in the quest for novel phyto-therapeutic drugs. The continuous exploration of new compounds in the medicinal plant is an auspicious strategy for the prevention of diseases. Therefore, the purpose of this research is to evaluate the cytotoxic and hepatoprotective compounds (betulinic acid and ricinine) isolated from T. conophorum seeds.

Bioactive constituents from seeds of Annona Senegalensis Persoon (Annonaceae).

Column chromatography led to the isolation and full characterization of N-cerotoyltryptamine (1) previously described in a mixture of 5 compounds, asimicin (2) and ent-19-Carbomethoxykauran-17-oic acid (3) isolated from this species for first time alongside stigmasterol glycoside (4) and lacceroic acid (5). The structures of the compounds were established by extensive EIMS, HRESIMS, 1 D and 2 D NMR studies. Compound 1 and the extract AS were more potent inhibitors of ROS with IC50 values of 2.7 ± 0.1 µg/mL and 8.7 ± 10.2 µg/mL respectively than Ibuprofen (11.2 ± 1.9 µg/mL) as a standard anti-inflammatory drug. Compound 2 showed high inhibition on nitric oxide (IC50 = 3.9 ± 0.2 µg/mL), almost 6 times more active than the standard compound L-NMMA (IC50 = 24.2 ± 0.8 µg/mL) used. Compounds showed relatively low toxicity on NIH-3T3 fibroblast cells compared to standard. The results indicate that compounds 1 and 2 are potent anti-inflammatory drug candidates.

Furan-Conjugated Tripeptides as Potent Antitumor Drugs.

Cervical cancer is among the leading causes of death in women. Chemotherapy options available for cervical cancer include highly cytotoxic drugs such as taxol, cisplatin, 5-florouracil, and doxorubicin, which are not specific. In the current study, we have identified a new peptide conjugate (Fur4-2-Nal3-Ala2-Phe1-CONH2) (conjugate 4), from screening of a small library of tripeptide-conjugates of furan, as highly potent anticancer compound against human cervical cancer cells (HeLa cells) (IC50 = 0.15 ± 0.05 µg/mL or 0.28 +/- 0.09 µM). Peptides were constructed on Rink amide resin from C- to N-terminus followed by capping by α-furoic acid moiety. The synthesized peptides were purified by recycling RP-HPLC, and structures of all the peptides were confirmed by using FABMS/ESIMS, 1H- NMR, 13C-NMR, and HR-FABMS. Conjugate 4 was furthermore found to be specifically active against human cervical cancer cells since it did not inhibit the proliferation of other human normal cells (HUVEC (human umbilical vein endothelial cells) and IMR-90 (normal human fibroblasts)), and cancer cells tested (HUVEC, MCF-7, and MDA-MB-231 cells), as well as in mice 3T3 cells (normal fibroblasts). This study revealed a good structure activity relationship of various peptide conjugates. Conjugate 4 in branched forms (4a and 4b) were also synthesized and evaluated against HeLa cells, and results revealed that both were inactive. Atomic force microscopy (AFM) studies and staining with rhodamine 123 and propidium iodide (PI) revealed that conjugate 4 possesses a membranolytic effect and causes the loss of mitochondrial membrane potential.

Effect of a dianthin G analogue in the differentiation of rat bone marrow mesenchymal stem cells into cardiomyocytes.

Loss of cardiomyocytes due to myocardial infarction results in ventricular remodeling which includes non-contractile scar formation, which can lead to heart failure. Stem cell therapy aims to replace the scar tissue with the functional myocardium. Mesenchymal stem cells (MSCs) are undifferentiated cells capable of self-renewal as well as differentiation into multiple lineages. MSCs can be differentiated into cardiomyocytes by treating them with small molecules and peptides. Here, we report for the first time, the role of a cyclic peptide, an analogue of dianthin G, [Glu2]-dianthin G (1) in the in vitro cardiac differentiation of rat bone marrow MSCs. In this study, [Glu2]-dianthin G (1) was synthesized using solid-phase total synthesis and characterized by NMR spectroscopy. MSCs were treated with two different concentrations (0.025 and 0.05 mM) of the peptide separately for 72 h and then incubated for 15 days to allow the cells to differentiate into cardiomyocytes. Treated cells were analyzed for the expression of cardiac-specific genes and proteins. Results showed significant upregulation of cardiac-specific genes GATA4, cardiac troponin T (cTnT), cardiac troponin I (cTnI), cardiac myosin heavy chain, and connexin 43 in the treated MSCs compared to the untreated control. For cardiac-specific proteins, GATA4, cTnT, and Nkx2.5 were analyzed in the treated cells and were shown to have significant upregulation as compared to the untreated control. In conclusion, this study has demonstrated the cardiac differentiation potential of [Glu2]-dianthin G (1)-treated rat bone marrow MSCs in vitro both at the gene and at the protein levels. Transplantation of pre-differentiated MSCs into the infarcted myocardium may result in the efficient regeneration of cardiac cells and restoration of normal cardiac function.

A new isoflavonol and other constituents from Cameroonian propolis and evaluation of their anti-inflammatory, antifungal and antioxidant potential.

Propolis is rich in diverse bioactive compounds. Propolis samples were collected from three localities of Cameroon and used in the study. Column chromatography separation of propolis MeOH:DCM (50:50) extracts yielded a new isoflavonol, 2-hydroxy-8-prenylbiochanin A (1) alongside 2',3'-dihydroxypropyltetraeicosanoate (2) and triacontyl p-coumarate (3) isolated from propolis for first time together with seven compounds: ß-amyrine (4), oleanolic acid (5), ß-amyrine acetate (6), lupeol (7), betulinic acid (8), lupeol acetate (9) and lupenone (10). These compounds were tested for their inhibitory effect on oxidative burst where intracellular reactive oxygen species (ROS) were produced from zymosan stimulated human whole blood phagocytes and on production of nitric oxide (NO) from lipopolysaccharide (LPS) stimulated J774.2 mouse macrophages. The cytotoxicity of these compounds was evaluated on NIH-3 T3 normal mouse fibroblast cells, antiradical potential on 2,2-diphenyl-1-picrylhydrazylhydrazyl (DPPH·) as well as their anti-yeast potential on four selected candida species. Compound 1 showed higher NO inhibition (IC50 = 23.3 ± 0.3 µg/mL) than standard compound L-NMMA (IC50 = 24.2 ± 0.8 µg/mL). Higher ROS inhibition was shown by compounds 6 (IC50 = 4.3 ± 0.3 µg/mL) and 9 (IC50 = 1.1 ± 0.1 µg/mL) than Ibuprofen (IC50 = 11.2 ± 1.9 µg/mL). Furthermore, compound 1 displayed moderate level of cytotoxicity on NIH-3 T3 cells, with IC50 = 5.8 ± 0.3 µg/mL compared to the cyclohexamide IC50 = 0.13 ± 0.02 µg/mL. Compound 3 showed lower antifungal activity on Candida krusei and Candida glabrata, MIC of 125 µg/mL on each strain compared to 50 µg/mL for fuconazole. The extracts showed low antifungal activities ranging from 250 to 500 µg/mL on C. albicans, C. krusei and C. glabrata and the values of MIC on Candida parapsilosis were 500 µg/mL and above. DPPH* scavenging activity was exhibited by compounds 1 (IC50 = 15.653 ± 0.335 µg/mL) and 3 (IC50 = 89.077 ± 24.875 µg/mL) compared to Vitamin C (IC50 = 3.343 ± 0.271 µg/mL) while extracts showed moderate antiradical activities with IC50 values ranging from 309.31 ± 2.465 to 635.52 ± 11.05 µg/mL. These results indicate that compounds 1, 6 and 9 are potent anti-inflammatory drug candidates while 1 and 3 could be potent antioxidant drugs.

Antibiofilm, antiquorum sensing and antioxidant activity of secondary metabolites from seeds of Annona senegalensis, Persoon.

The increasing resistance of bacteria to antibiotics has motivated the interest in potent natural compounds capable of disrupting bacterial cell-to-cell communication. Column chromatography of seed extract of Annona senegalensis afforded N-cerotoyltryptamine (1), asimicin (2) and ent-19-carbomethoxykauran-17-oic acid (3). The compounds were tested for their antimicrobial, antibiofilm, and anti-quorum sensing activities. The minimum inhibitory concentrations (MIC) values ranged from 50 µg/mL to 100 µg/mL for C. albicans ATCC 10239 and S. aureus ATCC 25923 E. coli ATCC 25922, C. violaceum CV026 and C. violaceum CV12472. All the compounds inhibited biofilm formations of all microorganisms tested in various percentages at MIC and MIC/2. Compound 2 also exhibited the highest antibiofilm activity against C. albicans (yeast) and E. coli with percentage inhibitions ranging from 6.3 ± 4.1 (MIC/4) to 37.9 ± 4.5 (MIC) for C. albicans and from 18.8 ± 1.1 (MIC/4) to 43.2 ± 0.5 (MIC) for E. coli. Compound 1, however, showed highest biofilm inhibition on S. aureus as the percentage inhibition varied from 26.7 ± 3.6 (MIC/4) to 43.8 ± 2.1 (MIC). Compound 2 showed highest percentage violacein inhibition on C. violaceum CV12472 ranging from 10.2 ± 0.5 (MIC/8), 65.76 ± 1.3 (MIC/2) and 100 (MIC). Compound 1 and 3 had percentage violacein formation inhibitions on C. violaceum CV12472 ranging from 9.66 ± 1.1 (MIC/4) to 100 (MIC), and from 17.4 ± 2.4 (MIC/4) to 100 (MIC), respectively. Swimming and swarming motility of P. aeruginosa PA01 strain was evaluated at three concentrations of 50, 75 and 100 µg/mL. The compounds inhibited the P. aeruginosa swimming and swarming motility at the three tested concentrations (50, 75 and 100 µg/ml) in a dose-dependent manner. The extents of inhibition of motility migration was relatively higher in the swimming model than in the swarming model for all compounds. Compound 1 exhibited the highest percentage inhibition of motility of 41.50 ± 3.5 and 39.73 ± 1.5 in swimming model and swarming model respectively at 100 µg/ml. Compound 3 showed the lowest percentage inhibition of 30.36 ± 2.0 and 23.50 ± 2.5 in swimming and swarming respectively at 100 µg/ml. At the lowest tested concentration of 50 µg/ml, it was compound 2 showing the highest inhibition of motility of 17.49 ± 0.5 and 14.29 ± 1.0 in swimming and swarming respectively. Compound 1 showed highest quorum sensing (QS) activity with QS inhibition zone of 20.0 ± 1.5 mm at MIC and 11.0 ± 1.0 mm at MIC/8 while compound 2 had the highest antimicrobial (AM) zone diameter amongst the compounds at MIC. Compound 3 was the QS inhibitory sample and did not show any QS inhibition at MIC/8 while showing its highest QS inhibition zone of 13.0 ± 1.6 mm at MIC. For antioxidant assays, no sample showed better activity than the standards. Compound 2 had highest activity with IC50 values of 87.79 ± 2.70 and 42.77 ± 1.53 µg/mL in DPPH and ß-carotene-linoleic acid assay respectively and was more active (IC50 of 97.69 ± 1.40 µg/mL) than standard quercetin (IC50 250.09 ± 0.87 µg/mL) in metal chelation assay.

In vivo anticonvulsant activity of 2-propanone-1,3,5,5-trimethyl-2-cyclohexen-1-ylidine in pilocarpine and strychnine induced-seizure models.

An imbalance between inhibitory (GABA) and excitatory (Glutamate) neurotransmission contribute to the development of epilepsy. Earlier studies reported that dysregulation of GABA and glutamatergic activities resulted in status epilepticus (SE) and ultimately support the development of temporal lobe epilepsy (TLE), a type of resistant epilepsy. In the earlier work, 2-propanone-1,3,5,5-trimethyl-2-cyclohexen-1-ylidine demonstrated anticonvulsant activity against pentylenetetrazole (PTZ)-induced seizures. Apart from the PTZ-induced TLE, the dysregulation muscaranic receptors and glycine receptors are also widely reported phenomena in the development of temporal lobe epilepsy. Keeping the role of these two receptors in epilepsy, the present work investigated the effect of 2-propanone-1,3,5,5-trimethyl-2-cyclohexen-1-ylidine in pilocarpine-induced and strychnine-induced seizure models. Our results demonstrated that 2-propanone-1,3,5,5-trimethyl-2-cyclohexen-1-ylidine significantly delayed the onset of seizure with maximum protection from SE in pilocarpine-induced seizure model. However, the test compound did not revealed any effect on strychnine-induced seizures in mice. Based on these observations, we suggest that 2-propanone-1,3,5,5-trimethyl-2-cyclohexen-1-ylidine could be a potential candidate in reduction of SE and treatment of temporal lobe epilepsy (TLE) in future.

Inhibitory Effects of Myrtucommuacetalone 1 (MCA-1) from Myrtus communis on Inflammatory Response in Mouse Macrophages.

(1) Introduction: Reactive oxygen species (ROS) and nitric oxide (NO) are key signaling molecules that play important roles in the progression of inflammatory disorders. The objective of this study was to explore the use of myrtucommuacetalone-1 (MCA-1), as a novel compound of natural origin and a potential anti-inflammatory agent. (2) Methodology: The anti-inflammatory potential of MCA-1, which was isolated from Myrthus communis Linn, was determined by assaying superoxide, hydrogen peroxide, and nitric oxide production in macrophages. Furthermore, the effects of the compound were analyzed via phosphorylation and translocation of the transcription factor NF kappa B, which is a key regulator of iNOS activation. The effect of MCA-1 on the inducible nitric oxide synthase (iNOS) enzyme was also examined using in silico docking studies. The anticancer potential for MCA-1 was evaluated with an MTT cytotoxic assay. (3) Results: In stimulated macrophages, MCA-1 inhibited superoxide production by 48%, hydrogen peroxide by 53%, and nitric oxide (NO) with an IC50 of <1 µg/mL. MCA-1 also showed a very strong binding pattern within the active site of the inducible nitric oxide synthase enzyme. Furthermore, 25 µg/mL of MCA-1 inhibited inducible nitric oxide synthase expression and abolished transcription factor (NFκB) phosphorylation and translocation to the nucleus. Cytotoxicity analyses of MCA-1 on 3T3 mouse fibroblasts, CC1 liver cell line, J774.2, macrophages and MDBK bovine kidney epithelial cell, yielded IC50 values of 6.53 ± 1.2, 4.6 ± 0.7, 5 ± 0.8, and 4.6 ± 0.7, µg/mL, respectively. (4) Conclusion: Our results suggest that MCA-1, a major phloroglucinol-type compound, shows strong anti-inflammatory activity and has a potential to be a leading therapeutic agent in the future.

Temporin-SHa and Its Analogs as Potential Candidates for the Treatment of Helicobacter pylori.

Helicobacterpylori is one of the most prevalent pathogens colonizing 50% of the world's population and causing gastritis and gastric cancer. Even with triple and quadruple antibiotic therapies, H. pylori shows increased prevalence of resistance to conventional antibiotics and treatment failure. Due to their pore-forming activity, antimicrobial peptides (AMP) are considered as a good alternative to conventional antibiotics, particularly in the case of resistant bacteria. In this study, temporin-SHa (a frog AMP) and its analogs obtained by Gly to Ala substitutions were tested against H. pylori. Results showed differences in the antibacterial activity and toxicity of the peptides in relation to the number and position of D-Ala substitution. Temporin-SHa and its analog NST1 were identified as the best molecules, both peptides being active on clinical resistant strains, killing 90-100% of bacteria in less than 1 h and showing low to no toxicity against human gastric cells and tissue. Importantly, the presence of gastric mucins did not prevent the antibacterial effect of temporin-SHa and NST1, NST1 being in addition resistant to pepsin. Taken together, our results demonstrated that temporin-SHa and its analog NST1 could be considered as potential candidates to treat H. pylori, particularly in the case of resistant strains.

Potent leishmanicidal and antibacterial metabolites from Olea ferruginea.

Inspired from the leishmanicidal and antibacterial potential of the fractions obtained from the crude extract of Olea ferruginea stem, the anti-leishmanial ethyl acetate fraction was subjected to chromatographic separation, leading to the isolation of a new compound ferruginan (1) and a known compound (+)- cycloolivil (2). The structures of 1 and 2 were determined by various spectroscopic techniques and were assayed for their in vitro antibacterial and leishmanicidal potential. Compound 1 showed 75% inhibition after 24 h of incubation and 98% inhibition after 48 h of incubation against Leishmania tropica KWH23 promastigotes at 100 µg/mL concentration, while compound 2 exhibited 73% and 96% inhibition at the same concentration and incubation time. Compound 1 also showed good activity against various bacterial pathogens.