Synthesis and bioactivities evaluation of oleanolic acid oxime ester derivatives as α-glucosidase and α-amylase inhibitors

Different oleanolic acid (OA) oxime ester derivatives (3a-3t) were designed and synthesised to develop inhibitors against α-glucosidase and α-amylase. All the synthesised OA derivatives were evaluated against α-glucosidase and α-amylase in vitro. Among them, compound 3a showed the highest α-glucosidase inhibition with an IC₅₀ of 0.35 µM, which was ∼1900 times stronger than that of acarbose, meanwhile compound 3f exhibited the highest α-amylase inhibitory with an IC₅₀ of 3.80 µM that was ∼26 times higher than that of acarbose. The inhibition kinetic studies showed that the inhibitory mechanism of compounds 3a and 3f were reversible and mixed types towards α-glucosidase and α-amylase, respectively. Molecular docking studies analysed the interaction between compound and two enzymes, respectively. Furthermore, cytotoxicity evaluation assay demonstrated a high level of safety profile of compounds 3a and 3f against 3T3-L1 and HepG2 cells.

Highlights

1. Oleanolic acid oxime ester derivatives (3a–3t) were synthesised and screened against α-glucosidase and α-amylase.
2. Compound 3a showed the highest α-glucosidase inhibitory with IC50 of 0.35 µM.
3. Compound 3f presented the highest α-amylase inhibitory with IC50 of 3.80 µM.
4. Kinetic studies and in silico studies analysed the binding between compounds and α-glucosidase or α-amylase.

     

In vitro and in silico studies of bis (indol-3-yl) methane derivatives as potential α-glucosidase and α-amylase inhibitors

In this paper, bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated for their inhibitory activity against α-glucosidase and α-amylase. All synthesised compounds showed potential α-glucosidase and α-amylase inhibitory activities. Compounds 5 g (IC₅₀: 7.54 ± 1.10 μM), 5e (IC₅₀: 9.00 ± 0.97 μM), and 5 h (IC₅₀: 9.57 ± 0.62 μM) presented strongest inhibitory activities against α-glucosidase, that were ∼ 30 times stronger than acarbose. Compounds 5 g (IC₅₀: 32.18 ± 1.66 µM), 5 h (IC₅₀: 31.47 ± 1.42 µM), and 5 s (IC₅₀: 30.91 ± 0.86 µM) showed strongest inhibitory activities towards α-amylase, ∼ 2.5 times stronger than acarbose. The mechanisms and docking simulation of the compounds were also studied. Compounds 5 g and 5 h exhibited bifunctional inhibitory activity against these two enzymes. Furthermore, compounds showed no toxicity against 3T3-L1 cells and HepG2 cells.

Highlights

1. A series of bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated inhibitory activities against α-glucosidase and α-amylase.
2. Compound 5g exhibited promising activity (IC₅₀ = 7.54 ± 1.10 μM) against α-glucosidase.
3. Compound 5s exhibited promising activity (IC₅₀ = 30.91 ± 0.86 μM) against α-amylase.
4. In silico studies were performed to confirm the binding interactions of synthetic compounds with the enzyme active site.

     

Enzymatic Analysis of an Amylolytic Enzyme from the Hyperthermophilic Archaeon Pyrococcus furiosus Reveals Its Novel Catalytic Properties as both an α-Amylase and a Cyclodextrin-Hydrolyzing Enzyme

Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF1939) similar to the enzymes in glycoside hydrolase family 13. This amylolytic enzyme, designated PFTA (Pyrococcus furiosus thermostable amylase), was cloned and expressed in Escherichia coli. The recombinant PFTA was extremely thermostable, with an optimum temperature of 90°C. The substrate specificity of PFTA suggests that it possesses characteristics of both α-amylase and cyclodextrin-hydrolyzing enzyme. Like typical α-amylases, PFTA hydrolyzed maltooligosaccharides and starch to produce mainly maltotriose and maltotetraose. However, it could also attack and degrade pullulan and β-cyclodextrin, which are resistant to α-amylase, to primarily produce panose and maltoheptaose, respectively. Furthermore, acarbose, a potent α-amylase inhibitor, was drastically degraded by PFTA, as is typical of cyclodextrin-hydrolyzing enzymes. These results confirm that PFTA possesses novel catalytic properties characteristic of both α-amylase and cyclodextrin-hydrolyzing enzyme.     

Gedunin and Azadiradione: Human Pancreatic Alpha-Amylase Inhibiting Limonoids from Neem (Azadirachta indica) as Anti-Diabetic Agents

Human pancreatic α-amylase (HPA) inhibitors offer an effective strategy to lower postprandial hyperglycemia via control of starch breakdown. Limonoids from Azadirachta indica known for their therapeutic potential were screened for pancreatic α-amylase inhibition, a known anti-diabetic target. Studies were carried out to reveal their mode of action so as to justify their hypoglycemic potential. Of the nine limonoids isolated/semi-synthesized from A.indica and screened for α-amylase inhibition, azadiradione and exhibited potential inhibition with an IC₅₀ value of 74.17 and 68.38 μM, respectively against HPA under in vitro conditions. Further screening on AR42J α-amylase secretory cell line for cytotoxicity and bioactivity revealed that azadiradione and gedunin exhibited cytotoxicity with IC₅₀ of 11.1 and 13.4μM. Maximal secreted α-amylase inhibition of 41.8% and 53.4% was seen at 3.5 and 3.3μM, respectively. Michaelis-Menten kinetics suggested a mixed mode of inhibition with maltopentaose (K _i 42.2, 18.6 μM) and starch (K _i ′ 75.8, 37.4 μM) as substrate with a stiochiometry of 1:1 for both azadiradione and gedunin, respectively. The molecular docking simulation indicated plausible π-alkyl and alkyl-alkyl interactions between the aromatic amino acids and inhibitors. Fluorescence and CD confirmed the involvement of tryptophan and tyrosine in ligand binding to HPA. Thermodynamic parameters suggested that binding is enthalpically and entropically driven with ΔG° of -21.25 kJ mol^-1 and -21.16 kJ mol^-1 for azadiradione and gedunin, respectively. Thus, the limonoids azadiradione and gedunin could bind and inactivate HPA (anti-diabetic target) and may prove to be lead drug candidates to reduce/control post-prandial hyperglycemia.     

Fijian medicinal plants and their role in the prevention of Type 2 diabetes mellitus

Medicinal plants (MPs) are natural sources of active compounds with potential therapeutic benefits in alleviating various illnesses for decades. Fijian people also are using these MPs for the management/prevention of Type 2 diabetes mellitus (T2DM) and associated complications. However, till date, none of these Fijian MP’s antidiabetic potential have been explored or evaluated. Here, we investigated the antidiabetic potential of Fijian MPs scientifically. Phytochemicals such as polyphenols were detected to inhibit the activity of α-amylase and α-glucosidase, the two key carbohydrate enzymes linked to T2DM. Therefore, in the present study, the total phenolic content (TPC), α-amylase and α-glucosidase inhibitory activity of five Fijian MPs: Vobo (Mussaenda raiateensis, MR), Vula walu (Blechnum orientale, BO), Gasau (Miscanthus floridulus, MF), Molikaro (Citrus limon, CL) and Beki ni sina (Dicranopteris caudate, DC) collected from mainland region of Vitilevu, Fiji Islands, were evaluated in vitro. The hydromethanolic (ME) and dichloromethane (DM) extracts of these selected MPs were investigated. The ME extracts of BO (0.102 ± 0.009 mM CE) and DC (0.098 ± 0.09 mM Catechin Equivalence [CE]) showed a higher TPC compared with the control [vanillic acid (0.052 ± 0.003 mM CE, *P value < 0.05)]. However, the TPC of MF, MR and CL were found in the range of 0.020 ± 0.009 to 0.009 ± 0.01 mM CE. The ME extracts of MF and MR inhibited α-glucosidase significantly in comparison with acarbose as evidenced from the IC₅₀ values (IC₅₀ of MF = 1.58 ± 0.03 ng/µl; IC₅₀ of MR = 1.87 ± 0.43 ng/µl and IC₅₀ of acarbose = 3.34 ± 0.15 ng/µl). Moreover, DM extracts of MR (IC₅₀ = 1.31 ± 0.29 ng/µl) also showed significantly higher α-glucosidase inhibitory activity. In contrary, MR (IC₅₀ = 16.18 ± 0.16 ng/µl) and CL (IC₅₀ = 9.21 ± 0.51 ng/µl) also showed significant α-amylase inhibitory activity in ME and DM extracts, respectively. These, results suggest that Fijian MPs could be a potential source of natural inhibitors of enzymes involved in carbohydrate digestion and thus may possibly be used in managing T2DM.     

In vitro alpha-amylase inhibition and in vivo antioxidant potential of Amaranthus spinosus in alloxan-induced oxidative stress in diabetic rats

Amaranthus spinosus Linn. (Amaranthaceae), commonly known as “Mulluharivesoppu” in Kannada, is used in the Indian traditional system of medicine for the treatment of diabetes. The present study deals with the scientific evaluation of alpha amylase and the antioxidant potential of methanol extract of A. spinosus (MEAS). The aim of this study was to investigate in vitro alpha-amylase enzyme inhibition by CNPG3 (2-chloro-4-nitrophenol α-d-maltotrioside) and in vivo antioxidant potential of malondialdehyde (MDA), glutathione (GSH), catalase (CAT) and total thiols (TT) in alloxan-induced diabetic rats of a methanolic extract of A. spinosus. Blood sugar was also determined in MEAS-treated alloxan-induced diabetic rats. MEAS showed significant inhibition of alpha-amylase activity and IC₅₀ 46.02 μg/ml. Oral administration of MEAS (200 and 400 mg/kg) for 15 days showed significant reduction in the elevated blood glucose, MDA and restores GSH, CAT and TT levels as compared with a diabetic control. The present study provides evidence that the methanolic extract of A. spinosus has potent alpha amylase, anti-diabetic and antioxidant activities.     

Combinatorial active-site variants confer sustained clavulanate resistance in BlaC β-lactamase from Mycobacterium tuberculosis

Bacterial resistance to β-lactam antibiotics is a global issue threatening the success of infectious disease treatments worldwide. Mycobacterium tuberculosis has been particularly resilient to β-lactam treatment, primarily due to the chromosomally encoded BlaC β-lactamase, a broad-spectrum hydrolase that renders ineffective the vast majority of relevant β-lactam compounds currently in use. Recent laboratory and clinical studies have nevertheless shown that specific β-lactam–BlaC inhibitor combinations can be used to inhibit the growth of extensively drug-resistant strains of M. tuberculosis, effectively offering new tools for combined treatment regimens against resistant strains. In the present work, we performed combinatorial active-site replacements in BlaC to demonstrate that specific inhibitor-resistant (IRT) substitutions at positions 69, 130, 220, and/or 234 can act synergistically to yield active-site variants with several thousand fold greater in vitro resistance to clavulanate, the most common clinical β-lactamase inhibitor. While most single and double variants remain sensitive to clavulanate, double mutants R220S-K234R and S130G-K234R are substantially less affected by time-dependent clavulanate inactivation, showing residual β-lactam hydrolytic activities of 46% and 83% after 24 h incubation with a clinically relevant inhibitor concentration (5 μg/ml, 25 µM). These results demonstrate that active-site alterations in BlaC yield resistant variants that remain active and stable over prolonged bacterial generation times compatible with mycobacterial proliferation. These results also emphasize the formidable adaptive potential of inhibitor-resistant substitutions in β-lactamases, potentially casting a shadow on specific β-lactam–BlaC inhibitor combination treatments against M. tuberculosis.     

Diosgenin from Dioscorea bulbifera: Novel Hit for Treatment of Type II Diabetes Mellitus with Inhibitory Activity against α-Amylase and α-Glucosidase

Diabetes mellitus is a multifactorial metabolic disease characterized by post-prandial hyperglycemia (PPHG). α-amylase and α-glucosidase inhibitors aim to explore novel therapeutic agents. Herein we report the promises of Dioscorea bulbifera and its bioactive principle, diosgenin as novel α-amylase and α-glucosidase inhibitor. Among petroleum ether, ethyl acetate, methanol and 70% ethanol (v/v) extracts of bulbs of D. bulbifera, ethyl acetate extract showed highest inhibition upto 72.06 ± 0.51% and 82.64 ± 2.32% against α-amylase and α-glucosidase respectively. GC-TOF-MS analysis of ethyl acetate extract indicated presence of high diosgenin content. Diosgenin was isolated and identified by FTIR, ¹H NMR and ¹³C NMR and confirmed by HPLC which showed an α-amylase and α-glucosidase inhibition upto 70.94 ± 1.24% and 81.71 ± 3.39%, respectively. Kinetic studies confirmed the uncompetitive mode of binding of diosgenin to α-amylase indicated by lowering of both Km and Vm. Interaction studies revealed the quenching of intrinsic fluorescence of α-amylase in presence of diosgenin. Similarly, circular dichroism spectrometry showed diminished negative humped peaks at 208 nm and 222 nm. Molecular docking indicated hydrogen bonding between carboxyl group of Asp300, while hydrophobic interactions between Tyr62, Trp58, Trp59, Val163, His305 and Gln63 residues of α-amylase. Diosgenin interacted with two catalytic residues (Asp352 and Glu411) from α-glucosidase. This is the first report of its kind that provides an intense scientific rationale for use of diosgenin as novel drug candidate for type II diabetes mellitus.     

Pancreatic α-Amylase Controls Glucose Assimilation by Duodenal Retrieval through N-Glycan-specific Binding,Endocytosis, and Degradation

α-Amylase, a major pancreatic protein and starch hydrolase, is essential for energy acquisition. Mammalian pancreatic α-amylase binds specifically to glycoprotein N-glycans in the brush-border membrane to activate starch digestion, whereas it significantly inhibits glucose uptake by Na⁺/glucose cotransporter 1 (SGLT1) at high concentrations (Asanuma-Date, K., Hirano, Y., Le, N., Sano, K., Kawasaki, N., Hashii, N., Hiruta, Y., Nakayama, K., Umemura, M., Ishikawa, K., Sakagami, H., and Ogawa, H. (2012) Functional regulation of sugar assimilation by N-glycan-specific interaction of pancreatic α-amylase with glycoproteins of duodenal brush border membrane. J. Biol. Chem. 287, 23104–23118). However, how the inhibition is stopped was unknown. Here, we show a new mechanism for the regulation of intestinal glucose absorption. Immunohistochemistry revealed that α-amylase in the duodena of non-fasted, but not fasted, pigs was internalized from the pancreatic fluid and immunostained. We demonstrated that after N-glycan binding, pancreatic α-amylase underwent internalization into lysosomes in a process that was inhibited by α-mannoside. The internalized α-amylase was degraded, showing low enzymatic activity and molecular weight at the basolateral membrane. In a human intestinal Caco-2 cell line, Alexa Fluor 488-labeled pancreatic α-amylase bound to the cytomembrane was transported to lysosomes through the endocytic pathway and then disappeared, suggesting degradation. Our findings indicate that N-glycan recognition by α-amylase protects enterocytes against a sudden increase in glucose concentration and restores glucose uptake by gradual internalization, which homeostatically controls the postprandial blood glucose level. The internalization of α-amylase may also enhance the supply of amino acids required for the high turnover of small intestine epithelial cells. This study provides novel and significant insights into the control of blood sugar during the absorption stage in the intestine.     

Design,synthesis, and evaluation of chalcone-Vitamin E-donepezil hybrids as multi-target-directed ligands for the treatment of Alzheimer’s disease

A novel series of chalcone-Vitamin E-donepezil hybrids was designed and developed based on multitarget-directed ligands (MTDLs) strategy for treating Alzheimer’s disease (AD). The biological results revealed that compound 17f showed good AChE inhibitory potency (ratAChE IC₅₀ = 0.41 µM; eeAChE IC₅₀ = 1.88 µM). Both the kinetic analysis and docking study revealed that 17f was a mixed type AChE inhibitor. 17f was also a good antioxidant (ORAC = 3.3 eq), selective metal chelator and huMAO-B inhibitor (IC₅₀ = 8.8 µM). Moreover, it showed remarkable inhibition of self- and Cu²⁺-induced Aβ_1–42 aggregation with a 78.0 and 93.5% percentage rate at 25 µM, respectively, and disassembled self-induced and Cu²⁺-induced aggregation of the accumulated Aβ_1–42 fibrils with 72.3 and 84.5% disaggregation rate, respectively. More importantly, 17f exhibited a good neuroprotective effect on H₂O₂-induced PC12 cell injury and presented good blood-brain barrier permeability in vitro. Thus, 17f was a promising multi-target-directed ligand for treating AD.     

Enzyme-Synthesized Highly Branched Maltodextrins Have Slow Glucose Generation at the Mucosal α-Glucosidase Level and Are Slowly Digestible In Vivo

For digestion of starch in humans, α-amylase first hydrolyzes starch molecules to produce α-limit dextrins, followed by complete hydrolysis to glucose by the mucosal α-glucosidases in the small intestine. It is known that α-1,6 linkages in starch are hydrolyzed at a lower rate than are α-1,4 linkages. Here, to create designed slowly digestible carbohydrates, the structure of waxy corn starch (WCS) was modified using a known branching enzyme alone (BE) and an in combination with β-amylase (BA) to increase further the α-1,6 branching ratio. The digestibility of the enzymatically synthesized products was investigated using α-amylase and four recombinant mammalian mucosal α-glucosidases. Enzyme-modified products (BE-WCS and BEBA-WCS) had increased percentage of α-1,6 linkages (WCS: 5.3%, BE-WCS: 7.1%, and BEBA-WCS: 12.9%), decreased weight-average molecular weight (WCS: 1.73×10⁸ Da, BE-WCS: 2.76×10⁵ Da, and BEBA-WCS 1.62×10⁵ Da), and changes in linear chain distributions (WCS: 21.6, BE-WCS: 16.9, BEBA-WCS: 12.2 DP_w). Hydrolysis by human pancreatic α-amylase resulted in an increase in the amount of branched α-limit dextrin from 26.8% (WCS) to 56.8% (BEBA-WCS). The α-amylolyzed samples were hydrolyzed by the individual α-glucosidases (100 U) and glucogenesis decreased with all as the branching ratio increased. This is the first report showing that hydrolysis rate of the mammalian mucosal α-glucosidases is limited by the amount of branched α-limit dextrin. When enzyme-treated materials were gavaged to rats, the level of postprandial blood glucose at 60 min from BEBA-WCS was significantly higher than for WCS or BE-WCS. Thus, highly branched glucan structures modified by BE and BA had a comparably slow digesting property both in vitro and in vivo. Such highly branched α-glucans show promise as a food ingredient to control postprandial glucose levels and to attain extended glucose release.     

Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β-Lactamase Inhibitor

The worldwide dissemination of CTX-M type β-lactamases is a threat to human health. Previously, we have reported the spread of bla _CTX-M-15 gene in different clinical strains of Enterobacteriaceae from the hospital settings of Aligarh in north India. In view of the varying resistance pattern against cephalosporins and other β-lactam antibiotics, we intended to understand the correlation between MICs and catalytic activity of CTX-M-15. In this study, steady-state kinetic parameters and MICs were determined on E. coli DH5α transformed with bla _CTX-M-15 gene that was cloned from Enterobacter cloacae (EC-15) strain of clinical background. The effect of conventional β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) on CTX-M-15 was also studied. We have found that tazobactam is the best among these inhibitors against CTX-M-15. The inhibition characteristic of tazobactam is defined by its very low IC₅₀ value (6 nM), high affinity (K _i = 0.017 µM) and better acylation efficiency (k ₊₂/K′ = 0.44 µM⁻¹s⁻¹). It forms an acyl-enzyme covalent complex, which is quite stable (k ₊₃ = 0.0057 s⁻¹). Since increasing resistance has been reported against conventional β-lactam antibiotic-inhibitor combinations, we aspire to design a non-β-lactam core containing β-lactamase inhibitor. For this, we screened ZINC database and performed molecular docking to identify a potential non-β-lactam based inhibitor (ZINC03787097). The MICs of cephalosporin antibiotics in combination with this inhibitor gave promising results. Steady-state kinetics and molecular docking studies showed that ZINC03787097 is a reversible inhibitor which binds non-covalently to the active site of the enzyme through hydrogen bonds and hydrophobic interactions. Though, it’s IC₅₀ (180 nM) is much higher than tazobactam, it has good affinity for CTX-M-15 (K _i = 0.388 µM). This study concludes that ZINC03787097 compound can be used as seed molecule to design more efficient non-β-lactam containing β-lactamase inhibitor that could evade pre-existing bacterial resistance mechanisms.     

Afatinib Reduces STAT6 Signaling of Host ARPE-19 Cells Infected with Toxoplasma gondii

Specific gene expressions of host cells by spontaneous STAT6 phosphorylation are major strategy for the survival of intracellular Toxoplasma gondii against parasiticidal events through STAT1 phosphorylation by infection provoked IFN-γ. We determined the effects of small molecules of tyrosine kinase inhibitors (TKIs) on the growth of T. gondii and on the relationship with STAT1 and STAT6 phosphorylation in ARPE-19 cells. We counted the number of T. gondii RH tachyzoites per parasitophorous vacuolar membrane (PVM) after treatment with TKIs at 12-hr intervals for 72 hr. The change of STAT6 phosphorylation was assessed via western blot and immunofluorescence assay. Among the tested TKIs, Afatinib (pan ErbB/EGFR inhibitor, 5 µM) inhibited 98.0% of the growth of T. gondii, which was comparable to pyrimethamine (5 µM) at 96.9% and followed by Erlotinib (ErbB1/EGFR inhibitor, 20 µM) at 33.8% and Sunitinib (PDGFR or c-Kit inhibitor, 10 µM) at 21.3%. In the early stage of the infection (2, 4, and 8 hr after T. gondii challenge), Afatinib inhibited the phosphorylation of STAT6 in western blot and immunofluorescence assay. Both JAK1 and JAK3, the upper hierarchical kinases of cytokine signaling, were strongly phosphorylated at 2 hr and then disappeared entirely after 4 hr. Some TKIs, especially the EGFR inhibitors, might play an important role in the inhibition of intracellular replication of T. gondii through the inhibition of the direct phosphorylation of STAT6 by T. gondii.     

Exploring the interaction of myricetin with human alpha-2-macroglobulin: biophysical and in-silico analysis

Myricetin (MYR) is a bioactive secondary metabolite found in plants that is recognized for its nutraceutical value and is an essential constituent of various foods and beverages. It is reported to exhibit a plethora of activities, including antioxidant, antimicrobial, antidiabetic, anticancer, and anti-inflammatory. Alpha-2-macroglobulin (α2M) is a major plasma anti-proteinase that can inhibit proteinases of both human and non-human origin, regardless of their specificity and catalytic mechanism. Here, we explored the interaction of MYR-α2M using various biochemical and biophysical techniques. It was found that the interaction of MYR brings subtle change in its anti-proteolytic potential and thereby alters its structure and function, as can be seen from absorbance and fluorescence spectroscopy. UV spectroscopy of α2M in presence of MYR indicated the occurrence of hyperchromism, suggesting complex formation. Fluorescence spectroscopy reveals that MYR reduces the fluorescence intensity of native α2M with a shift in the wavelength maxima. At 318.15 K, MYR binds to α2M with a binding constant of 2.4 × 10³ M⁻¹, which indicates significant binding. The ΔG value was found to be − 7.56 kcal mol⁻¹ at 298.15 K, suggesting the interaction to be spontaneous and thermodynamically favorable. The secondary structure of α2M does not involve any major change as was confirmed by CD analysis. The molecular docking indicates that Asp-146, Ser-172, Glu-174, and Tyr-180 were the key residues involved in α2M-MYR complex formation. This study contributes to our understanding of the function and mechanism of protein and flavonoid binding by providing a molecular basis of the interaction between MYR and α2M.     

Neuronal α‐amylase is important for neuronal activity and glycogenolysis and reduces in presence of amyloid beta pathology

Recent studies indicate a crucial role for neuronal glycogen storage and degradation in memory formation. We have previously identified alpha‐amylase (α‐amylase), a glycogen degradation enzyme, located within synaptic‐like structures in CA1 pyramidal neurons and shown that individuals with a high copy number variation of α‐amylase perform better on the episodic memory test. We reported that neuronal α‐amylase was absent in patients with Alzheimer''s disease (AD) and that this loss corresponded to increased AD pathology. In the current study, we verified these findings in a larger patient cohort and determined a similar reduction in α‐amylase immunoreactivity in the molecular layer of hippocampus in AD patients. Next, we demonstrated reduced α‐amylase concentrations in oligomer amyloid beta 42 (Aβ₄₂) stimulated SH‐SY5Y cells and neurons derived from human‐induced pluripotent stem cells (hiPSC) with PSEN1 mutation. Reduction of α‐amylase production and activity, induced by siRNA and α‐amylase inhibitor Tendamistat, respectively, was further shown to enhance glycogen load in SH‐SY5Y cells. Both oligomer Aβ₄₂ stimulated SH‐SY5Y cells and hiPSC neurons with PSEN1 mutation showed, however, reduced load of glycogen. Finally, we demonstrate the presence of α‐amylase within synapses of isolated primary neurons and show that inhibition of α‐amylase activity with Tendamistat alters neuronal activity measured by calcium imaging. In view of these findings, we hypothesize that α‐amylase has a glycogen degrading function within synapses, potentially important in memory formation. Hence, a loss of α‐amylase, which can be induced by Aβ pathology, may in part underlie the disrupted memory formation seen in AD patients.     

Factors affecting efficient in vitro micropropagation of Muscari muscarimi Medikus using twin bulb scale

Endemic Muscari muscarimi Medikus is the most fragrant plant among Muscari species and has a high ornamental potential. The natural populations of M. muscarimi, are severely affected by increased environmental pollution and urbanization. There is a need to develop a micropropagation method that should serve effectively for commercial propagation and conservation. Therefore, the study targeted to set up a strategy for efficient in vitro bulblet regeneration system of M. muscarimi using twin scale bulb explants on 1.0 × MS medium containing 4.44, 8.88, 17.76 μM BAP (6-Benzylaminopurine) plus 2.685, 5.37, 10.74 μM NAA (α-Naphthalene acetic acid). Maximum number of 19 daughter axillary bulblets and 16 daughter adventitious bulblets per twin bulb scale explant was regenerated on 1.0 × MS medium containing 17.76 μM BAP plus 10.74 μM NAA and 17.76 μM BAP plus 2.685 μM NAA respectively. The daughter bulblets regenerated on twin bulb scales on 8 out of 9 regeneration treatment could be easily rooted on 1.0 × MS medium containing 4.9 μM IBA (Indole-3-butyric acid). The daughter bulblets regenerated on 9th treatment (1.0 × MS medium containing 17.76 μM BAP plus 10.74 μM NAA) were transferred to 1.0 × MS medium containing 30 g/l sucrose to break negative carry over effect of this dose of BAP–NAA, where they grew 2–3 roots of variable length. Daughter bulblet diameter was increased by culturing them on 1.0 × MS medium containing 4.44 μM BAP plus 5.37 μM NAA. The results verified that both age and the source of explants had significant effect on regeneration. In another set of experiments, twin scales were obtained from in vitro regenerated daughter bulblets, although they induced bulblets, yet their bulblet regeneration percentage, mean number of bulblets per explant and their diameter were significantly reduced. In vitro regenerated bulblets were acclimatized in growth chamber under ambient conditions of temperature and humidity on peat moss, where they flowered. The study provides important information about selection of suitable micropropagation medium, strategies to improve bulblet diameter and rooting of M. muscarimi which offers a scope for commercial propagation.Abbreviations: MS medium, Murashige Skoog medium; BAP, 6-Benzylaminopurine; NAA, α-Naphthalene acetic acid; IBA, Indole-3-butyric acid     

Screening and Characterisation of Antimicrobial Properties of Semisynthetic Betulin Derivatives

Betulin (lup-20(29)-ene-3β, 28-diol) is a naturally occurring triterpene, which is found in substantial amounts from the outer bark of birch trees. A library of 51 structurally diverse semisynthetic betulin derivatives was screened against five bacterial strains, Enterobacter aerogenes, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus and a fungal strain Candida albicans, using broth microdilution assays. Primary antimicrobial screening at 50 µM concentration led to the identification of five compounds showing antimicrobial properties (inhibition of growth by >70% against one or more microbial strains). According to the dose-response results, 28-O-(N-acetylanthraniloyl)betulin (compound 5) was the most active, showing MIC₉₀ of 6.25 µM against two Gram-positive bacteria, E. faecalis and S. aureus. However, the activity of this compound was affected by albumin binding, which was demonstrated by the loss of activity in a host-pathogen co-culture assay as well as in the antibacterial assay in the presence of increased concentration of albumin. Furthermore, the effects on mammalian cells were evaluated by cytotoxicity assessment on hepatocyte cell culture after 24 h exposure to the compounds. Betulinic aldehyde (18), betulin-28-oxime (31) and hetero cycloadduct with acetoxy groups at carbon atoms 3 and 28 and ethyl substituent at the triazolo ring (43) displayed cytotoxicity towards hepatocytes, with IC₅₀ values of 47, 25 and 16 µM, respectively. The IC₅₀ value for 28-O-(N-acetylanthraniloyl)betulin (5) was 56 µM. The current study presents an insight into using betulin scaffold for developing derivatives with antibacterial potential, and furthermore the necessity of in-depth analysis of found actives through selectivity profiling and follow-up studies including in silico ADMET predictions.     

Anti-inflammatory components of the Vietnamese starfish Protoreaster nodosus

Background

In the present study, we examined the inhibitory effects of a methanolic extract, dichloromethane fraction, water layer, and polyhydroxylated sterols (1–4) isolated from the Vietnamese starfish Protoreaster nodosus on pro-inflammatory cytokine (IL-12 p40, IL-6, and TNF-α) production in LPS-stimulated bone marrow-derived dendritic cells (BMDCs) using enzyme-linked immunosorbent assays (ELISA).

Results

The methanolic extract and dichloromethane fraction exerted potent inhibitory effects on the production of all three pro-inflammatory cytokines, with IC₅₀ values ranging from 0.60 ± 0.01 to 26.19 ± 0.64 μg/mL. Four highly pure steroid derivatives (1–4) were isolated from the dichloromethane fraction and water layer of P. nodosus. Potent inhibitory activities were also observed for (25S) 5α-cholestane-3β,4β,6α,7α,8β,15α,16β,26-octol (3) on the production of IL-12 p40 and IL-6 (IC_50s = 3.11 ± 0.08 and 1.35 ± 0.03 μM), and for (25S) 5α-cholestane-3β,6α,8β,15α,16β,26-hexol (1) and (25S) 5α-cholestane-3β,6α,7α,8β,15α,16β,26-heptol (2) on the production of IL-12 p40 (IC_50s = 0.01 ± 0.00 and 1.02 ± 0.01 μM). Moreover, nodososide (4) exhibited moderate inhibitory effects on IL-12 p40 and IL-6 production.

Conclusion

This is the first report of the anti-inflammatory activity from the starfish P. nodosus. The main finding of this study is the identification oxygenated steroid derivatives from P. nodosus with potent anti-inflammatory activities that may be developed as therapeutic agents for inflammatory diseases.     

Synthesis,X-ray diffraction analysis,quantum chemical studies and α-amylase inhibition of probenecid derived S-alkylphthalimide-oxadiazole-benzenesulfonamide hybrids

Sulphonamide and 1,3,4-oxadiazole moieties are present as integral structural parts of many drugs and pharmaceuticals. Taking into account the significance of these moieties, we herein present the synthesis, single-crystal X-ray analysis, DFT studies, and α-amylase inhibition of probenecid derived two S-alkylphthalimide-oxadiazole-benzenesulfonamide hybrids. The synthesis has been accomplished in high yields. The final structures of both hybrids have been established completely with the help of different spectro-analytical techniques, including NMR, FTIR, HR-MS, and single-crystal X-ray diffraction analyses. In an effort to confirm the experimental findings, versatile quantum mechanical calculations and Hirshfeld Surface analysis have been performed. α-Amylase inhibition assay has been executed to investigate the enzyme inhibitory potential of both hybrids. The low IC₅₀ value (76.92 ± 0.19 μg/mL) of hybrid 2 shows the good α-amylase inhibition potential of the respective compound. Ultimately, the binding affinities and features of the two hybrids are elucidated utilising a molecular docking technique against the α-amylase enzyme.     

Genetic transformation of Indian isolate of Lemna minor mediated by Agrobacterium tumefaciens and recovery of transgenic plants

Transgenic plants of an Indian isolate of Lemna minor have been developed for the first time using Agrobacterium tumefaciens and hard nodular cell masses ‘nodular calli’ developed on the BAP - pretreated daughter frond explants in B₅ medium containing sucrose (1.0 %) with 2,4-D (5.0 μM) and 2-iP (50.0 μM) or 2,4-D (50.0 μM) and TDZ (5.0 μM) under light conditions. These calli were co-cultured with A. tumefaciens strain EHA105 harboring a binary vector that contained genes for β-glucuronidase with intron and neomycin phosphortransferase. Transformed cells selected on kanamycin selection medium were regenerated into fronds whose transgenic nature was confirmed by histochemical assay for GUS activity, PCR analysis and Southern hybridization. The frequency of transformation obtained was 3.8 % and a period of 11–13 weeks was required from initiation of cultures from explants to fully grown transgenic fronds. The pretreatment of daughter fronds with BAP, use of non-ionic surfactant, presence of acetosyringone in co-cultivation medium, co-culture duration of 3 d and 16 h photoperiod during culture were found crucial for callus induction, frond regeneration and transformation of L. minor. This transformation system can be used for the production of pharmaceutically important protein and in bioremediation.