Inhibitory evaluation of Curculigo latifolia on α-glucosidase,DPP (IV) and in vitro studies in antidiabetic with molecular docking relevance to type 2 diabetes mellitus

The inhibition of α-glucosidase and DPP enzymes capable of effectively reducing blood glucose level in the management of type 2 diabetes. The purpose of the present study is to evaluate the inhibitory potential of α-glucosidase and DPP (IV) activity including with the 2-NBDG uptake assay and insulin secretion activities through in vitro studies. The selected of active compounds obtained from the screening of compounds by LC-MS were docked with the targeted enzyme that involved in the mechanism of T2DM. From the results, root extracts displayed a better promising outcome in α-glucosidase (IC₅₀ 2.72 ± 0.32) as compared with the fruit extracts (IC₅₀ 3.87 ± 0.32). Besides, root extracts also displayed a better activity in the inhibition of DPP (IV), enhance insulin secretion and glucose uptake activity. Molecular docking results revealing that phlorizin binds strongly with α-glucosidase, DPP (IV) and Insulin receptor (IR) enzymes with achieving the lowest binding energy value. The present work suggests several of the compounds have the potential that contribute towards inhibiting α-glucosidase and DPP (IV) and thus effective in lowering post-prandial hyperglycaemia.     

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.

     

Rapid Identification of α-Glucosidase Inhibitors from Phlomis tuberosa by Sepbox Chromatography and Thin-Layer Chromatography Bioautography

Alpha-glucosidase inhibitors currently form an important basis for developing novel drugs for diabetes treatment. In our preliminary tests, the ethyl acetate fraction of Phlomis tuberosa extracts showed significant α-glucosidase inhibitory activity (IC₅₀ = 100 μg/mL). In the present study, a combined method using Sepbox chromatography and thin-layer chromatography (TLC) bioautography was developed to probe α-glucosidase inhibitors further. The ethyl acetate fraction of P. tuberosa extracts was separated into 150 individual subfractions within 20 h using Sepbox chromatography. Then, under the guidance of TLC bioautography, 20 compounds were successfully isolated from these fractions, including four new diterpenoids [14-hydroxyabieta-8,11,13-triene-11-carbaldehyde-18-oic-12-carboxy-13-(1-hydroxy-1-methylethyl)-lactone (1), 14-hydroxyabieta-8,11,13-triene-17-oic-12-carboxy-13-(1-hydroxy-1-methylethyl)-lactone (2), 14,16-dihydroxyabieta-8,11,13-triene-15,17-dioic acid (3), and phlomisol (15,16-eposy-8,13(16),14-labdatrien-19-ol) (4)], and 16 known compounds. Activity estimation indicated that 15 compounds showed more potent α-glucosidase inhibitory effects (with IC₅₀ values in the range 0.067–1.203 mM) than the positive control, acarbose (IC₅₀ = 3.72 ± 0.113 mM). This is the first report of separation of α-glucosidase inhibitors from P. tuberosa.     

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.

     

Drimanes from Drimys brasiliensis with leishmanicidal and antimalarial activity

This paper evaluates CHCl₃ and CH₃OH extracts of the stem bark, branches and leaves of Drimys brasiliensis and drimane sesquiterpenes isolated from the stem bark against strains of Leishmania amazonensis and Leishmania braziliensis promastigotes and Plasmodium falciparum trophozoites. All of the extracts and compounds were tested in cell lines in comparison with reference standards and cell viability was determined by the XTT method. The CHCl₃ and CH₃OH extracts from the stem bark and branches yielded promising results against two strains of Leishmania, with 50% inhibitory concentrations (IC₅₀ ) values ranging from 39-100 µg/mL. The CHCl₃ extract of the stem bark returned IC₅₀ values of 39 and 40.6 µg/mL for L. amazonensis and L. braziliensis, respectively. The drimanes were relatively effective: 1-β-(p-coumaroyloxy)-polygodial produced IC₅₀ values of 5.55 and 2.52 µM for L. amazonensis and L. braziliensis, respectively, compared with 1-β-(p-methoxycinnamoyl)-polygodial, which produced respective IC₅₀ values of 15.85 and 17.80 µM. The CHCl₃ extract demonstrated activity (IC₅₀ of 3.0 µg/mL) against P. falciparum. The IC₅₀ values of 1-β-(p-cumaroyloxyl)-polygodial and 1-β-(p-methoxycinnamoyl)-polygodial were 1.01 and 4.87 µM, respectively, for the trophozoite strain. Therefore, the results suggest that D. brasiliensis is a promising plant from which to obtain new and effective antiparasitic agents.     

Washingtonia filifera seed extracts inhibit the islet amyloid polypeptide fibrils formations and α-amylase and α-glucosidase activity

Washingtonia filifera seeds have revealed to possess antioxidant properties, butyrylcholinesterase and xanthine oxidase inhibition activities. The literature has indicated a relationship between Alzheimer’s disease (AD) and type-2 diabetes (T2D). Keeping this in mind, we have now evaluated the inhibitory properties of W. filifera seed extracts on α-amylase, α-glucosidase enzyme activity and the Islet Amyloid Polypeptide (IAPP) fibrils formation.Three extracts from seeds of W. filifera were evaluated for their enzyme inhibitory effect and IC₅₀ values were calculated for all the extracts. The inhibition mode was investigated by Lineweaver-Burk plot analysis and the inhibition of IAPP aggregate formation was monitored.W. filifera methanol seed extract appears as the most potent inhibitor of α-amylase, α-glucosidase, and for the IAPP fibril formation.Current findings indicate new potential of this extract that could be used for the identification or development of novel potential agents for T2D and AD.     

Antioxidant Activity and α-Glucosidase Inhibitory Activities of the Polycondensate of Catechin with Glyoxylic Acid

In order to investigate polymeric flavonoids, the polycondensate of catechin with glyoxylic acid (PCG) was prepared and its chemically antioxidant, cellular antioxidant (CAA) and α-glucosidase inhibitory activities were evaluated. The DPPH and ABTS radical scavenging activities and antiproliferative effect of PCG were lower than those of catechin, while PCG had higher CAA activity than catechin. In addition, PCG had very high α-glucosidase inhibitory activities (IC₅₀ value, 2.59 μg/mL) in comparison to catechin (IC₅₀ value, 239.27 μg/mL). Inhibition kinetics suggested that both PCG and catechin demonstrated a mixture of noncompetitive and anticompetitive inhibition. The enhanced CAA and α-glucosidase inhibitor activities of PCG could be due to catechin polymerization enhancing the binding capacity to the cellular membrane and enzymes.     

Antioxidant,inhibition of α-glucosidase and suppression of nitric oxide production in LPS-induced murine macrophages by different fractions of Actinidia arguta stem

In traditional systems of medicine, fruits, leaves, and stems of Actinidia arguta (Sieb. et Zucc.) Planch. ex Miq. have been used to treat various inflammatory diseases. The present study determined the proximate composition, antioxidant, anti-inflammatory, and hypoglycemic potential of A. arguta stem. Phenolic composition of hot water extract and its sub-fractions was determined by Folin–Ciocalteu’s reagent method. In vitro antioxidant activities of the samples were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging assays. Anti-inflammatory activity of different fractions was investigated through the inhibition of nitric oxide (NO) production in lipopolysaccharide (1 μg/ml) stimulated RAW 264.7 cells. In addition, inhibition of α-glucosidase activity of hot water extract was determined using p-nitrophenyl-α-d-glucopyranoside (pNPG) as a substrate. Ethyl acetate (557.23 mg GAE/g) fraction contains higher level of total phenolic content. The antioxidant activity evaluated by DPPH radical scavenging assay showed a strong activity for ethyl acetate (IC₅₀ of 14.28 μg/ml) and n-butanol fractions (IC₅₀ of 48.27 μg/ml). Further, ethyl acetate fraction effectively inhibited NO production in RAW 264.7 cells induced by lipopolysaccharide (LPS) than other fractions (nitrite level to 32.14 μM at 200 μg/ml). In addition, hot water extract of A. arguta stem exhibited appreciable inhibitory activity against α-glucosidase enzyme with IC₅₀ of 1.71 mg/ml. The obtained results have important consequence of using A. arguta stem toward the development of effective anti-inflammatory drugs.     

Anticholinesterse and antioxidant investigations of crude extracts,subsequent fractions,saponins and flavonoids of atriplex laciniata L.: potential effectiveness in Alzheimer’s and other neurological disorders

Background

Atriplex laciniata L. was investigated for phenolic, flavonoid contents, antioxidant, anticholinesterase activities, in an attempt to explore its effectiveness in Alzheimer’s and other neurological disorders. Plant crude methanolic extract (Al.MeF), subsequent fractions; n-hexane (Al.HxF), chloroform (Al.CfF), ethyl acetate (Al.EaF), aqueous (Al.WtF), Saponins (Al.SPF) and Flavonoids (Al.FLVF) were investigated for DPPH, ABTS and H₂O₂ free radical scavenging activities. Further these extracts were subjected to acetylcholinesterase (AChE) & butyrylcholinesterase (BChE) inhibitory activities using Ellman’s assay. Phenolic and Flavonoid contents were determined and expressed in mg Gallic acid GAE/g and Rutin RTE/g of samples respectively.

Results

In DPPH free radicals scavenging assay, Al.FLVF, Al.SPF and Al.MeF showed highest activity causing 89.41 ± 0.55, 83.37 ± 0.34 and 83.37 ± 0.34% inhibition of free radicals respectively at 1 mg/mL concentration. IC₅₀ for these fractions were 33, 83 and 82 μg/mL respectively. Similarly, plant extracts showed high ABTS scavenging potential, i.e. Al.FLVF (90.34 ± 0.55), Al.CfF (83.42 ± 0.57), Al.MeF (81.49 ± 0.60) with IC₅₀ of 30, 190 and 70 μg/ml respectively. further, H₂O₂ percent scavenging was highly appraised in Al.FLVF (91.29 ± 0.53, IC₅₀ 75), Al.SPF (85.35 ± 0.61, IC₅₀ 70) and Al.EaF (83.48 ± 0.67, IC₅₀ 270 μg/mL). All fractions exhibited concentration dependent AChE inhibitory activity as; Al.FLVF, 88.31 ± 0.57 (IC₅₀ 70 μg/mL), Al.SPF, 84.36 ± 0.64 (IC₅₀ 90 μg/mL), Al.MeF, 78.65 ± 0.70 (IC₅₀ 280 μg/mL), Al.EaF, 77.45 ± 0.46 (IC₅₀ 270 μg/mL) and Al.WtF 72.44 ± 0.58 (IC₅₀ 263 μg/mL) at 1 mg/mL. Likewise the percent BChE inhibitory activity was most obvious in Al.FLVF 85.46 ± 0.62 (IC₅₀ 100 μg/mL), Al.CfF 83.49 ± 0.46 (IC₅₀ 160 μg/mL), Al.MeF 82.68 ± 0.60 (IC₅₀ 220 μg/mL) and Al.SPF 80.37 ± 0.54 (IC₅₀ 120 μg/mL).

Conclusions

These results stipulate that A. laciniata is enriched with phenolic and flavonoid contents that possess significant antioxidant and anticholinestrase effects. This provide pharmacological basis for the presence of compounds that may be effective in Alzheimer’s and other neurological disorders.     

New Insights in Abdominal Pain in Paroxysmal Nocturnal Hemoglobinuria (PNH): A MRI Study

Introduction

Abdominal pain in PNH has never been investigated by in-vivo imaging studies. With MRI, we aimed to assess mesenteric vessels flow and small bowel wall perfusion to investigate the ischemic origin of abdominal pain.

Materials and Methods

Six PNH patients with (AP) and six without (NOP) abdominal pain underwent MRI. In a blinded fashion, mean flow (MF, quantity of blood moving through a vessel within a second, in mL·s^-1) and stroke volume (SV, volume of blood pumped out at each heart contraction, in mL) of Superior Mesenteric Vein (SMV) and Artery (SMA), areas under the curve at 60 (AUC₆₀) and 90 seconds (AUC₉₀) and K^trans were assessed by two operators.

Results

Mean total perfusion and flow parameters were lower in AP than in NOP group. AUC₆₀: 84.81 ± 11.75 vs. 131.73 ± 18.89 (P < 0.001); AUC₉₀: 102.33 ± 14.16 vs. 152.58 ± 22.70 (P < 0.001); K^trans: 0.0346 min^-1 ± 0.0019 vs. 0.0521 ± 0.0015 (P = 0.093 duodenum, 0.009 jejunum/ileum). SMV: MF 4.67 ml/s ± 0.85 vs. 8.32 ± 2.14 (P = 0.002); SV 3.85 ml ± 0.76 vs. 6.55 ± 1.57 (P = 0.02). SMA: MF 6.95 ± 2.61 vs. 11.2 ± 2.32 (P = 0.07); SV 6.52 ± 2.19 vs. 8.78 ± 1.63 (P = 0.07). We found a significant correlation between MF and SV of SMV and AUC₆₀ (MF:ρ = 0.88, P < 0.001; SV: ρ = 0.644, P = 0.024), AUC₉₀ (MF: ρ = 0.874, P < 0.001; SV:ρ = 0.774, P = 0.003) and K^trans (MF:ρ = 0.734, P = 0.007; SV:ρ = 0.581, P = 0.047).

Conclusions

Perfusion and flow MRI findings suggest that the impairment of small bowel blood supply is significantly associated with abdominal pain in PNH.     

Effects of Syzygium aromaticum-Derived Triterpenes on Postprandial Blood Glucose in Streptozotocin-Induced Diabetic Rats Following Carbohydrate Challenge

Purpose

Recent reports suggest that the hypoglycaemic effects of the triterpenes involve inhibition of glucose transport in the small intestine. Therefore, the effects of Syzygium spp-derived triterpenes oleanolic acid (OA) and maslinic acid (MA) were evaluated on carbohydrate hydrolyzing enzymes in STZ-induced diabetic rats and consequences on postprandial hyperglycaemia after carbohydrate loading.

Methods

We determined using Western blot analysis the expressions of α-amylase and α-glucosidase and glucose transporters SGLT1 and GLUT2 in the small intestine intestines isolated from diabetic rats treated with OA/MA for 5 weeks. In vitro assays were used to assess the inhibitory activities of OA and MA against α-amylase, α-glucosidase and sucrase.

Results

OA and MA ameliorated postprandial hyperglycemia in carbohydrate loaded diabetic rats as indicated by the significantly small glucose area under the curve (AUC) in treated diabetic animals compared with that in untreated diabetic rats. Western blotting showed that OA and MA treatment not only down-regulated the increase of SGLT1 and GLUT2 expressions in the small intestine of STZ-induced diabetic rats, but also inhibited small intestine α-amylase, sucrase and α-glucosidase activity. IC₅₀ values of OA against α-amylase (3.60 ± 0.18 mmol/L), α-glucosidase (12.40 ± 0.11 mmol/L) and sucrase (11.50 ± 0.13 mmol/L) did not significantly differ from those of OA and acarbose.

Conclusions

The results of suggest that OA and MA may be used as potential supplements for treating postprandial hyperglycemia.

Novelty of the Work

The present observations indicate that besides improving glucose homeostasis in diabetes, OA and MA suppress postprandial hyperglycaemia mediated in part via inhibition of carbohydrate hydrolysis and reduction of glucose transporters in the gastrointestinal tract. Inhibition of α-glucosidase and α-amylase can significantly decrease the postprandial hyperglycaemia after a mixed carbohydrate diet and therefore can be an important strategy in the management of postprandial blood glucose levels in NIDDM patients.     

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.     

Casein Fermentate of Lactobacillus animalis DPC6134 Contains a Range of Novel Propeptide Angiotensin-Converting Enzyme Inhibitors

This work evaluated the angiotensin-converting-enzyme (ACE)-inhibitory activities of a bovine sodium caseinate fermentate generated using the proteolytic capabilities of the porcine small intestinal isolate Lactobacillus animalis DPC6134 (NCIMB deposit 41355). The crude 10-kDa L. animalis DPC6134 fermentate exhibited ACE-inhibitory activity of 85.51% (±15%) and had a 50% inhibitory concentration (IC₅₀) of 0.8 mg protein/ml compared to captopril, which had an IC₅₀ value of 0.005 mg/ml. Fractionation of the crude L. animalis DPC6134 fermentate by membrane filtration and reversed-phase high-performance liquid chromatography (HPLC) generated three bioactive fractions from a total of 72 fractions. Fractions 10, 19, and 43 displayed ACE-inhibitory activity percentages of 67.53 (±15), 83.71 (±19), and 42.36 (±11), respectively, where ACE inhibition was determined with 80 μl of the fractions with protein concentrations of 0.5 mg/ml. HPLC and mass spectrometry analysis identified 25 distinct peptide sequences derived from α-, β-, and κ-caseins. In silico predictions, based on the C-terminal tetrapeptide sequences, suggested that peptide NIPPLTQTPVVVPPFIQ, corresponding to β-casein f(73-89); peptide IGSENSEKTTMP, corresponding to α_s1-casein f(201212); peptide SQSKVLPVPQ, corresponding to β-casein f(166-175); peptide MPFPKYPVEP, corresponding to β-casein f(124133); and peptide EPVLGPVRGPFP, corresponding to β-casein f(210-221), contained ACE-inhibitory activities. These peptides were chosen for chemical synthesis to confirm the ACE-inhibitory activity of the fractions. Chemically synthesized peptides displayed IC₅₀ values in the range of 92 μM to 790 μM. Additionally, a simulated gastrointestinal digestion confirmed that the ACE-inhibitory 10-kDa L. animalis DPC6134 fermentation was resistant to a cocktail of digestive enzymes found in the gastrointestinal tract.     

Flavonoid constituents,cytotoxic and antioxidant activities of Gleditsia triacanthos L. leaves

Gleditsia triacanthos L. is a deciduous tree belonging to the family Fabaceae. It possesses important biological activities as anti-mutagenic, anticancer, cytotoxic and treating rheumatoid arthritis. The total ethanol extract (EtOHE) and successive extracts (petroleum ether, chloroform, ethyl acetate, and aqueous ethanol) were prepared from the leaves. Eight flavone glycosides and two flavone aglycones named vicenin-I (1), vitexin (2), isovitexin (3), orientin (4), isoorientin (5), luteolin-7-O-ß-glucopyranoside (6), luteolin-7-O-ß-galactopyranoside (7), apigenin-7-O-ß-glucopyranoside (8), luteolin (9) and apigenin (10) were isolated from the aqueous ethanol extract of G. triacanthos L. leaves. Potent cytotoxic activity of the EtOHE extract was observed against the liver (IC₅₀ = 1.68 μg), breast (IC₅₀ = 0.74 μg), cervix (IC₅₀ = 1.28 μg), larynx (IC₅₀ = 0.67 μg) and colon (IC₅₀ = 2.50 μg) cancer cell lines. Cytotoxic activity of compounds 2, 4, 6 and 8 against, the liver, breast and colon cancer cell lines was also proved. Evaluation of the in-vivo antioxidant activity of the EtOHE and successive extracts revealed that the highest activity was exhibited by 100 mg of EtOHE (97.89% potency) as compared with vitamin E (100% potency). Compound 6 showed 91.8% free radical scavenging activity.     

Protective Effects of Mangosteen Extract on H2O2-Induced Cytotoxicity in SK-N-SH Cells and Scopolamine-Induced Memory Impairment in Mice

Mangosteen extracts (ME) contain high levels of polyphenolic compounds and antioxidant activity. Protective effects of ME against β-amyloid peptide (Aβ), induced cytotoxicity have been reported. Here, we further studied the protective effects of ME against oxidative stress induced by hydrogen peroxide (H₂O₂) and polychlorinated biphenyls (PCBs), and demonstrated the protection against memory impairment in mice. The cytoprotective effects of ME were measured as cell viability and the reduction in ROS activity. In SK-N-SH cell cultures, 200 μg/ml ME could partially antagonize the effects of 150 or 300 µM H₂O₂ on cell viability, ROS level and caspase-3 activity. At 200, 400 or 800 µg/ml, ME reduced AChE activity of SK-N-SH cells to about 60% of the control. In vivo study, Morris water maze and passive avoidance tests were used to assess the memory of the animals. ME, especially at 100 mg/kg body weight, could improve the animal’s memory and also antagonize the effect of scopolamine on memory. The increase in ROS level and caspase-3 activity in the brain of scopolamine-treated mice were antagonized by the ME treatment. The study demonstrated cytoprotective effects of ME against H₂O₂ and PCB-52 toxicity and having AChE inhibitory effect in cell culture. ME treatment in mice could attenuate scopolamine-induced memory deficit and oxidative stress in brain.     

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.     

Phytochemicals of Salacia oblonga responsible for free radical scavenging and antiproliferative activity against breast cancer cell lines (MDA-MB-231)

Salacia oblonga, an inhabitant of tropical regions has been used in traditional Indian medicinal systems. Phytochemicals were extracted in methanol from the plant and analyzed for various biological activities. The results of biochemical tests for total phenolics (297 ± 0.005 and 275 ± 0.006) and flavonoids (95 ± 0.004 and 61.6 ± 0.004) in the aerial and root parts were indicated as Gallic acid and quercetin equivalents respectively. The Aerial and root extracts showed strong reducing ability based on reducing power and FRAP assays. The extracts exhibited significant IC₅₀ values in DPPH, super oxide and nitric oxide radical scavenging assays. The extracts displayed low IC₅₀ values (<50 μg/ml) when assessed for antiproliferative activity against breast cancer cell lines using the MTT assay. GC-MS analysis of methanolic extracts have revealed the presence of compounds viz. n-Hexadecanoic acid, N-Methoxy-N-methylacetamide, Ursa-9(11), 12-dien-3-ol, Gamma-sitosterol etc., that might be potential candidates for the biological activity exhibited by the extract.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-015-0317-z) contains supplementary material, which is available to authorized users.Keyword: Salacia oblonga, Antioxidant activity, Free radical scavenging activity, Reactive oxygen species, Antiproliferative activity     

Effect of insulin-induced hypoglycemia on the serum concentrations of thyroxine,triiodothyronine and reverse triiodothyronine

The effect of insulin-induced hypoglycemia on serum thyroid hormone concentrations was studied in nine healthy individuals. Before, during and after the hypoglycemia blood samples were taken for measurement of the concentrations of glucose, thyroxine (T₄), triiodothyronine (T₃), reverse triiodothyronine (rT₃), catecholamines and pituitary hormones.There was no change in the mean serum T₄ level (± the standard error of the mean) of 67 ± 2 μg/l. However, the T₃ concentrations rose from a mean basal level of 1.86 ± 0.06 μg/l to a mean peak of 2.51 ± 0.21 μg/l (P < 0.01) at 45 minutes after the insulin injection, and the rT₃ concentrations fell from a mean basal level of 0.184 ± 0.008 μg/l to a mean nadir of 0.171 ± 0.022 μg/l (not a significant change). The mean peak epinephrine level was 545 ± 103 ng/l and it occurred between 30 and 45 minutes after the insulin injection; the mean peak norepinephrine level was 584 ± 114 ng/l and it occurred between 30 and 90 minutes after the injection. The growth hormone levels reached a mean peak of 26.1 ± 4.8 μg/l and the plasma cortisol levels rose to 215 ± 9 μg/l. The mean basal prolactin level was 8.5 ± 0.9 μg/l; in five subjects there was a rise to a mean peak of 50.6 ± 14.6 μg/l, whereas in the remaining four no significant increase occurred. No correlation was found between the changes in the serum T₃ concentration and any of the other factors studied.It was concluded that acute hypoglycemia is associated with a rapid increase in the serum T₃ concentration.     

Antioxidant and anticholinesterase investigations of Rumex hastatus D. Don: potential effectiveness in oxidative stress and neurological disorders

Background

Rumex species are traditionally used for the treatment of neurological disorders including headache, migraine, depression, paralysis etc. Several species have been scientifically validated for antioxidant and anticholinestrase potentials. This study aims to investigate Rumex hastatus D. Don crude methanolic extract, subsequent fractions, saponins and flavonoids for acetylcholinestrase, butyrylcholinestrase inhibition and diverse antioxidant activities to validate its folkloric uses in neurological disorders. Rumex hastatus crude methanolic extract (Rh. Cr), subsequent fractions; n-hexane (Rh. Hex), chloroform (Rh. Chf), ethyl acetate (Rh. EtAc), aqueous fraction (Rh. Aq), crude saponins (Rh. Sp) and flavonoids (Rh. Fl) were investigated against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at various concentrations (125, 250, 500, 1000 μg/mL) using Ellman’s spectrophotometric analysis. Antioxidant potentials of Rh. Sp and Rh. Fl were evaluated using DPPH, H₂O₂ and ABTS free radical scavenging assays at 62.5, 125, 250, 500, 1000 μg/mL.

Results

All the test samples showed concentration dependent cholinesterase inhibition and radicals scavenging activity. The AChE inhibition potential of Rh. Sp and Rh. Fl were most prominent i.e., 81.67 ± 0.88 and 91.62 ± 1.67 at highest concentration with IC₅₀ 135 and 20 μg/mL respectively. All the subsequent fractions exhibited moderate to high AChE inhibition i.e., Rh. Cr, Rh. Hex, Rh. Chf, Rh. EtAc and Rh. Aq showed IC₅₀ 218, 1420, 75, 115 and 1210 μg/mL respectively. Similarly, against BChE various plant extracts i.e., Rh. Sp, Rh. Fl, Rh. Cr, Rh. Hex, Rh. Chf, Rh. EtAc and Rh. Aq resulted IC₅₀ 165, 175, 265, 890, 92, 115 and 220 μg/mL respectively. In DPPH free radical scavenging assay, Rh. Sp and Rh. Fl showed comparable results with the positive control i.e., 63.34 ± 0.98 and 76.93 ± 1.13% scavenging at 1 mg/mL concentration (IC₅₀ 312 and 104 μg/mL) respectively. The percent ABTS radical scavenging potential exhibited by Rh. Sp and Rh. Fl (1000 μg/mL) were 82.58 ± 0.52 and 88.25 ± 0.67 with IC₅₀ 18 and 9 μg/mL respectively. Similarly in H₂O₂ scavenging assay, the Rh. Sp and Rh. Fl exhibited IC₅₀ 175 and 275 μg/mL respectively.

Conclusion

The strong anticholinesterase and antioxidant activities of Rh. Sp, Rh. Fl and various fractions of R. hastatus support the purported ethnomedicinal uses and recommend R. hastatus as a possible remedy for the treatment of AD and neurodegenerative disorders.     

Identification and Characterization of a Mucilaginibacter sp. Strain QM49 β-Glucosidase and Its Use in the Production of the Pharmaceutically Active Minor Ginsenosides (S)-Rh1 and (S)-Rg2

Here, we isolated and characterized a new ginsenoside-transforming β-glucosidase (BglQM) from Mucilaginibacter sp. strain QM49 that shows biotransformation activity for various major ginsenosides. The gene responsible for this activity, bglQM, consists of 2,346 bp and is predicted to encode 781 amino acid residues. This enzyme has a molecular mass of 85.6 kDa. Sequence analysis of BglQM revealed that it could be classified into glycoside hydrolase family 3. The enzyme was overexpressed in Escherichia coli BL21(DE3) using a maltose binding protein (MBP)-fused pMAL-c2x vector system containing the tobacco etch virus (TEV) proteolytic cleavage site. Overexpressed recombinant BglQM could efficiently transform the protopanaxatriol-type ginsenosides Re and Rg₁ into (S)-Rg₂ and (S)-Rh₁, respectively, by hydrolyzing one glucose moiety attached to the C-20 position at pH 8.0 and 30°C. The K_m values for p-nitrophenyl-β-d-glucopyranoside, Re, and Rg₁ were 37.0 ± 0.4 μM and 3.22 ± 0.15 and 1.48 ± 0.09 mM, respectively, and the V_max values were 33.4 ± 0.6 μmol min⁻¹ mg⁻¹ of protein and 19.2 ± 0.2 and 28.8 ± 0.27 nmol min⁻¹ mg⁻¹ of protein, respectively. A crude protopanaxatriol-type ginsenoside mixture (PPTGM) was treated with BglQM, followed by silica column purification, to produce (S)-Rh₁ and (S)-Rg₂ at chromatographic purities of 98% ± 0.5% and 97% ± 1.2%, respectively. This is the first report of gram-scale production of (S)-Rh₁ and (S)-Rg₂ from PPTGM using a novel ginsenoside-transforming β-glucosidase of glycoside hydrolase family 3.