In vitro anti-diabetic effect of flavonoids and pheophytins from Allophylus cominia Sw. on the glucose uptake assays by HepG2, L6, 3T3-L1 and fat accumulation in 3T3-L1 adipocytes.

BACKGROUND AND PURPOSE: Based on ethno-botanical information collected from diabetic patients in Cuba and firstly reported inhibition of PTP1B and DPPIV enzymes activities, Allophylus cominia (A. cominia) was identified as possible source of new drugs that could be used for the treatment of type 2 diabetes mellitus (T2-DM). EXPERIMENTAL APPROACH: in this study, the activity of the characterised extracts from A. cominia was tested on the glucose uptake using HepG2 and L6 cells, 3T3-L1 fibroblasts and adipocytes as well as their effect on the fat accumulation using 3T3-L1 adipocytes. KEY RESULTS: on 2-NBDG glucose uptake assay using HepG2 and L6 cells, extracts from A. cominia enhanced insulin activity by increasing glucose uptake. On HepG2 cells Insulin EC50 of 93 ± 21nM decreased to 13 ± 2nM in the presence of the flavonoids mixture from A.cominia. In L6 cells, insulin also produced a concentration-dependent increase with an EC50 of 28.6 ± 0.7nM; EC50 decreased to 0.08 ± 0.02nM and 5 ± 0.9nM in the presence of 100µg/ml of flavonoids and pheophytins mixtures, respectively. In 3T3-L1 fibroblasts, insulin had an EC50 of >1000nM that decreased to 38 ± 4nM in the presence of the flavonoids extract. However, in adipocytes, insulin produced a significant concentration-dependent increase and an EC50 of 30 ± 8nM was a further confirmation of the insulin responsiveness of the adipocytes to the insulin. At 100µg/ml, flavonoids and pheophytins extracts decreased fat accumulation in 3T3-L1 adipocytes by two folds in comparison to the control differentiated cells (p < 0.05). The crude extract of A. cominia did not show any enhancement of 2-NBDG uptake by 3T3-L1 adipocytes in the presence or absence of 100nM insulin. In addition, in fully differentiated adipocytes, both extracts produced significant decrease in lipid droplets in the cells and no lipid accumulation were seen after withdrawal of the extracts from the cell growth medium. However, there was no effect of both extracts on total protein concentration in cells as well as on Glut-4 transporters. CONCLUSIONS AND IMPLICATIONS: the pharmacological effects of the extracts from A. cominia observed in experimental diabetic models were shown in this study. A. cominia is potentially a new candidate for the treatment and management of T2-DM.

Dataset on the kinetics of the inhibition of PTP1B by the flavonoids and pheophytin A from Allophylus cominia.

The data presented in this article are related to the research article under the title "in vitro anti-diabetic activity of flavonoids and pheophytins from Allophylus cominia Sw. on PTP1B, DPPIV, alpha-glucosidase and alpha-amylase enzymes" (Semaan et al., 2017) [3]. This article defines the kinetics of inhibition of flavonoids and pheophytin A extracts from A. cominia which showed an inhibition of the PTP1B enzyme activity. The main reason to make these results public is to confirm that this study was followed up and no more experiments are needed, also to confirm that these compounds can be reported as PTP1B inhibitors.

In vitro anti-diabetic activity of flavonoids and pheophytins from Allophylus cominia Sw . on PTP1B, DPPIV, alpha-glucosidase and alpha-amylase enzymes.

BACKGROUND: Ethno-botanical information from diabetic patients in Cuba led to the identification of Allophylus cominia as a possible source of new drugs for the treatment of type 2 diabetes mellitus (T2-DM). EXPERIMENTAL: Chemical characterization of the extracts from A. cominia was carried out using chromatographic and spectroscopic methods. The extracts were tested for their activity on PTP1B, DPPIV, α-glucosidase enzymes and α-amylase. RESULTS: The flavonoid rich fractions from A. cominia inhibited DPPIV enzyme (75.3±2.33%) at 30µg/ml and produced a concentration-dependent inhibition against DPPIV with a Ki value of 2.6µg/ml. At 30µg/ml, flavonoids and pheophytins extracts significantly inhibited PTP1B enzyme (100±2.6% and 68±1% respectively). The flavonoids, pheophytin A and pheophytin B fractions showed significant concentration-dependent inhibition against PTP1B with Ki values of 3µg/ml, 0.64µg/ml and 0.88µg/ml respectively. At 30µg/ml, the flavonoid fraction significantly inhibited α-glucosidase enzyme (86±0.3%) in a concentration-dependent pattern with a Ki value of 2µg/ml. None of the fractions showed significant effects on α-amylase. Fatty acids, tannins, pheophytins A and B, and a mixture of flavonoids were detected in the methanolic extract from A. cominia. The identified flavonoids were mearnsitrin, quercitrin, quercetin-3-alloside, and naringenin-7-glucoside. CONCLUSION: The pharmacological effects of the extracts from A. cominia earlier observed in experimental diabetic models was confirmed in this study. Thus a new drug or formulation for the treatment of T2-DM could be developed from A. cominia.

The effects of cardamonin on lipopolysaccharide-induced inflammatory protein production and MAP kinase and NFkappaB signalling pathways in monocytes/macrophages.

BACKGROUND AND PURPOSE: In this study we examined the effect of the natural product cardamonin, upon lipopolysaccharide (LPS)-induced inflammatory gene expression in order to attempt to pinpoint the mechanism of action. EXPERIMENTAL APPROACHES: Cardamonin was isolated from the Greek plant A. absinthium L. Its effects were assessed on LPS-induced nitrite release and iNOS and COX-2 protein expression in two macrophage cell lines. Western blotting was used to investigate its effects on phosphorylation of the mitogen activated protein (MAP) kinases, ERK, JNK and p38 MAP kinase, and activation of the NFkappaB pathway, at the level of IkappaBalpha degradation and phosphorylation of NFkappaB. Also its effects on NFkappaB and GAS/GAF-DNA binding were assessed by EMSA. KEY RESULTS: Cardamonin concentration-dependently inhibited both NO release and iNOS expression but had no effect on COX-2 expression. It did not affect phosphorylation of the MAP kinases, degradation of IkappaBalpha or phosphorylation of NFkappaB. However, it inhibited NFkappaB DNA-binding in both LPS-stimulated cells and nuclear extracts of the cells (in vitro). It also inhibited IFNgamma-stimulated iNOS induction and GAS/GAF-DNA binding. CONCLUSIONS AND IMPLICATIONS: These results show that the inhibitory effect of cardamonin on LPS-induced iNOS induction is not mediated via effects on the initial activation of the NFkappaB or MAP kinase pathways but is due to a direct effect on transcription factor binding to DNA. However, although some selectivity in cardamonin's action is implicated by its inability to affect COX-2 expression, its exact mechanism(s) of action has yet to be identified.

Two new benzylisoquinoline alkaloids from Papaver triniifolium.

From the aerial parts of Papaver triniifolium Boiss. (Papaveraceae) collected from Erzincan (Eastern Anatolia), two new benzylisoquinoline alkaloids, miltanthoridine and miltanthoridinone were isolated. Their structures were established through the use of UV, EIMS, and NMR techniques.

Antimicrobial sesquiterpenes from Prostanthera aff. melissifolia and P. rotundifolia.

Aerial material of Prostanthera aff. melissifolia and P. rotundifolia yielded three sesquiterpenes, two known compounds and a novel sesquiterpene, prostantherol. Prostantherol was identified as (rel)-1a alpha,3a beta,7a alpha,7b alpha-decahydro-4 beta-hydroxy- 1,1,4 alpha,7 alpha-tetramethylcyclopropa[a]-naphthalene on the basis of a comprehensive spectroscopic analysis. All three compounds were investigated for antimicrobial activity against phytopathogens.

A non-covalently cross-linked chitosan based hydrogel.

Hydrogels are normally formed by the covalent cross-linking of linear polymers. In the case of chitosan based hydrogels this cross-linking is often achieved with glutaraldehyde, glyoxal or other reactive cross-linking agents. Such hydrogel materials have limited biocompatibility and biodegradability. However by the attachment of hydrophobic palmitoyl groups to glycol chitosan, a water soluble chitosan derivative, we have produced a version of the amphiphilic vesicle forming polymer-palmitoyl glycol chitosan (Uchegbu et al., 1998, J Pharm Pharmacol 58, 453-458). The level of palmitoylation in this variant of the polymer (GCP11), as determined by proton neutron magnetic resonance spectroscopy, is 19.62+/-2.42% (n=4). GCP11 has been used to prepare soft, slowly eroding hydrogels suitable for drug delivery by simply freeze-drying an aqueous dispersion of the polymer. Non-covalent cross-linking to form the gel matrix is achieved by the hydrophobic interactions of the palmitoyl groups. The resulting material, as examined by scanning electron microscopy, is porous and may be hydrated to up to 20x its weight in aqueous media without any appreciable change in volume-transforming from an opaque to a translucent solid. The slow erosion of this material in aqueous environments gives a biodegradable and ultimately more biocompatible material than covalently cross-linked hydrogels. Unlike most chitosan-based gels, the gel is hydrated to 20x its weight at alkaline pH but only 10x its weight at neutral and acid pH. This is as a result of the gradual erosion of the gel at lower pH values. Hydration is also reduced from 20x the dry gel weight in water to 10x the dry gel weight in the presence of dissolved salts such as sodium chloride. GCP11 hydrogels have been loaded to 0.1% w/w with a model fluorophore, rhodamine B, by simply freeze-drying an aqueous dispersion of GCP11 in the presence of a solution of rhodamine B dissolved in either water or phosphate buffered saline (PBS, pH=7.4). The release of this model fluorophore was retarded by between 8 and 12% when PBS was contained in the gel in accordance with the hydration profiles. Rhodamine B release was also reduced by between 13 and 25% in the presence of acid as a result of the reduced solubility of rhodamine B at acid pH.

Quaternary ammonium palmitoyl glycol chitosan--a new polysoap for drug delivery.

A new polysoap, quaternary ammonium palmitoyl glycol chitosan (GCPQ, M(w)=178,000 g mole(-1)) with drug solubilising potential has been synthesised and characterised. In solution hydrophobic domains of GCPQ polymeric micelles were identified by the hypsochromic shift in the lambda(max) of methyl orange and by the increase in the ratio of the fluorescence emission intensity of the third and first pyrene vibronic peaks (I(3)/I(1)). At high aqueous concentrations (>10 mg ml(-1)) GCPQ presents as a gel which solubilises pyrene (2.5 mM, normal solubility in water approximately 2 microM) on probe sonication. Dilution of the gel to a liquid solution of polymeric micelles (< or =3.75 mg ml(-1) of GCPQ), results in the observation of fluorescent pyrene excimers (excited dimers) and a high excimer to monomer fluorescence ratio (I(E)/I(M)). However, attempts to solubilise pyrene at a concentration of 2.5 mM in a liquid solution of GCPQ (3.75 mg ml(-1)) results in a reduced I(E)/I(M) value and pyrene precipitation. Viscometry measurements show a more compact conformation for the polymer solubilising pyrene than the polymer alone. The polymer is non-haemolytic when present as the liquid solution and relatively non cytotoxic. In conclusion, a new biocompatible polysoap (potential drug solubiliser) is described which forms hydrophobic domains in solution and shows hysteresis in its solubilisation of pyrene.

Novel weight-reducing activity of Galega officinalis in mice.

Galega officinalis (galega, Goat's Rue, French Lilac) is well known for its hypoglycaemic action and has been used as part of a plant mixture in the treatment of diabetes mellitus. During pharmacological investigations of an ethanolic extract of a powdered mixture of equal proportions of G. officinalis, Cressa cretica, Mangifera indica and Syzygium jambolanum, a weight reducing effect of galega was discovered. In this study we have investigated the novel weight reducing effect of galega in mice. Galega herb (10% w/w in the diet) caused a significant reduction in body weight in both normal and genetically obese (ob/ob) animals treated for 28 days when compared with respective controls (P < 0.01). In normal mice, the weight loss was reversible and initially associated with a transient reduction in food intake but was then maintained even in the presence of increased eating above the control level. Pair-fed normal mice receiving galega for seven days also showed significant weight loss (P < 0.01, compared with the control) in the presence of increasing food intake. In sharp contrast, weight loss in galega-treated ob/ob mice was accompanied by a persistent reduction in food intake over the 28-day treatment period. Post-mortem examinations of all galega-treated mice revealed a striking absence of body fat. Serum glucose was significantly reduced in both strains of mice receiving galega for 28 days (P < 0.01), whereas serum insulin was significantly reduced only in obese mice (P < 0.01). In summary, together with its established hypoglycaemic effects, galega has a novel weight reducing action that, in normal mice, is largely independent of a reduction in food intake. The mechanism of the weight reducing action of galega is unclear but involves loss of body fat.

Anti-inflammatory activity of Polygonum bistorta, Guaiacum officinale and Hamamelis virginiana in rats.

The aqueous ethanolic extracts of Polygonum bistorta L. Polygonaceae, Guaiacum officinale L. Zygophyllaceae and Hamamelis virginiana L. Hamamelidaceae were screened for anti-inflammatory activity. Administered (100 and 200 mg kg-1, p.o.) before the induction of carrageenan rat paw oedema, extracts of P. bistorta significantly suppressed both the maximal oedema response and the total oedema response (monitored as area under the time course curve). H. virginiana was inactive and G. officinale was only active at 200 mg kg-1. At 200 mg kg-1 administered before the induction of adjuvant arthritis, P. bistorta significantly inhibited both the acute and chronic phases of the adjuvant-induced rat paw swelling, while G. officinale and H. virginiana were only active against the chronic phase. Further studies on P. bistorta (100-800 mg kg-1) revealed a dose-dependent inhibition of the carrageenan-induced rat paw oedema over the dose range 100-400 mg kg-1, the E50 value being approximately 158.5 mg kg-1. The extract (200 mg kg-1), administered after the onset of the inflammatory responses reversed the course of both the carrageenan- and adjuvant-induced rat paw swelling. The results confirm that the extracts of P. bistorta, G. officinale and H. virginiana contain anti-inflammatory substances.

Isolation and identification of three potential impurities of pholcodine bulk drug substance.

Three previously unreported manufacturing impurities were isolated from a pholcodine mother liquor using preparative reversed-phase HPLC. The liquor was the residue remaining after recrystallisation of a production batch of pholcodine. The impurities, which are structurally related to pholcodine, were initially detected by thin-layer chromatography (TLC). Their structures were determined after separation by preparative HPLC (Econo-Prep 5 microm C18 column, 30 cm x 21.2 mm i.d.). Structure elucidation was carried out using nuclear magnetic resonance (NMR) spectroscopy, mass spectroscopy (MS) and ultra violet (UV) spectroscopy. The impurities were identified as alkylated derivatives of pholcodine possessing second 2-morpholinoethyl substituents at various positions.

Polymeric chitosan-based vesicles for drug delivery.

A simple carbohydrate polymer glycol chitosan (degree of polymerization 800 approx.) has been investigated for its ability to form polymeric vesicle drug carriers. The attachment of hydrophobic groups to glycol chitosan should yield an amphiphilic polymer capable of self-assembly into vesicles. Chitosan is used because the membrane-penetration enhancement of chitosan polymers offers the possibility of fabricating a drug delivery system suitable for the oral and intranasal administration of gut-labile molecules. Glycol chitosan modified by attachment of a strategic number of fatty acid pendant groups (11-16 mol%) assembles into unilamellar polymeric vesicles in the presence of cholesterol. These polymeric vesicles are found to be biocompatible and haemocompatible and capable of entrapping water-soluble drugs. By use of an ammonium sulphate gradient bleomycin (MW 1400), for example, can be efficiently loaded on to these polymeric vesicles to yield a bleomycin-to-polymer ratio of 0.5 units mg(-1). Previously polymers were thought to assemble into vesicles only if the polymer backbone was separated from the membrane-forming amphiphile by a hydrophilic side-arm spacer. The hydrophilic spacer was thought to be necessary to decouple the random motion of the polymer backbone from the ordered amphiphiles that make up the vesicle membrane. However, stable polymeric vesicles for use in drug delivery have been prepared from a modified carbohydrate polymer, palmitoyl glycol chitosan, without this specific architecture. These polymeric vesicles efficiently entrap water-soluble drugs.

The extraction, isolation and identification of the purgative component of Croton penduliflorus seed oil.

The purgative principles in Croton penduliflorus seed oil were isolated as white crystals by a bioassay-guided chromatographic separation process. The crystals were recovered in 7% w/w yield and identified (IR, 1H-NMR, GC-MS) as a mixture of palmitic, stearic and arachidic acids in approximately equimolar concentration.

A new dehydroaporphine alkaloid from Papaver fugax.

A new dehydroaporphine alkaloid (-)-6a,7-dehydrofloripavidine has been isolated from the aerial parts of Papaver fugax Poir. (Papaveraceae) collected from Erzurum (Eastern Anatolia). (-)-Floripavidine (aporphine) and (-)-mecambrine (proaporphine) were isolated as major alkaloids. Other bases present were (+)-salutaridine (promorphinane) and argentinine (phenanthrene). The presence of argentinine has been shown for the first time in the Papaveraceae.

Withanolides from the roots of Discopodium penninervium.

A new withanolide, 6alpha,7alpha-epoxy-5alpha,17beta-dihydroxy-1-oxowitha-2,24-dienolide, together with the known compounds withanolide A and withanone have been isolated from the roots of Discopodium penninervium. The identity of the new compound was established from spectroscopic data.

A new antibacterial sesquiterpene from Premna oligotricha.

A novel sesquiterpene, 7 alpha-hydroxy-6, 11-cyclofarnes-3(15)-en-2-one [1], has been isolated from the aerial parts of Premna oligotricha (Verbenaceae) using an antimicrobial bioassay-guided isolation procedure. The sesquiterpene was identified on the basis of spectroscopic data and showed weak activity against Gram-positive bacteria Bacillus pumilus, Bacillus subtilis, Staphylococcus aureus, and Streptococcus faecalis.

Triterpenoid Saponins from Caltha palustris.

Three triterpenoid saponins, hederagenin-3- O-alpha- L-arabinopyranoside, oleanolic acid-3- O-alpha- L-rhamnopyranosyl-(1-->2)-alpha- L-arabinopyranoside and hederagenin-3- O-alpha- L-rhamnopyranosyl-(1-->2)-alpha- L-arabinopyranoside, have been isolated from CALTHA PALUSTRIS (Ranunculaceae). The structures were determined by spectroscopic analysis of the acetates.

Comparative biotransformation of morphine, codeine and pholcodine in rat hepatocytes: identification of a novel metabolite of pholcodine.

1. Pholcodine (3-morpholinoethylmorphine), a semi-synthetic alkaloid, is widely used as an antitussive agent. 2. Norpholcodine [7,8-didehydro-4,5alpha-epoxy-3-(2-morpholinoethoxy)morphinan-6alpha-ol] (NP) and pholcodine-N-oxide [1(9a)-dehydro-(4aR,5S,7aR,9cS,12S)-4a,5,7a,8,9,9a-hexahydro-5-hydroxy-12-methyl-3-morpholinoethoxy-1H-8,9,c-(iminoethano)phenanthro[4,5-bcd] furan-12-oxide] (PNOX) were identified in incubations of pholcodine with freshly isolated rat hepatocytes by liquid chromatography/electrospray-mass spectrometry (LC/ESI-MS). 3. Synthesized NP and PNOX were characterized by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. 4. N-oxidation was the major metabolic pathway for pholcodine, producing a previously unreported metabolite. 5. The metabolism of morphine and codeine was also determined using freshly isolated hepatocytes. 6. For morphine, 3-glucuronidation was the major metabolic pathway, whilst for codeine it was dealkylation (O- and N-). 7. Neither morphine nor its metabolites were metabolites of pholcodine. 8. This observation supports the hypothesis that the absence of analgesic activity with pholcodine may be due to less O-dealkylation in vivo. 9. Together with the slow biotransformation of pholcodine (k(met) = 0.021 microM min(-1)) in comparison with morphine (k(met) = 0.057 microM min(-1)) and codeine (k(met) = 0.112 microM min(-1)), the results obtained were consistent with its low addiction potential and suggest that its antitussive efficacy is mediated by the parent drug or one of its metabolites other than morphine.