Cytotoxicity of iridals, triterpenoids from Iris, on human tumor cell lines A2780 and K562.

Six different triterpenoids (known iridals) extracted from Iris germanica L. were purified and bioassayed on two cultured human tumor cell lines: A2780 and K562 (and for each one a drug-sensitive and a drug-resistant cell line). All of the tested iridals had an IC50 in the 0.1 to 5.3 microg/ml range. Some of them were shown to be more effective than doxorubicine. Toxicity of iridals on multi-drug resistance (MDR) expressing cell lines seemed to indicate that the effects of these triterpenoids are less affected by MDR than those of doxorubicine or taxol.

Insulinotropic effect of Citrullus colocynthis fruit extracts.

Infusions of Citrullus colocynthis Schrad. (Cucurbitaceae) fruits are traditionally used as antidiabetic medication in Mediterranean countries, but to our knowledge no studies have been undertaken so far to determine the possible mechanisms involved in the antidiabetic properties of the fruit. The present study was designed to investigate whether these fruits possess insulinotropic effects. For this purpose, different extracts of Citrullus colocynthis seed components were obtained: RN II (crude extract), RN VI (hydro-alcoholic extract), RN X (purified extract) and RN XVII (beta-pyrazol-1-ylalanine), the major free amino acid present in the seeds. The insulin secretory effects of these different extracts were evaluated in vitro in the isolated rat pancreas and isolated rat islets in the presence of 8.3 mM glucose. All tested extracts, when perfused for 20 min at 0.1 mg/ml, immediately and significantly stimulated insulin secretion. This effect was transient. In addition, the purified extract (RN X) provoked a clear dose-dependent increase in insulin release from isolated islets. Moreover, a significant and persistant increase in pancreatic flow rate appeared during RN VI, RN X and RN XVII perfusions. In conclusion, our results show that different Citrullus colocynthis seed extracts have an insulinotropic effect which could at least partially account for the antidiabetic activities of these fruits.

4-Hydroxyisoleucine: effects of synthetic and natural analogues on insulin secretion.

4-Hydroxyisoleucine, a peculiar amino acid extracted from fenugreek seeds and never found in mammalian tissues, exhibits interesting insulinotropic activity. To investigate the structural requirements for this stimulating effect, the insulinotropic activity of the major isomer (2S,3R,4S) of 4-hydroxyisoleucine, in the presence of 8. 3 mM glucose, was compared to that of (1) its minor isomer (2R,3R, 4S) (2) its lactone form, (3) classical structurally related amino acids, and (4) synthetic monomethylated analogues. In the isolated, ex vivo, perfused rat pancreas, only the major isomer of 4-hydroxyisoleucine (200 microM) potentiated insulin release. On incubated isolated rat islets, the threshold concentration for a significant increase (P<0.05) in insulin release was 200 microM for (2S,3R,4S) 4-hydroxyisoleucine, 500 microM for (2S,4R) and (2S,4S) gamma-hydroxynorvalines as well as (2S,3S) and (2S,3R) gamma-hydroxyvalines, and 1 mM or more for other congeners. In conclusion, the insulinotropic properties of 4-hydroxyisoleucine, in the micromolar range, are seen only in the presence of the linear major isoform; they also require carbon alpha in S-configuration, full methylation and carbon gamma-hydroxylation.

4-Hydroxyisoleucine: experimental evidence of its insulinotropic and antidiabetic properties.

We have recently shown in vitro that 4-hydroxyisoleucine (4-OH-Ile), an amino acid extracted from fenugreek seeds, potentiates insulin secretion in a glucose-dependent manner. The present study was designed to investigate whether 4-OH-Ile could exert in vivo insulinotropic and antidiabetic properties. For this purpose, intravenous or oral glucose tolerance tests (IVGTTs and OGTTs, respectively) were performed not only in normal animals but also in a type II diabetes rat model. During IVGTT in normal rats or OGTT in normal dogs, 4-OH-Ile (18 mg/kg) improved glucose tolerance. The lactonic form of 4-OH-Ile was ineffective in normal rats. In non-insulin-dependent diabetic (NIDD) rats, a single intravenous administration of 4-OH-Ile (50 mg/kg) partially restored glucose-induced insulin response without affecting glucose tolerance; a 6-day subchronic administration of 4-OH-Ile (50 mg/kg, daily) reduced basal hyperglycemia, decreased basal insulinemia, and slightly, but significantly, improved glucose tolerance. In vitro, 4-OH-Ile (200 microM) potentiated glucose (16.7 mM)-induced insulin release from NIDD rat-isolated islets. So, the antidiabetic effects of 4-OH-Ile on NIDD rats result, at least in part, from a direct pancreatic B cell stimulation.

4-Hydroxyisoleucine: a novel amino acid potentiator of insulin secretion.

We report the characterization of a new insulinotropic compound, 4-hydroxyisoleucine. This amino acid has been extracted and purified from fenugreek seeds, which are known in traditional medicine for their antidiabetic properties. 4-Hydroxyisoleucine increases glucose-induced insulin release, in the concentration range of 100 micromol/l to 1 mmol/l, through a direct effect on isolated islets of Langerhans from both rats and humans. The stimulating effect of 4-hydroxyisoleucine was strictly glucose dependent; indeed, ineffective at low (3 mmol/l) or basal (5 mmol/l) glucose concentrations, the amino acid potentiated the insulin secretion induced by supranormal (6.6-16.7 mmol/l) concentrations of glucose. In addition, in the isolated perfused rat pancreas, we could show 1) that the pattern of insulin secretion induced by 4-hydroxyisoleucine was biphasic, 2) that this effect occurred in the absence of any change in pancreatic alpha- and delta-cell activity, and 3) that the more glucose concentration was increased, the more insulin response was amplified. Moreover, 4-hydroxyisoleucine did not interact with other agonists of insulin secretion (leucine, arginine, tolbutamide, glyceraldehyde). Therefore, we conclude that 4-hydroxyisoleucine insulinotropic activity might, at least in part, account for fenugreek seeds' antidiabetic properties. This secretagogue may be considered as a novel drug with potential interest for the treatment of NIDDM.

Composition of pulp, skin and seeds of prickly pears fruit (Opuntia ficus indica sp.).

The proximate composition of pulp, skin and seeds of prickly pear cactus (Opuntia ficus indica) was investigated and is reported on a dry weight basis. The most abundant component of the pulp and skin was ethanol-soluble carbohydrates. Pulp contained glucose (35%) and fructose (29%) while the skin contained essentially glucose (21%). Protein content was 5.1% (pulp), 8.3% (skin) and 11.8% (seeds). Starch was found in each of the three parts of the fruit. Pulp fibers were rich in pectin (14.4%), skin and seeds were rich in cellulose (29.1 and 45.1%, respectively). Skin was remarkable for its content of calcium (2.09%) and potassium (3.4%). Prickly pear is a neglected nutritional source which should be more widely used because of its potential nutrient contribution.

Characterization of a dioxygenase from Trigonella foenum-graecum involved in 4-hydroxyisoleucine biosynthesis.

A study was made of the enzyme in fenugreek implicated in the biosynthesis of 4-hydroxyisoleucine, which is an unusual amino acid known for its insulin stimulating effect. 4-Hydroxyisoleucine was detected by HPLC following isoleucine incubation with a cell-free extract from etiolated 6-day-old fenugreek seedlings in the presence of various cofactors. The reaction showed that 4-hydroxyisoleucine formation is dependent on the presence of Fe2+, 2-oxoglutarate, ascorbate and oxygen. This suggests that a 2-oxoacid dependent dioxygenase plays a key role in this biosynthetic pathway.

Protective effect of iridals from saponin injury in Candida albicans cells.

Spirostanol saponin permeabilization of Candida albicans cells resulted in phosphate (Pi) leakage from these cells. Pre-incubation of C. albicans suspension cultures with ergosterol or cycloiridals inhibited Pi leakage when cells were subjected to saponins. Saponins were shown to precipitate ergosterol but not cycloiridals. Inhibition of Pi leakage in the presence of cycloiridals could thus be due to their incorporation into cell membranes. Cycloiridals, which were previously shown to modify fluidity parameters in artificial phospholipid bilayers, seem in vivo to have a protective effect on membranes against surfactant (saponin) injury.

Steroid saponins from fenugreek seeds: extraction, purification, and pharmacological investigation on feeding behavior and plasma cholesterol.

The seeds of fenugreek (Trigonella foenum graecum L.) are traditionally assumed to have restorative properties. We have recently shown that a fenugreek seed extract containing steroid saponins increased food consumption and induced hypocholesterolemia in rats. This study aims to investigate the specific role of purified steroid saponins in these properties. For this purpose, an original technique for extraction and purification of steroid saponins was carried out. Thereafter, the effects of these steroid saponins were investigated on feeding behavior and metabolic endocrine changes in normal and diabetic rats. All the steroid saponins (furostanol type) were extracted from the seeds and separated from all other constituents of the entire extract by using several purification procedures to give an extract containing at least 90% of steroid saponins. Pharmcological experiments were performed in vivo in normal and streptozotocin diabetic rats: steroid saponins were administered chronically mixed with food (12.5 mg/day per 300 g body weight). Our data show that the treatment with steroid saponins significantly increased food intake and the motivation to eat in normal rats, while modifying the circadian rhythm of feeding behavior; it also stabilized the food consumption in diabetic rats, which resulted in a progressive weight gain in these animals, in contrast to untreated diabetic controls. Both in normal and diabetic rats, steroid saponins decreased total plasma cholesterol without any change in triglycerides. In conclusion, the present work reports a clear methodology to obtain all the steroid saponins and demonstrates that these saponins enhance food consumption and motivation to eat, and reduce plasma cholesterol levels in rats.

The protective function of the xanthophyll cycle in photosynthesis.

The rapid conversion of the carotenoid violaxanthin to zeaxanthin via antheraxanthin (xanthophyll cycle) in potato leaves exposed at 23 degrees C to a strong white light of 2000 microE.m-2.s-1 was associated with a slight inhibition of photosynthetic electron transport (as estimated from chlorophyll fluorescence measurements) and a low lipid peroxidation (as estimated from ethane measurements). When the xanthophyll cycle was blocked by dithiothreitol (3 mM) or low temperature (3 degrees C), photoinhibition of electron transport was exacerbated and pronounced lipid peroxidation occurred concomitantly. Accumulation of zeaxanthin and antheraxanthin in potato leaves by a non-photoinhibitory light treatment at 23 degrees C (900 microE.m-2.s-1 for 1 h) considerably reduced the level of lipid peroxidation during subsequent light stress at 3 degrees C. The presented results indicate that one of the functions of the xanthophyll cycle could be the protection of thylakoid membranes against lipid peroxidation, suggesting that zeaxanthin and antheraxanthin synthesized in strong light are present as free pigments in the membrane lipid bilayer.

Effects of a fenugreek seed extract on feeding behaviour in the rat: metabolic-endocrine correlates.

Fenugreek seeds (Trigonella foenum graecum L.) are assumed to have restorative and nutritive properties. The present work was designed to investigate the effects of a fenugreek seed extract on feeding behaviour. Experiments were performed to determine food consumption and motivation to eat as well as metabolic-endocrine changes in chronically treated animals. Male Wistar rats were given the seed extract orally (10 and 100 mg/day per 300 g body weight), mixed together with food, and control animals were monitored in parallel. The results show that chronic oral administration of the fenugreek extract significantly increases food intake and the motivation to eat. The treatment, however, does not prevent the anorexia nor the decreased motivation to eat induced by d-fenfluramine (2 mg/kg, IP). An increase in plasma insulin and a decrease in total cholesterol and very low-density lipoprotein (VLDL)-low-density lipoprotein (LDL) total cholesterol were also observed. In conclusion, chronic administration of a fenugreek seed extract enhances food consumption and motivation to eat in rats and also induces hyperinsulinemia as well as hypocholesterolemia.

Implication of steroid saponins and sapogenins in the hypocholesterolemic effect of fenugreek.

The transformation of fenugreek subfractions, rich in steroid saponins, was studied upon their passage through the digestive tract to determine the contribution of saponins and/or diosgenin and other steroid sapogenins to the hypocholesterolemic effect of fenugreek seeds. Feces of alloxan diabetic dogs fed fenugreek subfractions were analyzed, and diosgenin, smilagenin and gitogenin were identified and measured using capillary gas chromatography/mass spectrometry. Our results show that saponins are, in part (about 57%), hydrolyzed into sapogenins in the digestive tract. It appears that saponins may be implicated, alone or together with diosgenin, in the observed hypocholesterolemic effect of fenugreek seeds in diabetic dogs.

Improvement of Diosgenin Yield from Dioscorea deltoidea Plant Cell Cultures by Use of a Non-Traditional Hydrolysis Method.

The attractiveness of fermentor cultures of DIOSCOREA DELTOIDEA Wall. (Dioscoreaceae) as a source of diosgenin has been greatly improved by switching away from the traditional product recovery method that has been used in all previous studies. By using a known but little-used hydrolysis method involving 2N H (2)SO (4) in 70% isopropanol rather than using 2N aqueous HCl, diosgenin was found to be a growth-associated product instead of a non-growth-associated product as was formerly thought. This is an important improvement from a biotechnological standpoint because it means that diosgenin can be obtained directly from growth-phase tissue and that a non-growth phase is unnecessary. The reason that switching hydrolysis methods has this impact is that the non-traditional method gives high diosgenin yields from a broader group of steroidal glycosides. During the non-growth phase, steroidal glycosides were found to spontaneously change from furostanol saponins to spirostanol saponins. Whereas the nontraditional hydrolysis method gives high diosgenin yields from both types, the traditional method gives high yields only from the latter type.

Antidiabetic effects of subfractions from fenugreek seeds in diabetic dogs.

We have previously shown that the antidiabetic property of fenugreek seeds (Trigonella foenum graecum L.) is associated with the defatted seed material which is rich in fibers, saponins, and proteins. In the present work this defatted preparation was divided into two subfractions: subfraction "a" which contains the testa and endosperm and is rich in fibers (79.6%); and subfraction "b" which contains the cotyledons and axes and is rich in saponins (7.2%) and proteins (52.8%). We investigated the effects of each subfraction on hyperglycemia and the levels of pancreatic hormones when chronically administered to alloxan-diabetic dogs. Each subfraction was studied separately and was given to the dogs per os (mixed with the two daily meals), in addition to the insulin treatment (which was kept the same throughout the experiment) for a period of 21 days. The addition of subfraction "a" to insulin treatment resulted in a clear decrease of hyperglycemia and glycosuria accompanied by a reduction of the high plasma glucagon and somatostatin levels in diabetic dogs. The treatment also decreased the hyperglycemic response to the oral glucose tolerance test. In contrast the chronic administration of subfraction "b" had no effect on hyperglycemia or on the levels of pancreatic hormones in diabetic dogs. Our results show that the antidiabetic properties of fenugreek seeds are contained in the testa and endosperm. Although this subfraction is rich in fibers (high viscosity; 115 cP), it is not possible to exclude the existence of one or more unknown active pharmacological compounds in this subfraction of the seed.

Sterol composition and biosynthesis in sorghum: Importance to developmental regulation.

Sterol composition and biosynthesis have been examined in seeds, germinating seeds and blades from fally matured leaves ofSorghum bicolor in various stages of development'from seedlings (seven-day plants) to flowering (66-day) plants. The profile of the dominant free sterols of seeds was similar to that of leaf blades; both contained cholesterol, 24α-methylcholesterol (campesterol), 24ß-methylcholesterol (dihydrobrassicasterol), 24α-ethylcholesterol (sitosterol) and 24α-ethylcholesta-5,22-dienol (stigmasterol). Sufficient sterol intermediates were identified in the plant to indicate separate post-cycloartenol pathways to sterolic end products. The total free sterol content of the seed (µg/seed) increased somewhat during the 20 hr germination period. However, as the plant developed (seven to 48 days), there was a logarithmic increase in the leaf blade sterol content (µg/leaf blade) which plateaued at the onset of floral differentiation (ca. day 41). Over the next 18 days (48 to 66 days-period of inflorescense development), the sterol content rapidly decreased. In the early stages of plant development, the leaf blade pentacyclic triterpenoid (PT) content was negligible. With the onset of floral differentiation, PT content increased logarithmically, reaching a plateau level that surpassed the sterol content as flowering progressed. These results imply that a critical mass of sterol is associated with sorghum for floral induction. Sterol loss from the leaves of the flowering plants presumably was compensated for by the diversion of 2,3-oxidosqualene (SO) from sterol synthesis to PT production. Additional feeding and trapping experiments with [2-(14)C]mevalonic acid, [2-(3)H]cycloartenol, [24-(3)H]lanosterol [4-(14)C]sitosterol and [4-(14)C]cholesterol fed to germinating seeds and leaves from flowering plants demonstrated that sorghum possessed a cycloartenolbased pathway; germinating seeds synthesized 24-alkylsterols but not cholesterol, although cholesterol was identified in both dry and germinating seeds by gas chromatography-mass spectroscopy (GC-MS); and mature leaves synthesized cholesterol and 24α-alkylsterols but not 24ß-methylcholesterol.

Effects of fenugreek seeds on endocrine pancreatic secretions in dogs.

The components of fenugreek seeds were separated and analyzed to determine which fraction of the seed had hypoglycemic activity. These fractions were administered orally to normal or diabetic dogs for 8 days. The effect on blood glucose and pancreatic hormones was studied in normal dogs. The lipid extract had no effect; the defatted fraction (50.2% fibers: gum 17.7%, hemicellulose 22%, cellulose 8.3%, lignin 2.2%) lowered basal blood glucose level, plasma glucagon and somatostatin levels and reduced the orally induced hyperglycemia. The addition of this fraction to the insulin treatment resulted in a decrease of hyperglycemia and glycosuria in diabetic dogs. The results indicate that the defatted part is responsible for the antidiabetic action. However, the present study does not permit one to know whether the effects are caused by an unknown pharmacological compound or by the gastrointestinal action of fibers.

Hypocholesterolaemic effect of fenugreek seeds in dogs.

Fractions of fenugreek seed were added to the diet of normal or diabetic hypercholesterolaemic dogs for 8 days. The effects on levels of blood glucose, plasma glucagon and plasma cholesterol were investigated. The lipid extract had no effect. The defatted fraction which is rich in fibres (53.9%) and contains 4.8% of steroid saponins significantly lowered basal blood glucose (P less than 0.02), plasma glucagon (P less than 0.01) and plasma cholesterol (P less than 0.02) levels in normal dogs. The addition of this fraction to the food of diabetic hypercholesterolaemic dogs caused a decrease of cholesterolaemia (P less than 0.02) and reduced hyperglycaemia. In conclusion, the defatted portion of fenugreek seed induces a hypocholesterolaemic effect.