Elevated plasma fibrinogen caused by inadequate alpha-linolenic acid intake can be reduced by replacing fat with canola-type rapeseed oil.

The effects of canola-type rapeseed oil (RSO) on serum lipids, plasma fibrinogen, lipid oxidation and fatty acids were studied in three groups of subjects, two of which had not been consuming fish in their habitual diets. Forty-two volunteers (35 women, 7 men, 16-62 years) replaced fat with RSO for 6 weeks in a parallel design. The average cholesterol and fibrinogen concentrations were 5.0 mmol/l and 2.6 g/l, respectively. The intake of alpha-linolenic acid (alpha-LLA) was doubled. Efficient competitive inhibition by alpha-LLA was seen as a decrease in long-chain (LC) n-6 PUFA at 3 weeks. Elevated fibrinogen (2.6-3.9 g/l) decreased by 0.95 g/l at 6 weeks. Docosahexaenoic acid (22:6n-3) in plasma phospholipids increased at low fibrinogen levels only. The associations and changes in plasma C18 and LC PUFA followed the competitive and metabolic principles of the body, and especially in the case of n-3 PUFA according to the recycling pathway.

Preliminary antiproliferative effects of some species of Terminalia, Combretum and Pteleopsis collected in Tanzania on some human cancer cell lines.

Methanolic extracts (25 microug/ml) of species belonging to the genera of Combretum, Terminalia and Pteleopsis, collected during a field expedition in Tanzania in 1999, were screened for their antiproliferative and cytotoxic effects against three human cancer cell lines (HeLa, cervical carcinoma; T 24, bladder carcinoma; and MCF 7, breast carcinoma). A leaf extract of Combretum fragrans and a fruit extract of C. zeyheri gave the strongest antiproliferative and cytotoxic effects of all the twenty-four extracts screened in this investigation. In contrast to the highly powerful leaf extract of C. fragrans, the root extract of this species gave no cytotoxic effects against the investigated cancer cell lines at a concentration of 25 microg/ml. The other investigated species of Combretum and Terminalia differed greatly in their cytotoxic potential. Root extracts of Terminalia sambesiaca and T. sericea gave the strongest cytotoxic effects of the five species of Terminalia used in this study. Eight of the twenty-four investigated plant extracts showed pronounced cytotoxic effects (<30% proliferation compared to the control) against the T 24 bladder cancer cells, seven against the HeLa cells and four against the MCF 7 cells.

Effect of beta-sitosterol on precipitation of cholesterol from non-aqueous and aqueous solutions.

The aim of the present work was to study the solubility and phase behaviour of the beta-sitosterol-cholesterol mixed crystals in the presence and absence of water. Cholesterol, beta-sitosterol and 3:1, 1:1 and 1:3 mixtures of these were co-precipitated from acetone and acetone-water solutions. Precipitated crystals were analysed using powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), optical microscopy and Karl-Fischer titrimetry. The quantification of the sterols in solutions was preformed using GC-MS. The solubility of the sterols was mutually limiting. In the aqueous system, the solubility of both the sterols were significantly lower than in the absence of water, but the decrease in the solubility was considerably greater with the more hydrophobic beta-sitosterol. In the aqueous system, the total sterol solubility decreased with the increasing proportion of beta-sitosterol. The formation of new crystal structures, solid solutions of cholesterol and beta-sitosterol, was observed in non-aqueous as well as in aqueous environments except with the lowest cholesterol proportion in the system, in which case mixed crystals with eutectic behaviour were formed.

Ethnobotanical and antimicrobial investigation on some species of Terminalia and Combretum (Combretaceae) growing in Tanzania.

An ethnobotanical investigation on the medicinal uses of some species of Terminalia and Combretum (Combretaceae) was carried out in Mbeya, Tanzania during a 5-weeks field expedition. Of the sixteen species collected, Combretum fragrans F. Hoffm., Combretum molle G. Don., Combretum psidioides Welw., Combretum zeyheri Sond., Terminalia kaiserana F. Hoffm. and Terminalia sericea Burch ex. DC. have medical applications against various bacterial infections, such as gonorrhoea and syphilis, and against symptoms like diarrhoea, hypertension and even cancer. Antimicrobial screening of the crude extracts of the selected Combretum and Terminalia species was performed by the agar diffusion method. Among the most effective extracts were methanol extracts of the roots of Terminalia sambesiaca Engl. & Diels., T. kaiserana Guill. & Perrott., T. sericea Burch. ex DC., C. fragrans F. Hoffm. and Combretum padoides Engl. & Diels., all of which showed marked inhibition against Gram-positive bacteria, and were also good inhibitors of Enterobacter aerogenes. All four of the extracts of the roots of T. sericea tested, (methanol, ethanol, acetone and hot water) had good antimicrobial activity. A methanolic leaf extract of T. kaiserana was the only extract to have a bacteriocidic effect on Escherichia coli. Methanol root extracts of T. sambesiaca and methanol leaf extracts of T. sericea were the most effective against Candida albicans. The results of the antimicrobial screening support the ethnomedical uses of these plants.

Cholesterol-lowering effect of spreads enriched with microcrystalline plant sterols in hypercholesterolemic subjects.

BACKGROUND: Plant sterols have been shown to reduce serum lipid concentrations. The effectiveness is highly dependent on the physical state of the plant sterols. By means of a new crystallizing method, plant sterols can be added into dietary fats and oils homogeneously. In this fat ingredient, plant sterols are in a microcrystalline form. AIMS OF THE STUDY: We investigated the cholesterol-lowering effect and possible side effects of vegetable oil-based spreads fortified with two different doses of microcrystalline plant sterols. METHODS: This double-blind randomized, placebo-controlled study consisted of a 6-wk run-in and a 6-month experimental period. During the run-in period, all 155 hypercholesterolemic subjects received rapeseed oil-based control spread. In the beginning of the experimental period subjects were randomly assigned into one of three experimental groups. The control group continued to use control spread, and the two test groups used spreads with added plant sterols of either 1.5 g/d or 3.0 g/d. The subjects consumed test spreads as a part of their normal diet without any restrictions in lifestyle and diet. RESULTS: Plasma total- and LDL-cholesterol concentrations were significantly reduced by 7.5-11.6% (0.46-0.62 mmol/1) in groups consuming margarine enriched with free plant sterols, compared with the control group. The effects were similar between the two groups consuming either 1.5g or 3.0 g plant sterols per day. No effect on HDL-cholesterol or triacylglycerol concentrations occurred. The test spreads did not induce any adverse effects in blood clinical chemistry, hematology or decreases in serum concentrations of lipid soluble vitamins. CONCLUSIONS: Microcrystalline plant sterols are effective in lowering serum total- and LDL-cholesterol concentrations without obvious side effects. The daily dose of 1.5 g plant sterols is enough to reach the maximum effect.

Near infrared reflectance spectroscopy for the fast identification of PVC-based films.

Near infrared (NIR) reflectance spectroscopy was used to develop a non-destructive and rapid qualitative method for the analysis of plastic films used by the pharmaceutical industry for blistering. Three types of films were investigated: 250 microm PVC [poly(vinyl chloride)] films, 250 microm PVC films coated with 40 g m(-2) of PVDC [poly(vinylidene dichloride)] and 250 microm PVC films coated with 5 g m(-2) of TE (Thermoelast) and 90 g m(-2) of PVDC. Three analyses were carried out using different pre-treatment options and a PLS (partial least squares) algorithm. Each analysis was aimed at identifying one type of film and rejecting all types of false sample (different thickness, colour or layer). True and false samples from four plastics manufacturers were included in the calibration sets in order to obtain robust methods that were suitable regardless of the supplier. Specificity was demonstrated by testing validation sets against the methods. The tests showed 0% of type I (false negative identification) and 1% of type II errors (false positive identification) for the PVC method, 13 and 3%, respectively, for the PVC-PVDC method and no error for the PVC-TE-PVDC method. Type II errors, mostly due to the slight sensitivity of the methods to film thickness, are easily corrected by simple thickness measurements. This study demonstrates that NIR spectroscopy is an excellent tool for the identification of PVC-based films. The three methods can be used by the pharmaceutical industry or plastics manufacturers for the quality control of films used in blister packaging.

Major human plasma lipid classes determined by quantitative high-performance liquid chromatography, their variation and associations with phospholipid fatty acids.

An HPLC method with evaporative light-scattering detection (ELSD) was optimized and validated for the simultaneous quantitation of cholesteryl esters (CEs), triacylglycerols (TGs), free cholesterol (FC) and phosphatidylcholine (PC) in human plasma. The separation of CEs from TGs, the most variable plasma lipid class, was improved by speeding up the gradient steps and by increasing the re-equilibration time between runs. The calibrations were made at levels of 0.14-14 microg lipid/injection. The intra- and inter-day precision values of the method ranged between 1.9 and 4.5 and 2.3-7.2% (RSD, n=6), respectively, including determinations at two concentration levels. In comparison to other lipid classes, quantitation of PC proved to be equally repeatable despite its lowest detector response. The relative recoveries varied from 97.0 to 110.3%, showing good accuracy of the method. The methodological variation of the lipid classes covered 0.6-3.1% of their total variation in the study population (n=48). The CE/FC ratio showed an even closer relationship with phospholipid linoleic acid (18:2n-6; r=0.65, P<0.001) than with serum cholesterol levels, while eicosapentaenoic acid (20:5n-3) was significantly associated with PC (r=0.41, P<0.01). The CE/FC ratio increased (P<0.01) during soyabean oil substitution and the level of PC increased (P<0.01) during cold-pressed rapeseed oil substitution.

Antimicrobial activity of some coumarin containing herbal plants growing in Finland.

Antimicrobial screening against selected Gram-positive and Gram-negative bacteria, yeasts, mold, as well as plant pathogenic fungi, with emphasis on method optimization was carried out on methanol extracts prepared from seven plants grown in Finland. Sensitivity to the extracts was found to vary considerably among the micro-organisms, the extract from Petroselinum crispum and Ruta graveolens showing the highest toxicity against Rhizoctonia solani. The growth of Heterobasidium annosum was inhibited, whereas that of Phytophtora (cactorum) was promoted by all the extracts. The antibacterial and antifungal activities of six natural coumarin compounds were weak, except for the inhibitory effect against Fusarium culmorum.

Aging in Pinus sylvestris L. seeds: changes in viability and lipids.

Aging of Scots pine seeds (Pinus sylvestris L.) leads to changes in seed quality, such as loss of germinability, delayed growth and abnormality in developing seedlings. The knowledge of biochemical changes responsible for these aging processes is plentiful in some seeds, which are of world-wide interest, but for pine seeds these studies are rare. The aim of the present study, was to analyse pine seeds of varying ages in order to identify biochemical changes occurring in aged pine seeds, and to see if a correlation existed between these results and traditionally used seed-quality parameters, such as germinability and electrolyte leakage.

A bioassay using Artemia salina for detecting phototoxicity of plant coumarins.

Artemia salina (brine shrimp) has been successfully used for toxicity testing, and a screening test for phototoxicity has been developed based on this method. The ability of the method to test the phototoxic potential of seven known compounds was investigated. Athamantin (an angular furanocoumarin) and umbelliferone (a simple coumarin) showed no phototoxicity, while linear furanocoumarins exhibited phototoxic activity in the following order: psoralen > bergapten > peucedanin > xanthotoxin. The applicability of this method was also tested in screening the phototoxicity of plant material. Six plants from Apiaceae [Aegopodium podagraria L., Anethum graveolens L., Angelica archangelica L., Levisticum officinalis Koch, Petroselinum crispum (P. Mill) A. W. Hill., and Peucedanum palustre (L.) Moench] and one from Rutaceae (Ruta graveolens L.) were selected, all of them known to contain furanocoumarins. Extracts from leaves collected at different times during the growth period were used in the screening. Our results were in accordance with the furanocoumarin content of these plants and with the results of other phototoxicity tests. The Artemia salina method proved to be rapid, simple and inexpensive, and is therefore ideal in the initial biological screening of large numbers of samples for simultaneous detection of both toxicity and phototoxicity.

Somaclonal variation in transformed roots and protoplast-derived hairy root clones of Hyoscyamus muticus.

Substantial somaclonal variation in growth rate, morphology, and alkaloid production of Hyoscyamus muticus L. hairy root clones obtained by transformation with four Agrobacterium strains was shown. The hyoscyamine content of the root clones (n = 100) obtained from the same origin varied from 0.03 to 0.59% of dry weight. The clones produced 25-320 times less scopolamine than hyoscyamine. The best producing root clone was used as a starting material for protoplast isolation. The hyoscyamine content of protoplast-derived hairy root clones (n = 171) ranged from 0.04 to 1.45 % of dry weight. Most clones showed improved alkaloid production in relation to the parent clone, but the mean hyoscyamine content of the clones was the same as that of the parent clone. All the studied hairy root clones showed relatively stable alkaloid production during long-term cultivation. No correlation was found between alkaloid production and growth rate or morphology of the clones.

Isolation and immunomodulatory effect of flavonol glycosides from Epimedium hunanense.

The ethyl acetate and n-butanol fractions from aerial parts of E. hunanense were initially screened to find active fractions with immunomodulatory activity. Nine compounds, tricin (1), luteolin (2), thalictoside (3), icariin I (4), baohuoside I (5), quercitrin (6), icariin (7), epimedin C (8), and B (9) were isolated from this species for the first time, and 3 was isolated for the first time from flavonoid extracts of the genus. Their structures were established by chemical and spectroscopic methods. The immunomodulatory effects of the n-butanol fraction and epimedin C isolated from the fraction were investigated. Hydrocortisone acetate (HCA) was used as an immunosuppressant to inhibit the immune response of mice. The n-butanol fraction and epimedin C significantly enhanced the response of spleen antibody-forming cells (SAFC) to near normal in the mice treated with HCA. They also significantly enhanced lymphocyte proliferation and caused a significant recovery of interleukin-2 (IL-2) production in the mice inhibited with HCA. In conclusion, they are active principles with immunoenhancing effects.

Characterization of transgenic plants derived from hairy roots ofHyoscyamus muticus.

Mature plants were regenerated via protoplasts fromAgrobacterium rhizogenes-transformed root cultures ofHyoscyamus muticus L., and chemical analyses were performed on 34 individual plants. The regenerated plants showed strong phenotypic differences from clone to clone as well as from the control plants. Polymerase chain reaction studies revealed that the plants exhibiting the strongest phenotypic alterations contained therol (A, B and C) genes, whereas the plants with fewer alterations had lost them. The plants produced hyoscyamine, scopolamine and a range of different calystegins, and considerable somaclonal variation was observed. Alkaloid production in the plants transgenic for therol genes was clearly reduced. The pattern of calystegins was similar within all the regenerated plants lackingrol genes. Among the plants withrol genes, the calystegin B1 was not detectable. It seems clear that the presence ofrol genes is detrimental to the alkaloid accumulation in the transgenic plants in contrast to hairy root cultures.

Optimization of the HPLC analysis of biogenic amines in Peucedanum palustre plants and cell culture lines.

The HPLC separation of dansylated amides including mono-, di-, and polyamines from a Peucedanum palustre (L.) Moench plant and an embryogenic cell culture line was studied. The flowers, stems, leaves, and roots of the plant were analyzed separately. The mobile phase was optimized with the "PRISMA" model, which is a multi-solvent optimization system. Twenty-five standard amines were tested, twelve of which were present in the plant samples. The amine concentrations varied considerably in the different organs of the plant. There were also differences in amine concentrations between the wild plant and the embryogenic cell line and plantlets. The "PRISMA" optimization system proved to be a rapid and efficient method in the HPLC analysis of biogenic amines present in plant samples.

Efficient plant regeneration from hairy root-derived protoplasts of Hyoscyamus muticus.

Successful plant regeneration was achieved for the first time from hairy root-derived protoplasts of Hyoscyamus muticus. High yields (7 × 10(6) / g fresh weight) of protoplasts were isolated directly from the transformed roots of Hyoscyamus muticus using an enzyme mixture comprising 1 % macerozyme and 2 % cellulase in an osmoticum consisting of 0.2 M CaCl2 and 0.6 M mannitol. Protoplasts were first cultured in liquid NT/PRO I medium and further on semi-solid NT/PRO II agar medium. The procedure permits highly efficient formation of colonies. The plating efficiency varied from 1-9 %. The small individual colonies regenerated easily into shoots and roots at frequencies of 18 % and 70 %, respectively. The time required for the development of small plantlets from protoplasts was 8-11 weeks. The regenerated plants contained rolB from Ri-T-DNA and exhibited an altered phenotype compared to the control plants.

Virulence of different Agrobacterium strains on hairy root formation of Hyoscyamus muticus.

Hyoscyamus muticus accession was evaluated for its response to inoculation with Agrobacterium rhizogenes strains LBA9402, A4, 15834, and Agrobacterium tumefaciens strain C58 CI pRT GUS104. The Agrobacterium strain used for the transformation has a significant influence on the phenotype of the clone as well as on the growth rate and hyoscyamine production of these root culture clones. The most virulent strains were C58 CI pRT GUS104 and LBA9402. More roots were obtained on LSO medium than on B50 medium. Acetosyringone addition and the time from wounding affected root formation. The alkaloid content was highest in clones C58 and A4 (≈90mg·l(-1)). There are great differences between individual hairy root clones, and hence they are not as uniform as has often been speculated. The Agrobacterium strain used for the transformation has a great influence in this respect.

Volatile constituents of wild and in vitro cultivated Gloeophyllum odoratum.

The brown-rot fungus Gloeophyllum odoratum was collected from spruce stumps in southern Finland. The volatiles in the fruiting body and fungal cultures grown in malt extract and liquid medium were investigated. Chitin, chitosan and D-(+)-glucosamine at a concentration of 450 mgl-1 medium were used as elicitors. Chitosan completely inhibited growth in the solid medium. The main volatile(s) according to GC and GC-MS analysis were either linalool, citronellol, geraniol and methyl p-methoxyphenylacetate or drimenol depending on the culture type and elicitor. The composition of volatiles in the natural fungus differed slightly from that of the cultivated fungus since the major compound was methyl p-methoxyphenylacetate. The volatile oils were toxic to larvae of the brine shrimp, Artemia salina, indicating that they may possess insecticidal and cytotoxic activity.

Does potassium and magnesium supplementation lower the blood pressure of spontaneously hypertensive rats?

The effects of an orally administered potassium and magnesium supplementation on the systolic blood pressure of 42 spontaneously hypertensive rats were determined for 10 weeks. The experimental preparation was mixed with standard rat pellets and this mixture was given to three rat groups at three concentrations: 6%, 9%, and 12%. Potassium- and magnesium-containing nutrient statistically significantly inhibited the normal increase in systolic blood pressure in these rats. It could even decrease the systolic blood pressure below the initial level and its effect was similar to the known calcium antagonist verapamil. The systolic blood pressure of rats fed with the standard pellet diet containing 6% sodium chloride (NaCl) increased from 191 to 209 mmHg, whereas that of animals on 6% K-Mg supplementation decreased from 197 to 181 mmHg. 9% and 12% K-Mg supplementation produced an almost equally significant decrease in systolic blood pressure, according to Student's unpaired t-test. The experimental nutrient could possibly be used in human studies on the feasibility of normalizing blood pressure by reducing the harmful effects of dietary salt.

Bioactivity of certain Egyptian Ficus species.

The fruit extracts of Ficus sycomorus L., F. benjamina L., F. bengalensis L. and F. religiosa L. were screened for bioactivity. F. bengalensis and F. religiosa demonstrated activity in the brine shrimp test (Artemia salina) which indicates toxicity, whereas F. sycomorus and F. benjamina showed no activity. All the fruit extracts exhibited antitumor activity in the potato disc bioassay. None of the tested extracts showed any marked inhibition on the uptake of calcium into rat pituitary cells GH4C1. The extracts of the four tested Ficus species had significant antibacterial activity, but no antifungal activity. The results of this preliminary investigation support the traditional use of these plants in folk medicine for respiratory disorders and certain skin diseases.

Spontaneous somatic embryogenesis and plant regeneration from root cultures of Peucedanum palustre.

The regeneration of Peucedanum palustre (L.) Moench (milk parsley) was established for the first time via somatic embryogenesis from primary root cultures. Callus formation occurred on the root cultures and showed spontaneous embryogenic capability on B5 basal medium supplemented with a low concentration of indoleacetic acid (5.5 × 10(-7) M). 2,4-Dichlorophenoxyacetic acid was not needed for the initiation of embryogenesis. The somatic embryos germinated and formed plantlets on hormone-free B5 medium. These plantlets were easily transferable to pots, and are presently passing their second growing season in the greenhouse.Development of the somatic embryos progressed through the globular, heart-shaped, torpedo-shaped, and cotyledonary stages, typical of zygotic embryos. Synchronization performed by sieving the embryos did not affect the development time. The culture has retained its embryogenic capacity for 25 months.