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.

Natural products in the process of finding new drug candidates.

Natural derived or originated compounds still play a major role as drugs, and as lead structures for the development of synthetic molecules. About 50% of the drugs introduced to the market during the last 20 years are derived directly or indirectly from small biogenic molecules. In the future, natural products will continue to play a major role as active substances, model molecules for the discovery and validation of drug targets. A multidisciplinary approach to drug discovery involving the generation of truly novel molecular diversity from natural product sources, combined with total and combinatorial synthetic methodologies provides the best solution to increase the productivity in drug discovery and development. Screening for new drugs in plants implies the screening of extracts for the presence of novel compounds and an investigation of their biological activities. It is currently estimated that approximately 420,000 plant species exist in nature. For the purpose of lead discovery, or for the scientific validation of a traditional medicinal plant or a phytopharmaceutical, active principals in complex matrices need to be identified. Therefore, the interfacing of biological and chemical assessment becomes the critical issue. Drug discovery from plants can be guided by epidemiologic studies facilitated with computer assisted HPLC microfractionation and microplate technology. Epidemiologic studies have shown that high dietary flavonoid intake may be associated with decreased risk for cardiovascular disease. Chlamydia pneumoniae is a common human pathogen and epidemiological and clinical studies have shown a connection between chronic C. pneumoniae infection, atherosclerosis and the risk of myocardial infarction. We will present here the detection of natural compounds active against C. pneumoniae as an example.

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.

The coumarin osthol attenuates the binding of thyrotropin-releasing hormone in rat pituitary GH4C1 cells.

The influence of two plant coumarins, osthol and xanthotoxin, on intracellular Ca2+ ([Ca2+]i) transients evoked by TRH were studied in clonal rat pituitary GH4C1 cells. Osthol, but not xanthotoxin, decreased the TRH-induced transient increase in [Ca2+]i in Fluo-3 loaded cells incubated in Ca(2+)-free buffer. Binding experiments with [3H]TRH showed that osthol decreased the binding of TRH to its receptor, whereas the affinity of the receptor for TRH increased. This resulted in a decreased TRH-evoked production of IP3 in cells treated with osthol, and a decreased mobilization of sequestered calcium. Osthol did not inhibit the release of calcium evoked by exogenous IP3 in permeabilized cells. Furthermore, osthol decreased the uptake of 45Ca2+ in response to high K+. Xanthotoxin had no effects in these experiments. The results show that osthol modulates TRH-evoked responses by interacting with the TRH receptor.

Characterization of the polyphenolic composition of purple loosestrife (Lythrum salicaria).

Phenolic compounds of purple loosestrife (Lythrum salicaria L.) were analysed by the use of liquid chromatography-mass spectrometry (LC/MS) equipped with atmospheric pressure chemical ionisation (APCI) and electrospray ionisation (ESI). The presence of vitexin and orientin as well as their isomers, isovitexin and isoorientin, were confirmed using ion trap multiple stage LC/MS3 analysis. Several phenolic acids and tannins were also detected. Ellagitannins, vescalagin and pedunculagin, are reported from the plant for the first time.

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.

Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds.

Plant phenolics, especially dietary flavonoids, are currently of growing interest owing to their supposed functional properties in promoting human health. Antimicrobial screening of 13 phenolic substances and 29 extracts prepared from Finnish plant materials against selected microbes was conducted in this study. The tests were carried out using diffusion methods with four to nine microbial species (Aspergillus niger, Bacillus subtilis, Candida albicans, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa, Saccharomyces cerevisiae, Staphylococcus aureus and Staphylococcus epidermidis). Flavone, quercetin and naringenin were effective in inhibiting the growth of the organisms. The most active plant extracts were purple loosestrife (Lythrum salicaria L.) against Candida albicans, meadowsweet (Filipendula ulmaria (L.) Maxim.), willow herb (Epilobium angustifolium L.), cloudberry (Rubus chamaemorus L.) and raspberry (Rubus idaeus L.) against bacteria, and white birch (Betula pubescens Ehrh.), pine (Pinus sylvestris L.) and potato (Solanum tuberosum. L.) against gram-positive Staphylococcus aureus.

Antioxidant activity of plant extracts containing phenolic compounds.

The antioxidative activity of a total of 92 phenolic extracts from edible and nonedible plant materials (berries, fruits, vegetables, herbs, cereals, tree materials, plant sprouts, and seeds) was examined by autoxidation of methyl linoleate. The content of total phenolics in the extracts was determined spectrometrically according to the Folin-Ciocalteu procedure and calculated as gallic acid equivalents (GAE). Among edible plant materials, remarkable high antioxidant activity and high total phenolic content (GAE > 20 mg/g) were found in berries, especially aronia and crowberry. Apple extracts (two varieties) showed also strong antioxidant activity even though the total phenolic contents were low (GAE < 12.1 mg/g). Among nonedible plant materials, high activities were found in tree materials, especially in willow bark, spruce needles, pine bark and cork, and birch phloem, and in some medicinal plants including heather, bog-rosemary, willow herb, and meadowsweet. In addition, potato peel and beetroot peel extracts showed strong antioxidant effects. To utilize these significant sources of natural antioxidants, further characterization of the phenolic composition is needed.

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.

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.

Anti-inflammatory activity of extracts from leaves of Phyllanthus emblica.

Leaves and fruits of Phyllanthus emblica L. have been used for the anti-inflammatory and antipyretic treatment of rural populations in its growing areas in subtropical and tropical parts of China, India, Indonesia, and the Malay Peninsula. In the present study, leaves of Ph. emblica were extracted with ten different solvents (n-hexane, diethyl ether, methanol, tetrahydrofuran, acetic acid, dichloromethane, 1,4-dioxane, toluene, chloroform, and water). The inhibitory activity of the extracts against human polymorphonuclear leukocyte (PMN) and platelet functions was studied. Methanol, tetrahydrofuran, and 1,4-dioxane extracts (50 micrograms/ml) inhibited leukotriene B4-induced migration of human PMNs by 90% and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-induced degranulation by 25-35%. The inhibitory activity on receptor-mediated migration and degranulation of human PMNs was associated with a high proportion of polar compounds in the extracts as assessed by normal phase thin layer chromatography. Diethyl ether extract (50 micrograms/ml) inhibited calcium ionophore A23187-induced leukotriene B4 release from human PMNs by 40%, thromboxane B2 production in platelets during blood clotting by 40% and adrenaline-induced platelet aggregation by 36%. Ellagic acid, gallic acid and rutin, all compounds isolated earlier from Ph. emblica, could not explain these inhibitory activities on PMNs or platelets by Ph. emblica extracts. These results show that the leaves of Ph. emblica have inhibitory activity on PMNs and platelets, which confirm the anti-inflammatory and antipyretic properties of this plant as suggested by its use in traditional medicine. The data suggest that the plant leaves contain as yet unidentified polar compound(s) with potent inhibitory activity on PMNs and chemically different apolar molecule(s) which inhibit both prostanoid and leukotriene synthesis.

Calcium channel blocking activity: Screening methods for plant derived compounds.

Calcium channel blockers are a heterogenous group of substances that inhibit influx of Ca(2+) into the cell. Their main therapeutical influence is on the function of heart and blood circulation. A vast number of pure natural compounds with calcium antagonistic activity, mainly coumarins, have been isolated from plants and identified. Screening natural products for calcium channel blockers has been done through classical in vitro aorta or ileum strip assays, but recently in vitro screens using animal cell lines also have been established. The number of in vivo assays on natural compounds is quite limited so far. Much more research is needed to clarify the basic pharmacology and to determine the possible clinical use of pure compounds discovered from plants.

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.

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.

Choice of solvent in the extraction of Angelica archangelica roots with reference to calcium blocking activity.

Twenty solvents were tested in the extraction of compounds from the roots of Angelica archangelica L. (Apiaceae), and the calcium-antagonistic activity of the extracts was investigated. Special attention was paid to the physical and chemical properties of the solvents and their extraction abilities. The calcium antagonistic effect of the extracts was investigated by measuring the inhibition of depolarization-induced Ca2+ uptake in rat pituitary GH4C1 cells. The criteria used in determining the best solvents for the extraction were the yield and the biological activity of the extract, as well as the amount of nonpolar compounds in the extract. The final criterion used in selecting the solvent was its usability with reference to boiling point, chemical interactions (e.g. methylation), etc. Chloroform was found to be the best solvent for the extraction of nonpolar, biologically active compounds from the roots of A. archangelica.

HPLC Analysis of the Main Furanocoumarins from Peucedanum palustre.

The main furanocoumarins from the roots of PEUCEDANUM PALUSTRE were separated with a RP-HPLC method. The mobile phase for the separation of the eight main compounds (oxypeucedanin hydrate, bergapten, oxypeucedanin, isobyakangelicin angelate, ostruthol, imperatorin, isoimperatorin, and columbianadin) was optimized with the "PRISMA" model, which is a multi-solvent optimization system. The chromatographic method can be applied for quantitative determination, as was demonstrated for different samples of P. PALUSTRE. The purity and identity of the peaks were controlled by diode-array detection and comparison with standards.