aDepartment
of Pharmaceutical Sciences, University of Antwerp,
Belgium
bDepartment of Pharmacy, University of
Regensburg, Regensburg, Germany
cDepartment of
Chemistry, University of Antwerp, Belgium
Angiogenesis or neovascularization plays a role in normal physiological processes such as wound healing, but also in a number of pathological processes, for instance diabetic retinopathy, arthritis, and the growth of solid tumours. Therefore angiogenesis is a potential target for antitumoral therapy. Among the known angiogenesis inhibitors, natural compounds such as sulphated carbohydrates or triterpenoids play a prominent role [1]. In the present communication the antiangiogenic activity of a synthetic dihydrobenzofuran lignan 1, examined in the CAM (chorioallantoic membrane) assay, an in vivo model for angiogenesis, is discussed.
Synthesis of 1 from caffeic acid methyl ester has been described before [2]. The CAM assay is based upon the formation of a chorioallantoic membrane, in which neovascularization takes place, in fertilised chicken’s eggs at a certain stage of the development of the embryo [3]. Agarose pellets impregnated with the test compound are placed onto the vascular membrane of opened eggs, and the influence on angiogenesis is evaluated by observing the avascular zone surrounding the pellet. Antiangiogenic activity is expressed as a score where 0 = no or weak effect, 1 = medium effect and 2 = strong effect (capillary free zone is at least twice as large as the pellet). Also membrane irritation and embryotoxicity can be evaluated. Suramin and a b-1,4-galactan sulphate (LuPS S5) [4] with an average molecular weight of 20 kDa were used as positive control, and an agarose pellet as blank. Results are listed in Table 1. Racemic 1 was tested at a dose of 20 mg/pellet, corresponding to about 50 nmol/pellet. When evaluating both enantiomers separately, it turned out that especially the (2R,3R)-isomer exhibited a strong antiangiogenic activity, with a score of 1.5. However, also a membrane irritating effect (about 50%) was observed at 20 mg/pellet. The (2R,3R)-isomer still showed antiangiogenic properties (score 0.8) at 5 mg/pellet. Dihydrobenzofuran 1 can be considered as a new lead for antiangiogenic agents, which deserves further exploration.
Table 1. Antiangiogenic activity of 1 in the CAM-assay.
1. Paper, D.H. Planta Med. 1998, 64, 686-695.
2. Pieters, L. et al., J. Med. Chem. 1999, 42, 5475-5481.
3. Paper, D.H. et al., in Relevance of Tumor Models for Anticancer Drug Development. Contributions to Oncology, Vol. 54; Fiebig, H.H.; Burger, A.M., Eds.; Karger-Verlag: Basel, 1999, pp. 191-199
4. Hoffman, R.; Paper, D.; Donaldson, J.; Vogl, H. Br. J. Cancer 1996, 73, 1183-1186
Abt. Pharmazeutische Biologie und Mikrobiologie, Universität Hamburg, Bundesstrasse 45, 20146 Hamburg, Germany
The world-wide increase in the prevalence of antibiotic resistant bacteria requires the rapid development of novel, more potent drugs. However, neither the modification of known lead structures nor the genomics-based approach yielded a rapid solution by identification of new targets. Instead, a more promising approach is the search for new lead structures for well characterized targets. One such target is DNA-gyrase, a bacterial type II topoisomerase which in combination with topoisomerase I maintains a constant DNA supercoiling degree. For a rapid detection of inhibitors of bacterial topoisomerases a high throughput screening system was developed.
The luc gene for firefly luciferase was used as reporter gene and translationally fused to a supercoiling-dependent promoter, either ptopA or pgyrA by a PCR-based technique. The recombinant reporter gene was inserted into a broad host-range plasmid and introduced into different E. coli strains. Since the activities of the two promoters is reciprocally affected by alterations in the supercoiling degree, e.g. due to inhibition of one topoisomerase, the quotient of the respective luciferase activities designated as Qsc = activity of ptopA / activity of pgyrA, is altered. The activities of the luc gene product is determined by a luminometer.
The system responds to subinhibitory concentrations of known inhibitors of DNA gyrase, like nalidixic acid and novobiocin interacting with subunit A and B, respectively. In addition to nalidixic acid which decreases Qsc, alterations in temperature as well as different growth phases or various toposiomerase mutations have an impact on the Qsc.
Chemistry of Medicinal Plants Lab., National Research Center, Dokki, Egypt
The genus Acacia ehrenbergiana belongs to Leguminosae family. The previous studies of the chloroform extract of Acacia ehrenbergiana revealed anti-inflammatory activity (1). Here, we investigate Acacia ehrenbergiana collected in Qatar to identify the anti-inflammatory principle.
1.5 Kg of ground air-dried plant was extracted with chloroform (3 L X 3 times). The combined chloroform extract was evaporated under reduced pressure at 40°C and weighed 20 g. 19.5 g was dissolved in hexane and partitioned with 80% methanol in water. The aqueous methanol layer was concentrated under Vacuo at 40°C till half of its volume. The aqueous residue was partitioned with methylene chloride. The methylene chloride layer was evaporated under reduced pressure at 40°C. The residue was dissolved in acetone and kept at 10°C for 24 hours. The acetone soluble fraction was subjected to C-18 "Sep-Pak" cartridge and eluted with acetone. The collected fraction was concentrated and subjected to vacuum liquid chromatography (VLC) using hexane as eluant followed by gradual increasing of methylene chloride till 100% then gradual increasing by acetone till 100%. Nine fractions were collected and subjected to the bioassay described (1).
Fraction VII (50% acetone in methylene chloride) and Fraction VIII (75% acetone in methylene chloride) showed anti-inflammatory activity. The two fractions were collected and rechromatographed by VLC eluted with methylene chloride-acetone (40: 60). Five fractions were collected and subjected to bioassay.
The third fraction showed high anti-inflammatory activity. TLC showed that this fraction contained two components. The two components were separated using preparative thin layer chromatography using silica gel eluted with cyclohexane-acetone (6:4). The substance has Rf = 3.9 showed the anti-inflammatory activity. The mass spectral analysis of it showed the typical fragmentation of a-Spinasterol, a diterpene whose anti-inflammatory properties have already reported (2).
(1) Rizk, A.M., Williamson, E.M. and Evans, F.J.; Intern. J. Crude Drug. Res., 23: 1 (1985).
(2) Tang, X., Ma, Y. and Li, P. Zhangguo Zhang Yao Za Zhi, 20: 231, 253 (1995).
1Department of Biocybernetics, Tokyo Medical and Dental University, 2-3-10 Kanda Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
2Department of Pharmacology, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria
3Department of Pharmacognosy, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria
4Department of Forensic Medicine and Morbid Anatomy, Faculty of Health Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria
The pharmacological and toxicological studies of the aqueous infusion of pods of Cassia fistula (Family: Caesalpiniaceae) were studied in rodents. The aim of this study was to estimate medium lethal doses (LD50) after oral administration in mice and also to identify the target organs such as liver, kidney and testis for its possible toxic effects after oral sub-chronical administration. Pharmacologically, investigations were carried out on laxative activity in rats and effects of the aqueous infusion were also studied on the isolated guinea pig ileum in order to study the possible presence of gut stimulating constituents to rationalize the dual function in folkloric use of the plant. Results showed that the extract of pods possess significant dose-dependent laxative activity between 100 and 500 mg/kg, per os in rats. The extract also produced a concentration-dependent inhibition of the normal rhythmic contraction of the isolated guinea pig ileum at concentrations of 4-8 mg/ml, which was not blocked by the a-adrenergic receptor antagonist (phentolamine) at concentration of 10-8 M. The calculated LD50 value was 6.6 g/kg per os. The sub-chronical histopathological analysis of the essential target organs showed no adverse effect. Clinical observations and histopathological findings did not show significant differences between control and treated groups. These data corroborate the traditional use of this plant and establishes the pharmacological effect of the pods extract and may help to explain the possible dual effects of the plant depending on the level of doses employed in terms of its laxative and anti-diarrheic properties. It is concluded that the aqueous infusion of C. fistula pods is considered to be safe and have minimal side effects. The results further show that Cassia fistula pods can be used to substitute for Cassia acutifolia (Senokot® tablet), presently being imported to Nigeria as a laxative drug because of its strong laxative activity and low level of toxicity.
Elujoba A.A., Iweibo G.O. (1988) Planta Med. 4: 372
Wasfi, I.A., Bashir, A.K., et al (1994) J. Ethnopharmacology. 43:141-149
* Pharmacognosy Dept., Faculty of Pharmacy, Assiut University, Assiut, Egypt
** Medicine Dept. Faculty of Medicine, Assiut University, Assiut, Egypt
Ambrosia maritima is used in folklore medicine to treat diabetes mellitus. In the present investigation extracts of the herb were subjected to both experimental screening and a detailed clinical study to verify the reputed effects. To trace its active constituents, the drug was extracted with solvents of gradually increasing polarity followed by fractionation of these extracts and testing their hypoglycaemic activity, and detailed chemical study was performed. The esquiterpene damsin was positive although as present we can not consider it strictly for responsible for the recorded activity so the aqueous extract was used in the clinical.
Unfortunately the decoction of the herb has intense bitter taste and was rejected by all patients. To overcome this the decoction was dried and packed into gelatin capsules. The clinical evaluation of the capsulated dried decoction was conducted on 2 groups of non-insulin diabetic patients (NIDD). The investigations included blood levels, kidney functions, serum electrolytes blood pressure and liver functions. Highly significant reductions in blood glucose levels were observed. In addition the herb might be benificial for those NIDD patients who have concamitant hypertension or renal troubles.
1Department of Pharmacognosy
2Departmentof Pharmaceutical Toxicology
Faculty of Pharmacy, Hacettepe University, 06100, Ankara Turkey
The biological activities of the flavonoids have been the subject of extensive studies for the last fifteen years. Quercetin is the most abundant flavonoid in human diet. Although it has been reported to be carcinogenic and inducer of DNA damage, it has also been suggested that quercetin can effectively protect cells and tissues against the deleterious effects of reactive oxygen species. According to our recent studies, quercetin can modulate the effects of food mutagens such as 3-amino-1-methyl-5H-pyrido (4,3b) indole (Trp-P-2) and 2-amino-3-methylimidazo (4,5f) quinolone (IQ) and exhibit antigenotoxic effects since DNA damage was found to be reduced in the Comet assay in human lymphocytes(1).
Like flavonoids, phenylethanoid glycosides also represent a large group of natural products mainly spread in several botanical families of Tubiflorae (Verbenaceae, Lamiaceae, Scrophulariaceae) (2, 3).
Considering the widespread occurrence of phenylethanoids in plants, relatively little work has been done on the toxicity of this group of compounds. As a continuation of our phytochemical studies on the isolation of secondary metabolites from medicinal Lamiaceae plants, a phenylpropanoid fraction (Figure) rich in lavandulifolioside, verbascoside, leucoseptoside A and martynoside were isolated from Stachys macrantha (4 ).
To investigate the mutagenic effects of the phenylethanoid fraction a new technique Comet assay is used which provides a relatively simple assay for measuring DNA strand breakage in human lymphocytes (5). For this purpose, phenylethanoid fraction is dissolved in DMSO, which does not exceed 0.1% and studied in 4 different concentrations (10, 50, 100, 200 g/mL). The results are compared with quercetin.
General Structure of Phenylethanoids of the Fraction.
The frequency of strand breaks is not increased at the concentrations studied showing that phenylethanoids do not seem to be the inducer of DNA damage like quercetin but further studies are needed to evaluate its anti-mutagenic potential.
1. Anderson, D., Dobrzynska, M.M., Basaran, N., Basaran, A.A., Yu,T-W. Mutation Research (1998). 402, 269.
2. Cometa, F., Tomassini, I., Nicoletti M., Pieretti, S. (1993). Fitoterapia 64 (3): 195.
3. Jimenez, C., Riguera, R. (1994). Nat. Prod. Rep. 11: 591.
4. Çalýþ, I., Basaran, A.A., Saracoðlu, Ý, Sticher, O., (1992) Phytochemistry 31(1), 167.
5. Basaran, A.A., Akbay, P., Ündeðer, Ü., Basaran, N., (2001). Phytotherapy Research, in press
Department of Pharmacology, Faculty of Pharmacy, University Complutense, Madrid, Spain
Tanacetum microphyllum, an endemic species of the Iberian peninsula, has been widely used for a variety of diseases such as inflammmatory and digestive diseases in traditional medicine. We have shown that extracts of this plant exert anti-inflammatory actions in vivo, in the rat adjuvant arthritis model (1), mouse carrageenan oedema test (2), and in the PMA-induced mouse ear oedema (3). These investigations led to the identification and isolation of the most active compounds from the dichloromethane extract of T. microphyllum: four flavonoids, centauredin , 5,3-dihidroxy-4-methoxy-7-methoxycarbonylflavonol, santin and ermanin (2, 3) and one sesquiterpene lactone, hidroxyachillin (4). These compounds have shown anti-inflammatory activity in vivo (2-5) and in vitro inhibition of arachidonic acid metabolism by some of them (4, 6).
NO is a mediator implicated in inflammation. This prompted us to study the possible inhibition by these five compounds on NO production in LPS activated mouse peritoneal macrophages as a part of their anti-inflammatory mechanism. The compounds were added simultaneously or 18 h after LPS-stimulation. The concentration range was choosen after MTT assay and we tested 0-100 µM for hidroxyachillin, centauredin and 5,3'-dihydroxy-4'-methoxy-7-methoxycarbonylflavonol and 0-25 µM for santin and ermanin. We have found that all substances inhibited the production of NO in LPS-activated macrophages in a dose dependent manner, showing the following inhibition percentages: hydroxyachillin 59 % at 100 µM , 5,3'-dihydroxy-4'-methoxy-7-methoxycarbonylflavonol 80 % at 100 µM, centaureidin 72 % at 100 µM , santin 65 % at 25 µM and ermanin near 100 % at 25 µM. These results led us to study the mechanism of action of this inhibition and we have found that it was due to i-NOs inhibition.
1 Abad, M.J., Bermejo, P., Villar, A. (1991) Phytother. Res. 5, 179-181.
2 Abad, M.J., Bermejo, P., Villar, A., Valverde, S. (1993) J. Nat. Prod. 56, 1164-1167.
3 Martinez, J., Silván, A. M., Abad, M.J., Bermejo, P., Villar, A. (1997) J. Nat. Prod. 60, 142-144.
4 Abad, M.J., Bermejo, P., Valverde, S., Villar, A. (1994) Planta Med. 60, 228-231.
5 Silván, A. M., Abad, M.J., Bermejo, P., Villar, A. (1996) Inflamm. Res. 45, 289-292.
6 Abad, M.J., Bermejo, P., Villar, A. (1995) Gen. Pharmacol. 26, 81
Department of Biology, Faculty of Science, Jerash Private University, P.O. Box: 311, Jerash 26115, Jordan
Jordanian traditional medicine is characterised by a unique combination of knowledge and practice of Arabic and Islamic heritage. Medicinal plants represent one of the major components of the traditional medicine in Jordan. This work was designed to document the most commonly used medicinal plants for: (1) Childbirth and reduction of delivery pain. (2) Female fertility promotion. (3) Stop uterine bleeding. (4) Galactopoiesis.
A questionaire was prepared to fulfil the requirements of this work. The form contains data on plant material involving the vernacular name, part used, method of preparation, approximate dosage, and administration route. The data were collected from traditional healers, herbalists and midwives. All the informants were above the age of 50 years. In summary, results are:
1. Medicinal plants used for childbirth: Althaea setosa, Anisum vulgare, Carum carvi, Cassia acutifolia, Coriandrum sativum, Foeniculum vulgare, Marticaria aurea, Nigella sativa, Phoenix dactylifera, Salix fragilis, Salvia triloba and Trigonella foenum gracum.
2. Medicinal plants used for female fertility promotion: Alpinia officinarum or / Langnas officinarum, Eruca sativa, Foeniculum vulgare, Linum usitatissmum, Malva parviflora, Phoneix dactylifera, Raphanus sativus and Trigonella foenum gracum.
3. Medicinal plants used for stopping uterine bleeding: Achillea fragrantissima, Capsella bursa pastoris and Ceratonia siliqua.
4 Medicinal plants used for galactopoiesis: Anisum vulgare, Arachis hypogaea, Carum carvi, Foeniculum vulgare, Nigella sativa, Pistacia atlantica, Sesamum indicum, Trigonella foenum gracum and Urtica pilulifera.
The medicinal plants used were frequently prepared as decoction and taken orally.
In the light of the data presented in this work, the implementation of research programs to study the physiology, pharmacology and toxicity potentials of these herbs would make a real contribution to the welfare of Jordanian population.
Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran
A survey in the field of traditional Iranian, Indian and Chinese medicine indicates the therapeutic benefits of consumption of leaves and stems of Viscum album and their extensive use for the treatment of many disorders. However the therapeutic effects of Viscum album fruits are not evaluated in detail. The present research was designed to study the anti-diabetic effects of the aqueous extract of mistletoe fruits.
By subcutaneous injection of Alloxan (l60 mg/kg body weight) 14 male Wistar rats were become diabetic and then randomly divided into experimental and control groups (n=7). During the experimental period, the experimental rats were received aqueous extract of mistletoe (0.5 g/L) through drinking water. Mistletoes were collected from Mazanderan forest in north of Iran. Blood samples were collected weekly from rats of both groups and the blood pH, the glucose, urea, triglyceride and cholesterol levels of blood serum and acetoacetic acid level of urine were measured (table).
Statistical comparison of the results obtained from experimental and control groups
The symptoms of diabetes mellitus disease are including metabolic alterations which preliminary recognized by hyperglycemia and glycosuria and then by disorders of protein and fat metabolism, followed by body weight lose, alteration of blood pH (acidosis) and other biochemical factors in internal environment. The results obtained from the present research indicate the obvious anti-diabetic effects of mistletoe consumption. Because Alloxan destroys Beta cells of Langerhans islets, mistletoe consumption by lowering the blood glucose level, probably by increasing the permeability of cells plasma membrane to glucose, prevent the progressive disorders and correct the metabolic alterations due to diabetes.
1Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
2Centro de Biología Fundamental, Instituto Carlos III, Crt. Majadahonda a Pozuelo, 28220 Majadahonda, Madrid, Spain
In our search for new classes of antiviral agents, we have examined extracts of several South American medicinal plants, some of them used in traditional medicine. The results presented here concern several Ecuatorian and Bolivian medicinal plants from the following species: Baccharis trinervis Pers., Baccharis teindalensis L., Baccharis genistelloides (Lam.) Pers, Baccharis rubricaulis Rusby, Ambrosia arborescens Miller., Eupatorium articulatum Hort., Eupatorium glutinosum Lam., Tagetes pusilla H.B.& K., Neurolaena lobata R. Br. and Conyza floribunda H.B.& K. from the Compositae; Phoradendron crassifolium (Polh.) Eicher from the Loranthaceae; Rumex obtusifolius L. from the Polygonaceae; Plantago australis Lam. from the Plantaginaceae and Satureja boliviana (Benth.) Briquet from the Lamiaceae.
Ethanolic and aqueous extracts were tested for inhibitory activity on human immunodeficiency virus (HIV). Both types of extracts were relatively non-toxic to human lymphocytic MT-2 cells, but only the aqueous extract of Baccharis trinervis exhibited potent anti-HIV activity in an in vitro MTT assay. To delineate the extract-sensitive phase, some studies of the antiviral properties of the active extract are described in this paper. Based on the results presented here, a separation scheme was devised, which permitted the preliminary fractionation of the extract, with the aim of finding an inhibitor of this virus.
Acknowledgement: Supported by PR182-6738/96 UCM, CAM 08.2/0006/1998, Fundación Caja Madrid, FIPSE 3074/99 and Ministerio de Educación y Ciencia (SAF 98/0186).
1Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
2Centro de Biología Fundamental, Instituto Carlos III, Crt. Majadahonda a Pozuelo, 28220 Majadahonda, Madrid, Spain
In an ethnopharmacological screening of selected medicinal plants, folk medicinal plants were assayed in vitro to detect anti-HIV activity. In order to find antiviral compounds, ethanolic and aqueous extracts from Spanish medicinal plants have been tested for their antiviral activity against Human Inmunodeficiency Virus type I (HIV-1). We evaluated the antiviral activity by the cytopathic effect (CPE), the cellular viability using a colorimetric assay (MTT) and a bioluminometric detection (luciferase assay). Aqueous extract of Tuberaria lignosa (Sweet) Samp. showed moderate activity against HIV-1 and a strong cytotoxicity. Lyophilized aqueous extract was divided in soluble and insoluble ethanolic 70% fractions, and the ethanolic insoluble fraction showed a potent activity, reaching more than 60% cytoprotective effect. The effective aqueous extracts were fractionated by gel filtration (Sephadex LH-20) in order to study the active compounds. We evaluated the activity of the five fractions obtained.
Acknowledgement: Supported by PR182-6738/96 UCM, CAM 08.2/0006/1998, Fundación Caja Madrid, FIPSE 3074/99 and Ministerio de Educación y Ciencia (SAF 98/0186).
Institute of Pharmaceutical Biology and Phytochemistry, Hittorfstr. 56, D-48149 Münster, Germany
The serin proteinase elastase plays an important role in the degradation of connective tissue in that it disintegrates dead or defective tissue. In healthy tissue, the activity of elastase is limited by endogenous inhibitors. An increasing concentration of the elastase activity supports various disorders such as arthrithis and rheumatism.
The secondary roots of Harpagophytum procumbens (Pedaliaceae), a herbaceous plant from the South-West parts of Africa, are known for their anti-inflammatory and antirheumatic activity. However, the active substances are not completely known. Most of the targets hitherto used for identification of active constituents stem from the arachidonic cascade. Yet, another effect may be the inhibition of the elastase activity.
We investigated the aqueous extract of Harpagophyti radix for its ability to inhibit this enzyme. Furthermore, we tested several secondary metabolites as shown in the table. Percentage of compounds is based on the dry matter of the aqueous extract of H. procumbens. Inhibition by the crude extract was observed at a concentration of 371µg/mL. 6’-O-acetylacteoside shows the most potent effect with an IC50 of 47 µg/mL followed by caffeic acid at 85 µg/mL.
Institute of Pharmacognosy, University of Graz, Universitaetsplatz 4/1, A-8010 Graz, Austria
Rhus aromatica AIT. (sumac; Anacardiaceae) is widely distributed in North America, East Asia and South Africa. Previous phytochemical studies on the root bark revealed fatty acids, triterpenes, sterols, essential oil, flavonoids, tannins and orcin-b-D-glucoside (1). Rhois aromaticae rad. cort. is used as herbal drug and in pharmaceutical preparations against disturbances of the urinary tract. Extracts have been reported to possess antiviral (2) and antimicrobial activity (3). This study was carried out as part of our search for new antioxidant, antiinflammatory and antitumor agents from plant sources. The antioxidative properties were measured by the DPPH assay (4), the xanthine oxidase test (5), and the assay for non-enzymatic lipid peroxidation in liposomes (6). The antiinflammatory activity was investigated by the HET-CAM assay (7, mod.) and the croton-oil test (8). The antitumor effect was estimated by the EBV-EA activation test (9). The EtOH 30% and MeOH extracts were tested by the DPPH assay in concentrations of 0.01-1mg/ml. The EtOH extract exhibited a strong dose-dependent inhibition of DPPH activity (IC50 10.9 ± 0.45µg/ml) which was similar to catechin and epicatechin. The MeOH extract was less potent. In the xanthine oxidase assay both extracts did not show any activity up to the concentration of 60µg/ml. The EtOH extract, BHT and quercetin were investigated by the non-enzymatic lipid peroxidation assay for their ability to act as -OH radical scavenging agents. The results showed that the EtOH extract exhibited Fe(III)-ascorbate-induced peroxidation of phospholipid liposomes in a dose-related manner (IC50 20.2 ± 1.42µg/ml). The presence of Fe(II) and Cu(II) led to a less potent activity (IC50 34.6 ± 2.02µg/ml and IC50 25.0 ± 1.52µg/ml, resp.). In the HET-CAM assay only the EtOH extract showed a pronounced reduction of inflammation (score value 31.0 ± 1.55; conc 200µg/pellet). The results were confirmed by the croton–oil test as in vivo test model. These antiinflammatory properties support the traditional use of Rhus aromatica for the treatment of cystitis and nephritis. The anticarcinogenic activity of the extracts was evaluated involving Epstein-Barr early antigen activation in Raji cells promoted by 12-O-tetradecanoylphorbol-13 acetate. At a concentration of 10µg/ml the inhibition of antigen activation was found to be 33.2% while 100µg/ml gave 85.6% of inhibition.
Acknowledgement: The authors express sincere gratitude to Prof. Dr. H. Tokuda, Dept of Biochemistry, Kyoto Prefactoral University of Medicine, Kyoto / Japan, for performing the EBV-EA activation test. Thanks go also to Prof. Dr. R. Della Loggia, DEMREP, University of Trieste / Italy, for ongoing biological testing (Croton-oil test).
1. Effenberger S. (1990) Inaugural-Dissertation, Fachbereich Pharmazie der FU Berlin
2. May G.et al. (1978) Arzneim.Forsch. 28: 1-7.
3. Brantner AH. (1999) Drogenreport 21: 27-28
4. Hatano T.et al. (1988) Chem.Pharm.Bull. 36: 2090-2097
5. Noro T.et al. (1983) Chem.Pharm.Bull. 31: 2708-2711
6. Houghton PJ.et al. (1995) Planta Med. 61: 33-36
7. D’Arcy PF et al. (1967) Br. J. Pharmac.Chemother. 29:378-387
8. Tubaro A.et al. (1985) Agents Actions 17: 347-349
Tokuda H.et al. (1996) Cancer Letters 104: 91-95
1 Institute of Pharmacology and Toxicology, Domagkstrasse 12, 48149 Muenster, Germany
2 Section on Functional Neuroanatomy, National Institute of Mental Health, Bethesda, Maryland, 20892, USA
The effects of long-term (once daily for 7 weeks) oral imipramine (15 mg/kg) and St. John’s wort (Hypericum, 500 mg/kg) administration on changes in gene transcription induced by repeated immobilization stress have been studied in stress-responsive rat brain circuits. In situ hybridization was used to measure mRNA levels of corticotropin-releasing hormone (CRH) and vasopressin (AVP) in the hypothalamic paraventricular nucleus (PVN); proopiomelanocortin (POMC) in the pituitary; glucocorticoid receptors (MR, type I and GR, type II), serotonin 1A receptors (5-HT1A), brain-derived neurotrophic factor (BDNF), and cyclic AMP response element binding protein (CREB) in the hippocampus; glutamic acid decarboxylase (GAD) in the bed nucleus of the stria terminalis (BST); and tyrosine hydroxylase (TH) in the locus coeruleus. Our results show that repeated immobilization stress (2 h daily for 7 days) produced a marked increase in mRNA expression of CRH in the PVN, POMC in the anterior pituitary, GAD65/67 in the BST, CREB in the hippocampus, and TH in the locus coeruleus. It produced a significant decrease in mRNA expression of 5-HT1A and BDNF in the hippocampus and no changes in mRNA expression of AVP in the PVN or MR and GR in the hippocampus. Long-term pre-treatment with either imipramine or St. John’s wort reduced to control levels the stress-induced increases in gene transcription of GAD65 and GAD67 in the BST, CREB in the hippocampus, and POMC in the anterior lobe of the pituitary. The stress-induced increases in mRNA levels of CRH in the PVN and TH in the locus coeruleus were reduced by imipramine but not by the plant extract. The stress-induced decreases in BDNF and 5-HT1A mRNA levels in hippocampus were not prevented by either imipramine or St. John’s wort. These data show that select stress-induced changes in gene transcription in particular brain areas can be prevented by long-term treatment with either the prototypic tricyclic antidepressant imipramine or the herbiceutical St. John’s wort. However, imipramine appears to be more effective in blocking stress effects on the HPA axis than the plant extract.
1 Institute of Pharmacology and Toxicology, Domagkstrasse 12, 48149 Muenster, Germany
2 Institute of Anatomy, Vesaliusweg 2-4, 48149 Muenster, Germany
Chronic effects of St. John’s wort and hypericin, one of its major active compounds, on regional brain amine metabolism have not been reported. We used a HPLC system to examine the effects of short-term and long-term administration of imipramine, Hypericum extract, and hypericin on regional levels of serotonin (5-HT), norepinephrine (NE), dopamine (DA) and their metabolites in the rat brain. We focussed our interest on hypothalamus and hippocampus as these brain regions are thought to be major targets of antidepressant drug action.
Imipramine, Hypericum extract, and hypericin given daily for 8 weeks significantly increased 5-HT levels in hypothalamus. 5-hydroxyindolicacetic acid (5-HIAA) levels showed no change in both brain regions. The [5-HIAA] / [5-HT] ratio was significantly lowered in the hypothalamus and in the hippocampus after 8 weeks of daily treatment with the Hypericum extract. Consistent changes in catecholamine levels were only detected in hypothalamus tissues after long-term treatment. Comparable to the plant extract, imipramine as well as hypericin significantly decreased DOPAC and HVA levels in the hypothalamus. The dopamine turnover ratio in hypothalamus tissues was reduced after eight weeks by Hypericum extract and hypericin, it was only slightly reduced by imipramine. Hypericin selectively reduced NE concentrations in the hippocampus after two as well as after eight weeks. A similar effect was observed for imipramine in the hypothalamus but only after the eight weeks treatment period.
Our data clearly show that only long-term administration of St. John’s wort and its acitve constituent hypericin modify levels of neurotransmitters in brain regions involved in the pathophysiology of depression.
Institute of Pharmacognosy, University of Graz, Universitaetsplatz 4/1, A-8010 Graz, Austria
Bacterial infections are a world wide problem. In the last decade antibiotic resistant infections occurred demanding new therapeutic strategies. For people living in developing countries mainly medicinal plants and natural substances are available for the treatment of infectious diseases. Medicinal plants and plant compounds usually show a weak antimicrobial activity. The gram-positive methicillin resistant Staphyllococcus aureus (MRSA) is one of the bacteria causing nosocomial infections by the expression of multidrug resistant (MDR) efflux pump. Very few antimicrobial agents are available so far to treat MRSA infections. The objective of this study was to investigate the synergistic effects of natural compounds against MRSA and Staphyloccocus aureus in comparison. The investigation was carried out as a part of our search for plants and compounds with antimicrobial activities (1).
The use of berberine alkaloids from berberine species as a MDR pump inhibitor is well documented (2). It has also been reported that a compound without any activity can improve the effect of berberine.
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berberine |
harmine |
yohimbine |
Therefore four alkaloids (berberine.HCl, berberine.SO4, harmine, yohimbine) alone and in combinations were tested in a dilution serie against the methicillin resistant Staphyloccocus aureus NCTC 10442 and Staphylococcus aureus ATCC 25923. The combined biocide action was investigated by a dilution method (3). The results were expressed as fractional inhibitory concentration (FIC) index which indicates the ratio of the MIC for the substance in combination to that of the substance alone. By this index synergistic, antagonistic and indifferent effects were evaluated.
From the results it is confirmed that none of the alkaloids alone has any activity against MRSA up to a concentration of 600µg/ml. Harmine and yohimbine were tested in combinations with berberine.HCl in different concentrations against MRSA. It has been observed that harmine improved the activity of berberine.HCl. Berberine.HCl alone did not show any effect against MRSA in the concentrations tested.
1. Chakraborty A., Brantner A H., J. Ethnopharmacol. 68: 339, (1999)
2. Stermitz, F.R., Tawara Matsuda; J; Lorenz, P., Lewis, K., Proc. Natl. Acad. Sci. 97, 1433, (2000)
3. European Pharmacopoeia (1997) 3rd ed.
Institute of Pharmacy, Humboldt-University of Berlin, Goethestr. 54, D-13086 Berlin, Germany
Reactive oxygen species such as superoxide anion, hydroxyl radical, single oxygen and hydrogen peroxide can induce peroxidative degradation of biomembranes and have been related to different diseases as inflammatory diseases, myocardial infarction, atherosclerosis, as well as aging and cell death.
Endothelial cells as an element of the vascular system show a high risk for oxidant injury because of the close contact with flowing blood as well as the location of xanthine oxidase. Therefore they seem to be a target of the cytotoxicity by oxidant stress induced by hypoxanthine-xanthine oxidase.
We tested the decline of endothelial cells (ECV-304 cell-line) after oxidant injury with hypoxanthine-xanthine oxidase and determined protection provided by flavone and flavonol glycosides as well as by caffeic acid because of their well known antioxidative and radical scavenger activity.
We tested different concentrations of hypoxanthine and xanthine oxidase as well as different times of exposure. Suitable effects (cytotoxicity of about 80 %) were achieved with 100 µM hypoxanthine and 2 mU/ml xanthine oxidase.
To test the effect of a variety of flavonoids and caffeic acid on the hypoxanthine-xanthine oxidase-induced toxicity we investigated different concentrations of these compounds. In these experiments the cells were incubated with both hypoxanthine-xanthine oxidase and test substance for 1 h. After the period of cell damage the buffer was removed and the cells were cultivated with fresh medium for further 4 days.
Quercetin had the strongest effect on the inhibition of hypoxanthine-xanthine oxidase-induced toxicity and was able to inhibit toxicity almost completely at a concentration of 6 µM. The beneficial effect ofthe flavonol glycosides rutin and hyperoside was quite weak (40% restitution at 200 µM and 400 µM, respectively). The flavone glycoside diosmin showed a better effect of protection (50% restitution at 10 µM). We determined the effect of caffeic acid and its phenyethylether at 100 µM and 10 µM, respectively for a 50 % restitution of cell growth.
Our results suggest that flavonoids and phenolcarbonic acids in our nutrition as well as in phytotherapeutical supplements of human diet are expected as preventive agents. These substances protect endothelial cells against free oxygen radicals often involved in the pathogenesis of diseases.
Department of Pharmacy, Division of Pharmacognosy, FIN-00014 University of Helsinki, Finland
Mentha aquatica, M. arvensis var. piperascens, M. crispa, M. x dalmatica, M. sp. "Frantsila", M. haplocalyx, M. "Native Wilmet" and M. x verticillata deodorised water-soluble extracts were assessed for their respective abilities to reduce iron from ferric to ferrous as quantified indirectly from the spectrophotometric measurement of Prussian blue, a coloured complex. The extracts clearly demonstrated variable degrees of ferric to ferrous reducing power across the concentration range [0.01 to 1.00 mg/mL] assessed however, with the exception of Mentha “frantsila” [1.00 mg/mL, p > 0.20]. None of the extracts exhibited comparable activity to Pycnogenol® (positive control), a patented procyanidin-rich water-soluble extract obtained from the bark of French maritime pine (P. maritima L.). Although the ferric to ferrous iron reduction assay is not necessarily a robust indicator of the overall antioxidant efficacy of a natural product-rich plant-derived extract, its use in a battery of antioxidant bioactivity screening techniques has merit. Thus, the extracts isolated from these Mentha species will undergo further investigation to characterise their overall antioxidant potential.
1Hacettepe University, Faculty of Pharmacy, Dept. of Pharmacognosy, Ankara, Turkey
2Atatürk University, Faculty of Medicine, Dept. of Pharmacology, Erzurum, Turkey
3Atatürk University, Faculty of Pharmacy, Dept. of Pharmacognosy, Erzurum, Turkey
The genus Centhranthus (Valerianaceae) is represented by three species in the flora of Turkey (l). Plants of this family are well known for their sedative properties (2). Centranthus longiflorus ssp. longiflorus ia a perennial herb and is traditionally used as sedative (3). In a previous paper we reported the chemical constituents of this plant. Some of them were iridoids and valepotriates whereas other compounds possessed flavonoid and triterpene structures (4).
In this study, iridoid containing aqueous extract, potentially possessing sedative properties were investigated for their sedative, anticonvulsant and antidepressant activities using the thiopental sleeping test, caffeine induced convulsion test and forced swimming depression test and compared to diazepam. Mice and Sprague-Dawley rats were used as the experimental animals. The aqueous extract of Centranthus longiflorus ssp. longiflorus (Cle-1) was administered by gavage 60 min before the thiopental sleeping test in doses of 25, 50, 100 and 200 mg/kg and for the convulsion and forced swimming depression tests in doses of 50, 100, 200 mg/kg. Diazepam was dissolved in distilled water and used as a reference in a dosis of 5 mg/kg given by gavage. The 100 mg/kg dose level of Cle-1 was found more active than other doses tested. When the effect of Cle-1 was compared with diazepam, the aqueous extract (100 mg/kg) showed sedative, anticonvulsant and antidepressant effects similar to the effect of diazepam at 5 mg/kg dose level.
Student's t test and probability level of p<0.05 were chosen as the criterion of statistical significance. These results imply the potential use of Centranthus longiforus ssp. longiflorus preparations as herbal medicines with sedative activity.
1. Davis, P. H., Flora ofT urkey and East Aegean Islands Vol. 4, University Press, 1972, Edinburgh p. 558-559
2. Hölzl, J., Fink, C., Arznm.-Forsch., 1984, 34 (1) 44-47
3. Baytop, T., Türkiye'de Bitkiler ile Tedavi, Sanal Matbaasi, 1984, Istanbul, p.282
4. Demirezer, L. Ö., Güvenalp, Z., Schiewe, H-J., Strietzel, I., Harmandar. M., Zeeck, A., Phytochemistry, 1999, 51, 909-912
1Dept. of Pharmacology, Faculty of Medicine, Atatürk University, Erzurum,Turkey
2Dept. of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey
Rumex (Polygonaceae) species are widely spread plants in the flora of Turkey [l]. Several Rumex species are ethnomedically used for inflammation and fever [2]. In our previous study, antiinflammatory effect of R. patientia roots was reported [3]. It is known that antiinflammatory drugs (NSAID) have analgesic and antipyretic activities [4]. In this study the aqueous extract from the roots of R. patientia L. (D-1) was investigated for its analgesic and antipyretic effects by using paw licking and pyrogene induced hyperthermia assays on mice and rabbits. D-1 (10 mg/kg) decreased the pain induced by formadehyde 1.9 times in comparison with control the group in early phase (0-5 min). Morphine, ASA and indomethacin reduced the pain 2.7, 1.6 and 2.5 times more, respectively, than control group. D-1 inhibited the pain, which occurred in the late phase (15-30 min) of the formaldehyde test, 5.3 times more than the control group. According to the antipyretic activity results, 150 mg/kg D-1 reduced hyperthermia. The maximal inhibitory effects of Rumex extract were obtained at different times after treatment, the average temperature being reduced by 0.3 °C (p<0.008 after l hour), 0.8 °C (p<0.001 after 2 hours), 1.5 °C (p<0.0001 after 3 hours) and 1.8 °C (p<0.0006 after 4 hours) as compared to hyperthermia rabbits. ASA and indomethacin exerted an activity profile similar to that obtained with Rumex extract. All findings together confirmed the ethnomedical data concerning the analgesic and antipyretic effects of the herbal remedies from this plant species.
l. Davis, P.H. (1967) Flora of Turkey and the East Aegean Islands, Edinburgh, University Press.
2. Baytop, T. (1984) Therapy with Medicinal Plants (Past and Present), Istanbul, Nobel Tip Kitabevleri.
3. Süleyman, H., Demirezer, L.Ö., Kuruüzüm, A., Banoglu, Z.N., Göcer, F., Özbakir, G, Gepdiremen, A. (1999) J. Ethnopharmacology, 65, 141-8.
4. Insel, A. (1996) Analgesic-antipyretic and antiinflammatory agent and drugs employed in the treatment of gout. In: Gilman AG (ed) Godman and Gilman's - The Pharmacological Basis of Therapeutics, New-York, McGraw-Hill, 617-657.
1Dept. of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, Ankara,Turkey
2Dept. of Pharmacology, Faculty of Medicine, Atatürk University, Erzurum.Turkey
3Dept. of Biochenustry Faculty of Medicine, Atatürk University, Erzurum-Turkey
The genus Rumex (Polygonaceae) is representcd by 25 species in the flora of Turkey (1,2). The dried roots of R. palientia have been used as purgative, constipate, depurative and tonic m traditional medicine in Anatolia (3). Previously, we reported that the aqueous extract of the roots of R. palientia (D-1) exhibited strong antiinflammatory, antipyretic and analgesic activities (4, 5). All our investigations showed that D-1 has an effect similar to that of NSAIDs.
The aim of this study was to investigate the effect of D-1 on normal stomach tissue and stress-induced ulcer model in rats in comparison to NSAIDs such as nimesulid, indomethacin, diclofenac, naproxen and ibuprofen. For stress-induced ulcer assay, after one hour of drug administration, the animals were waited 24 hours at a prone position and killed. Finally their stomachs were removed and the ulcerative zones were macroscopically evaluated. The numbers and sizes of ulcers were measured. Statistical significance was determined by Mann Whitney-U test and a p<0.05 was considered as significant Although, oral administration of indomethacin, dichlophenac, naproxen and ibuprofen resulted in the production of damage on stomach, but D-1 (150 and 500 mg/kg) and nimesulid (100 and 300 mg/kg) did not damage the stomach. Additionally, in the 150 mg/kg dose of D-1 administered rats numbers of stress ulcers were exactly identical to that of the control. However, 500 mg/kg dose of D-1 reduced the number of stress ulceration 1.9 times compared to the control group, and decreased the size of ulceration areas 1.1 times compared to the control group. High doses of NSAIDs caused stomach damage, in this study nimesulid, like D-1, was shown for the first time to possess a significant antiulcerogenic effect on stress-ulcer.Further studies are necessary to elucidate their exact mechanisms of action.
l. Davis, P.H. (1967) Flora of Turkey and the East Aegean Islands, Edinburgh, University Press.
2. Davis, P.H- (l 988) Flora of Turkey and the East Aegean Islands, Edinburgh, University Press,
3. Baytop, T. (1984) Therapy with Medicinal Plants (Fast and Present), Istanbul, Nobel Tip Kitabevleri
4. Süleyman, H., Demirezer, L.Ö., Kuruüzüm, A,, Banoglu, Z.N., Göcer, F., Özbakir, G., Gepdiremen, A. (1999) J. Ethnopharmacology, 65,141-8
5. Süleyman, H., Demirezer, L.Ö., Kuruüzüm, A. (2001) Pharmazie (in press).
SEARCHING FOR SOME Terminalia AND Combretum SPECIES (COMBRETACEAE) WITH ANTIMICROBIAL ACTIVITY
1 Division of Pharmacognosy, Department of Pharmacy, P.O. Box 56, FIN-00014 University of Helsinki
2 Viikki Drug Discovery Technology Center, Department of Pharmacy, P.O. Box 56, FIN-00014 University of Helsinki
3 Department of Ecology and Systematics, P.O. Box 7, FIN-00014 University of Helsinki, Finland
4 Department of Botany, University of Dar es Salaam, P.O. Box 35060, Dar es Salaam, Tanzania
The genera Combretum and Terminalia (Combretaceae) comprise many species which are well known as medicinal plants in the tropical parts of the world. At least 24 species of Combretum are used as medicinal plants in Africa, and many of them against microbial infections [1]. Cold water extracts of C. molle G. Don. are used for the treatment of diarrhoea. Terminalia species are frequently used as medicinal plants in different African countries; roots of T. macroptera can be found on market places in West Africa, and the decoction of the roots of this plant have been found to possess strong antimicrobial activity against Shigella dysentheriae [2]. Terminalia avicennoides Guill. & Perrott., which is used in West-African traditional medicine was found to show strong antifungal activities against Candida albicans, and it is believed that the antifungal activities are due to saponines and tannins, which can be found in large quantities in many species of Terminalia [3]. In this study the ethnomedical uses of 16 species of Terminalia and Comhretum were documented during a field excursion to Mbeya, Tanzania in spring 1999. Species collected during the excursion have been screened for antimicrobial activity using the agar diffusion method. 21 crude extracts of 11 species, of which some are used in Tanzanian traditional medicine, were screened and we found that all species possessed antimicrobial activities. Terminalia sambesiaca Engl. & Diels. was the antimicrobially most efficient species in this investigation. Root extracts in methanol of this species gave MIC values of 0.9 mg/ml against the Gram-negative Enterobacter aerogenes.
Table 1. MIC values (mg/ml) of species of Combretum and Terminalia collected in Tanzania.
r,
entire root; s, stem bark. Ampicillin and streptomycine as
positive controls. * not tested.
1. Hedberg, I., Hedberg, H. (1982) J. Ethnopharmacol. 6: 29-60.
2. Silva, 0., Duarte, A., Cabrita, J., Pimentel, M., Dinix, A., Gomes, E. (1996) J. Ethnopharmacol 59: 55-59.
3. Baba-Moussa, F., Akpagana, K., Bouchet, P. (1999) J. Ethnopharmacol. 66: 335-338.
Institut für Pharmazie, Humboldt-Universität zu Berlin, Goethestr. 54, 13086 Berlin, Germany
Lettuce opium, the condensed brown latex from Lactuca virosa L. (Prickly Lettuce) has been used for many years as an analgesic and antitussive agent since its invention by Kore in 1792. Some hundred years ago the effects of Lactuca virosa L. or other Lactuca species have been known in ancient Egypt and Greece. Lactucin and lactucopicrin, two guaiane-type sesquiterpene lactones, were isolated in 1939 by Schenck et al. [l] and recent phytochemical examination of the plant led to the isolation of other sesquiterpene lactones, namely jacquinelin, 8-desoxylactucin, 11b, 13-dihydrolactucin and lactuside A [2, 3]. The analgesic potency of those substances has been shown by Gromek et al. [4].
With a view to find a possibility to account for pharmacological effects of the drug, the material was tested for its ability to inhibit the activity of NEP (enkephalinase), which is the common name given to the enzyme that cleaves methionine5- or leucine5- enkephalin at the Gly3- Phe4- bond. For determination of NEP activity we used a two-step assay according to Bormann et al. [5]. Tests with aqueous and methanolic extracts of the dried latex showed a distinct inhibiting effect on NEP in a concentration-dependent way. We tested two types of lettuce opium, one gathered from the annual plant and the other from the biannual plant. Differences were found between the two types: Annual Lactucarium showed a more pronounced inhibition, in particular the aqueous extract. The aqueous extract of the annual plant was the more active one with an IC50 value of 90 µg/ml. Tests with aqueous extracts of the biannual plant led to an IC50 value of 530 µg/ml.
On account of well-known inhibitory potency of flavonoids on NEP the extract was tested for their presence by TLC. We could exclude the presence of flavonoids. Additionally, we tested opioid receptor binding in mice brain membranes by receptor binding assay but no effects of lettuce opium on opioid receptors were seen.
1. Schenck, G., Graf, H., Schreber, W. (1939) Archiv der Pharmazie 277: 137-145
2. Gromek, D. (1989) Polish Journal of Chemistry 63: 297 - 301
3. Gromek, D. (1991) Polish Journal of Chemistry 65: 1979 - 1981
4. Gromek, D., Kisiel, W., Klodziñska, A., Chojnacka - Wójcik, E. (1992) Phytotherapy Research 6: 285 - 287
5. Bormann, H., Melzig, M.F. (2000) Pharmazie 55: 129-132
1 Institut für Pharmazie, Humboldt-Universität zu Berlin, Goethestr. 54, D-13086 Berlin, Germany
2 Institut für Pharmazie, Universität Leipzig, Johannisallee 21-23, D-04103 Leipzig, Germany
The tall growing plant of great burdock (Arctium lappa L., Asteraceae) with large leaves and purple flower heads and fruits with hooked bristles is native to Europe and Asia and has been naturalized in the United States [1]. Preparation from burdock roots are used against gastrointestinal diseases, gout, rheumatism, bile and bladder disorders, externally against skin complaints. Commission E mentions minor gastrointestinal complaints and application with ichthyosis, psoriasis, and seborrhea of the scalp [2-4].
Anti-oxidative, anti-inflammatory, and radical scavenging effects have been reported for the roots and crude extracts of burdock [5, 6]. The aim was to apply a simple and rapid method to demonstrate these effects.
A methanolic extract, obtained by ultra turrax extraction was fractionated by solid phase elution using SephadexTM LH-20 as solid phase and methanol : water of different proportions as eluent. Fractionation was TLC-guided by applying the horizontal Desaga H-chamber. Fractions of interest were further submitted to a TLC assay in order to control their anti-oxidative effect using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical as spray reagent. Positive fractions were controlled for purity by additional TLC and submitted to subsequent solid phase elution. Compounds of sufficient purity such as chlorogenic acid and quinic acid derivatives (e. g. 1,3-di-O-[E]-caffeoyl-quinic acid) were analyzed by various chromatographic and spectroscopic methods (Fig. 1: 1H- and 13C-NMR spectra) for structure elucidation.
Fig. 1: 1H- and 13C-NMR spectra from 1,3-di-O-[E]-caffeoyl-quinic acid
V. E. Tyler (1993) The Honest Herbal, 3rd ed., The Haworth Press, New York, 63-64
G. Willuhn in M. Wichtl (ed.) (1997) Teedrogen & Phytopharmaka, Wissenschaftliche Verlags-Gesellschaft, Stuttgart, 98-100
K. Horz in W. Blaschek, R. Hänsel, K. Keller, J. Reichling, H. Rimpler, G. Schneider (eds.) Hagers Handbuch der Pharmazeutischen Praxis, 5th ed. (1998), Springer Verlag, Berlin, Heidelberg, New York, Drogen A-K, Folgeband 2, 140-158
B. Gehrmann, W.-G. Koch, C. O. Tschirch, H. Brinkmann (2000) Arzneidrogenprofile, Deutscher Apotheker Verlag, Stuttgart, 130
Y. Maruta, J. Kawabata, R. Niki (1995) Antioxidative caffeoylquinic acid derivatives in the roots of burdock (Arctium lappa L.), J. Agric. Food Chem. 43(10), 2592-2595
C. C. Lin, J. M. Lu, J. J. Yang, S. C. Chuang, T. Ujiie (1996) Anti-inflammatory and radical scavenge effects of Arctium lappa, Am. J. Chin. Med. 24 (2), 127-137
1Institute of Pharmacy, Assam Medical College, Dibrugarh 786002, Assam, India
2Department of Zoology, Gauhati University, Guwahati 781 014, India
The rhizome of Costus speciosus Koen. (Zingiberaceae) is used in the treatment of variety of diseases in Indian health care remedies. The rhizome of C. speciosus is used in the treatment of fever, urinary problems, rheumatism, dyspepsia and menstrual disorders in women. The juice and extract obtained from the rhizomes is extensively used by the different ethnic groups of Assam in the treatment of a variety of hepatic disorders including infective hepatitis.
The antihepatotoxic activity of aqueous and methanolic extracts of rhizomes of C. speciosus have been investigated using CCI4 and acetaminophen induced liver damage in albino rats. Two extracts were tested in five models with three different treatment schedules. Five liver specific biochemical parameters viz. serum glutamic oxalacetate transaminase (SGOT), serum glutamic pyruvate transaminase (SGPT), serum alkaline phosphatase (SALP), serum sorbitol dehydrogenase (SSDH) and serum glutamic dehydrogenase (SGLDH) and histopathological examination of the liver were undertaken to monitor the status of the liver. Silymarin, a natural antihepatotoxic agent has been used as a standard compound to compare the activity of the extract.
The experimental animals were treated with aqueous and methanolic extract of C. speciosus before and after administration of carbon-tetrachloride and acetaminophen in three different treatment protocols. Both the extracts of C. speciosus at a dose of 300 mg and 500 mg/kg. p.o. exhibited significant inhibition of SGOT, SGPT, SALP, SSDH and SGLDH elevated due to liver necrosis produced by CC14 (1.5m1/kg, s.c.) and acetaminophen (3.0 g/kg, p.o.). Comparison of the percentage reduction of individual enzymes and overall percentage reduction of all biochemical parameters suggested that the potency of both extracts are well comparable (78.2-96.0 %) to equal dose of silymarin. The liver biopsy of experimental rats showed significant restoration of normal histomorphological pattern of liver cells.
These observations support the ethnopharmacological claims of folklore uses of rhizomes of C. speciosus in the treatment of liver diseases and the presence of antihepatotoxic bioactive phytoconstituent(s) in both extracts.
1Institut für Pharmazie, Universität Leipzig, Johannisallee 21-23, D-04103 Leipzig
2Institut für Pharmazie, Humboldt-Universität zu Berlin, Goethestr. 54, D-13086 Berlin
The small growing plant of Madeira sorrel (Rumex maderensis LOWE, Polygonaceae) with small leaves and nearly invisible flowers and tiny winged nut fruits is also known as azedas as well as labaça [1]. Preparations are used as diuretics and against inflammatory eye diseases in folk medicine. The first area of application is reported from Portugal, the latter from Africa [1]. Only limited information is known about phytochemical studies on Madeira sorrel [2].
A methanolic extract was obtained from the aerial parts of ground plant material by maceration at room temperature. For purification, the resulting sirupy extract was repeatedly shaken with methylene chloride. The aqueous phase was diluted in methanol : water and fractionated by solid phase elution using Sephadex LH-20 as solid phase and methanol : water of different proportions as eluent [3].
The fractionation was TLC-guided applying the horizontal DESAGA H-chamber and using 2,2diphenyl-1-picrylhydrazyl (DPPH) as spray reagent in order to control anti-oxidative effects of the fractions obtained.
Thus, several polyphenolic compounds such as vitexin, isovitexin, and the trimeric proanthocyanidine epicatechine-[4->6]-epicatechine-[4->8]-epicatechine [4] were isolated, identified, and DPPH-tested.
1. D. Rivera, C. Obon (1995) Joumal of Ethnopharmacology 46, 73-93
2. P. H. List, L. Hörhammer (eds.): Hagers Handbuch der Pharmazeutischen Praxis, 4h ed. (1979), Springer Verlag, Berlin, Heidelberg, New York, vol. VI, part B, 200
3. B. Branse-Passek (2000) Dissertation, University of Leipzig
4. D. Inkrot, B.Branse-Passek, B. Gehrmann, J.-W.Rauwald (2001) Congress Issue 42 d Annual Meeting of the Society of Pharmacognosy
BIOACTIVE NUTRIENTS AND NON-NUTRIENTS FROM ROSEROOT (RHODIOLA ROSEA L.) AS COMPONENTS OF FUNCTIONAL FOOD.
Research Institute of Medicinal Plants, Poznan 61-707, Poland
Roseroot is distributed in the arctic regions of eastern Siberia. It is the plant’s root that provides adaptogenic characteristics. An adaptogen offers varied physiological support to numerous systems in the body. Thus roseroot is supposed to increase the body’s non-specific resistance, normalize the functions of the body, enhance energy level, increase memory and improve body resistance to numerous environmental stressors. Rhodiola rosea has been used for hundreds of years in Russian traditional medicines to treat cold and flu-like symptoms, improving mental and physical capacities and promote longevity and may hence be regarded as a “functional food”.
The purpose of our investigation was to carry out complex research of roseroot root as a potential raw material for functional food. Plant material, water and water–alcoholic extracts derived from it were investigated.
Tab.1 Roseroot – nutritive value Tab.2 Roseroot – salidroside content
|
Nutrient |
Unit |
in 100 g |
|
Plant mat./ Extract |
Yield [%] |
Salidroside [%] |
|
Energy |
kcal |
169 |
|
Root |
- |
0.5 – 1.1 |
|
Protein |
g |
5.9 |
|
Water |
23 |
1.4 |
|
Fat |
g |
1.7 |
|
25% EtOH |
20 |
1.5 |
|
Carbohydrates |
g |
69 |
|
50% EtOH |
19 |
1.9 |
|
Dietary fiber |
g |
42 |
|
75% EtOH |
16 |
2.1 |
|
Calcium |
mg |
1614 |
|
95% EtOH |
10 |
2.3 |
Acknowledgement: This research was supported by State Committee for Scientific Research (Grant No 4 P05F 00115)
Energy and nutritive value of the root were established, including nutrients like fat, total protein, total carbohydrates, dietary fiber, minerals, vitamin, amino acids and fatty acids (Tab.1).
Non-nutrients were estimated by colorimetric method for presence of salidroside in root and extracts. Content of salidroside in commercial samples of root amounted from 0.5 to 1.1 % and in extracts from 1.4 to 2.3 % (Tab.2). Acute toxicity of root was estimated, LD50 (oral, rat) = 5g/kg b.w.
Departamento de Farmacología, Facultad de Farmacia, U.C.M. Madrid, Spain
*Centro de Biología Molecular Severo Ochoa, CSIC, Madrid, Spain
Within the scope of the ethnopharmacological screening of selected medicinal plants, several species from the Iberian Peninsula were assayed in vitro to detect antiviral activity by using assays previously described (1). The ethanolic and aqueous extracts from the Spanish medicinal plant, Nepeta tuberosa, Lamiaceae, have been tested for their antiviral activity against herpes simplex type 1 (HSV-1), vesicular stomatitis virus (VSV) and polio virus type 1 (polio). The aqueous extract exhibited moderate anti-HSV-1 activity and strong anti-VSV activity but no anti-polio activity, whereas the ethanolic extract was ineffective against all viruses. The aqueous extract was lyophilised and fractionated with solvents of increasing polarity( hexane, dichloromethane, ethyl acetate, butanol). The organic fractions and the remaining water soluble principles were assayed for their antiviral activity against HSV-1 and VSV.
Confluent HeLa cells were infected with the viruses mentioned above at multiplicities of infection of 1 and incubated at 37 ºC (5% CO2) 24 h (VSV-1 and polio) and 48 h. (HSV-1). Just after addition of the virus, the fractions were added, and tested at concentrations ranging from 25-500 ìg/ml. To evaluate the antiviral activity the cytopathic effect (CPE) was determined and protein synthesis, evaluated by means of [35S]-methionine incorporation.
Among all fractions obtained from the initial aqueous extract, only the water-soluble residue was active at concentrations ranging from 25 to 250 µg/ml. This particular fraction inhibited HSV-1 replication (45% at 250 µg/ml) and also showed anti-VSV activity (85 % at 250 µg/ml).
The antiviral activity of the aqueous extract of N. tuberosa is exhibited only by the water-soluble compounds tentatively identified as polysaccharides. Further investigations are being undertaken to elucidate the structure(s).
Acknowledgement: Supported by DGICYT PB94-0148 and PR182-6738/96 UCM
Abad MJ, Guerra JA, Bermejo P, Irurzun A, Carrasco L (2000) Phytoter. Res., 14, 604
INFLUENCE OF PHLOROGLUCINOL DERIVATES ON THE PRODUCTION OF OXYGEN RADICALS BY POLYMORPHONUCLEAR LEUCOCYTES AND IN THE HORSERADISH PEROXIDASE/H2O2 SYSTEM
Department of Applied BioSciences, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH) Zurich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland
Hitherto pharmacological investigations on prenylated acylphloroglucinol derivatives focused mainly on their antibacterial and cytotoxic activities [1] or in the case of hyperforin and its analogues on their antidepressant effects [2]. Continuing our research on the radical scavenger and antioxidative activity of natural products [3] we investigated for the first time a series of 22 prenylated bi- and tricyclic acylphloroglucinol derivatives for 1.) their radical scavenger activity against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2.) their radical scavenger activity in an in-vitro enzymatic test system using horseradish peroxidase/H2O2 and 3.) their influence on the ex-vivo production of reactive oxygen species (ROS) by human polymorphonuclear leukocytes (PMNs) stimulated with formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ).
Despite of the low or lacking radical scavenger activity of the phloroglucinol derivatives against the DPPH radical, some of these compounds showed a strong and dose dependent inhibition of the ex-vivo production of ROS by PMNs in the micro molar range after stimulation with FMLP. The most potent compounds were 1 (IC50 = 2.5 mM), 2 (IC50 = 3.5 mM) and hyperforin (3, IC50 = 1 mM).
In contrast, the signal transduction pathway evoked by OZ is not influenced by compounds 1 and 2 leading to the assumption, that their activity is due to specific activation or inhibition of enzymes and/or receptors involved in the FMLP mediated pathway of the oxidative burst. This is corroborated by the fact that different stereochemistry and varying substitution reduced the activity of these compounds significantly.
1. Winkelmann, K., Thesis No. 14137, Swiss Federal Institute of Technology Zurich, 2001, pp. 11
2. Kaul, R., Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, 2000, pp. 75
3. Heilmann, J., Çalis, I., Kirmizibekmez, H., Schühly, W.; Harput, S.; Sticher, O. Planta Med. 2000; 66: 746
1Dipartimento di Scienze Farmaceutiche, Universita di Padova, Italy
2Dipartimento di Biologia, Universita di Padova, Italy.
Lignans such as podophyllotoxin or their semisynthetic derivatives Etoposide and Teniposide have important antineoplastic and antiviral properties (1). The latter are included in a wide variety of anticancer chemotherapy protocols. Natural sources of podophyllotoxin are mainly Podophyllum emodi and Podophyllum peltatum. Researches on alternative sources of aryltetraline derivatives are proceeding. We are actually considering Anthriscus sylvestris Hoffm. (Apiaceae), a perennial herb growing in Europe, Asia and north east of United States (5). The root of this plant has been used in Korean folk medicine as diuretic and antitussive (6); a recent review (4) indicates this plant as a potential source of anticancer compounds. Cytotoxic lignans and phenyl propanoids have been demonstrated to be present in the ground parts (e. g. 2,6) and in the fruits (3).
In this paper we report an approach for the extraction and the finding of biological active compounds on the basis of cytotoxic activity of sequential extracts.
Our researches have pointed out the presence of cytotoxic constituents in the apolar extract of the ground parts of Anthriscus sylvestris in agreement with other authors (6). Extracts obtained with petroleum ether and chloroform showed activity against two solid human tumors cells lines (LoVo and LoVo/Doxo), however, no activity has been observed with more polar extracts. A significant different response was observed in cells expressing the multi drug resistant factor (MDRF).
The chloroform extract has been fractionated by chromatographic techniques and the peaks of activity are related to the presence of four main components. The isolation and the characterization of these compounds are in progress.
1. Gordaliza M., Castro M.A., Miguel del Corral J.M., San Feliciano A. (2000) Current. Pharmaceutical Design,6, 1811-1839
2. Ikeda R., Nagao T., Okabe H., Nakano Y., Matsunaga H., Katano M., Mori M., (1998a) Chem. Pharm. Bull. 46(5) 871-874
3. Ikeda R., Nagao T., Okabe H., Nakano Y., Matsunaga H., Katano M., Mori M., (1998b) Chem. Pharm. Bull. 46(5) 875-878
4. Imbert T. F. (1998) Biochimie 80,207-222
5. Kozawa M., Morita N., Hata K., (1978) Chem. Pharm. Bull. 26, 1337-1338.
6. Young-Hee L., Moon-Jeong L., Dong-Hyuk S., Hwan-Bong C., Seung-Woo H., Eun-Yi M., Sung-June Y., Won-Sick W. (1999) Arch. Pharm. Res., 22 2, 208-212.
TRANSPORT OF ALKAMIDES FROM Echinacea SPECIES THROUGH CACO-2 MONOLAYERS
aDepartment of Applied BioSciences, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH) Zurich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland
bInstitute of Pharmaceutical Biology, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany
Echinacea preparations are commonly used for the prevention and treatment of colds and infections. Different primary and secondary plant metabolites are discussed to contribute to the activity of these phytopharmaceuticals. In vitro tests showed immunostimulatory and anti-inflammatory effects for polysaccharides, caffeic acid derivatives and alkamides, but data on the bioavailability of these compounds are scarce [1]. Recently we reported for the first time on the detection of the alkamides dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (1/2) in human blood after oral intake of E. purpurea mother tincture [2].
To
gain more insights into the human intestinal absorption of these
alkamides, transport studies were performed with human adenocarcinoma
colonic cell line Caco-2 (ATCC) as a model to assess the epithelial
transport of 1/2. In a first step, a HPLC system was
established to quantitatively detect the alkamides on the apical and
basolateral side of the cell layers. Second, cytotoxic effects
against Caco-2 cells were excluded for a broad concentration range (1
- 50 mg/ml) of
1/2, using a MTT based cytotoxicity assay.
After apical loading of 25 or 50 mg/ml of dodeca-2E,4E,8Z,10E/Z-tetraenic acid isobutylamides (1/2), about 10 % of these compounds could be detected on the basolateral side within 15 min. Close monitoring of the transport during 6 hours revealed a nearly complete transport of 1/2 to the basolateral side after 4 hours. No significant metabolism was observed. These results support the assumption that the alkamides can be easily transported from the intestinum and hence may contribute to the in vivo effects of Echinacea preparations. Further absorption studies on structural related alkamides are in progress.
[1] Bauer R. Chemistry, analysis and immunological investigations of Echinacea phytopharmaceuticals. In: Wagner H., Ed. Immunomodulatory agents from plants: Birkhäuser Verlag, 1999: 41-88
[2] Dietz, B.; Heilmann, J.; Bauer, R. Planta Medica, in press 2001
Institut für Pharmazie, Humboldt Universität zu Berlin, Berlin, Germany
Agrostemma githago L. var. githago is a well known toxic member of the Caryophyllaceae family. To date three triterpenoid saponins have been isolated from the corn cockle saponin mixture with gypsogenin (3b-hydroxy olean 12 en 23 al 28 oic acid) as aglycone, called githagoside [1], a monodesmosidic tetraglycoside and agrostemmasaponins 1 and 2, both 3, 28 bisdesdesmosides of gypsogenin [2]. Aqueous extracts of the seeds were found to show remarkably higher toxicity in cultivated endothelial cells in comparison with the isolated saponins. To all appearances, ribosome inactivating proteins (RIPs) like agrostin [3], lectin-like compounds which occur in the seeds of the corn-cockle, can be held responsible for this observation. A combination of particular saponins with a formyl function in triterpene position 4 (conc.: 3µg/ml) together with agrostin (Mr: 27,000) showed comparable toxicity against an ECV 304 cell line (Agrostin IC50=2.6 ng/ml). In order to confirm this result we combined agrostin with helianthussaponin 2 (oleanane type, bisdesmosidic, no formyl function in triterpene position 4) in equal concentrations and determined an IC50 of 3.5 µg/ml. In addition we investigated the Saponinum album saponin mixture with similar structural features (triterpenoid saponins of oleanane type, bisdesmosides, formyl function in triterpene position 4), which is isolated from Gypsophila fastigata L. and Gypsophila paniculata L.. Chromatographic analysis confirmed the presence of two main saponin fractions with IC50 values which are comparable to the agrostemmasaponin values.
In order to draw an analogy between the well known toxic mechanism of action of lectins which can be inhibited by certain sugars with a specific configuration and affinity to the lectin's haptomer, and, as a result, leading to a prevention of toxicity, we tested seven different sugars known for their affinity to different lectins. None of them diminished toxicity values significantly.
Another experiment concerning the mechanism of toxicity was carried out. Our experiments demonstrated that the toxic effect was induced after an incubation time of 1h.
1. Tschesche, R., Schulze, H. (1974) Chem. Ber. 107: 2710 2719.
2. Siepmann, C., Bader, G., Hiller, K., Wray, V., Domke, T., Nimtz, M. (1998) Planta Med. 64: 159 164.
3. Stirpe, F., Gasperi Campani, A., Barberi, L., Falasca, A., Abbondanza, A., Stevens,
W. (1983) Biochem. J. 216: 617 625.
Departments of aPharmacology & Toxicology, bQuality Control, cR & D and dScientific Res., Steigerwald Arzneimittelwerk GmbH, Havelstr. 5, 64295 Darmstadt, Germany
A test system for the study of the absorption kinetic of plant extracts and their fixed combinations is a requirement for the evaluation of pharmacological effects and clinical levels of efficacy. Besides, the proof of absorption is also demanded by licensing agencies in toxicological studies.
As plant extracts contain a large number of compounds, of which the serum concentrations after oral application often do not reach the limits of detection, the model of the everted gut sac of the rat (1, 2) was adapted as a test system for the study of the absorption of several extracts, which are used in the therapy of gastrointestinal functional diseases. The absorption of "marker" substances of the extracts and of their fixed combination product STW 5* was assayed and quantified. 4–6 concentrations of each extract corresponding to 1–100 µl of the original extract/ml were tested. Absorption rates, in relation to the extract concentrations, were determined. The tested extracts and their marker substances were:
|
Iberis amara totalis : |
glucoiberine, cucurbitacines E and I (HPLC) |
|
Matricariae flos : |
bisabolonoxide A (GC) |
|
Carvi fructus : |
carvone (GC) |
|
Cardui Mariae fructus : |
silybine A and B (HPLC) |
|
Melissae folium : |
rosmarinic acid (HPLC) |
|
Menthae folium : |
menthol (GC) |
|
Chelidonii herba : |
chelidonic acid (HPLC) |
|
Liquiritiae radix : |
glycyrrhicinic acid (HPLC) |
A strong concentration dependency of the absorption could be shown (linear correlation, R² > 0,9). Accordingly, the ex vivo/in vitro test system of the everted gut sac can be seen as a reliable model for the quantitative study of intestinal absorption of phytomedicinal products.
(1) Wilson T.H., Wiseman G, J. Physiol. 123:116-125 (1954)
(2) Barthe L., Woodley J.F., Kenworthy S., Houin G., Eur. J. Drug Metab. Pharm. 23: 313-323 (1998)
*Iberogast, Steigerwald Arzneimittelwerk GmbH
Department of Pharmacology, College of Medicine, Institute of Natural Medicine, Hallyrn, University, Chunchon, 200-702, S. Korea
Oxidative stress and inflammation are postulated to play important roles in the pathogenesis of Alzheimer‘s disease (AD). Ferulic acid (4-hydroxy-3-methoxycinnamic acid), a phenolic compound present in a variety of plants and food derived from them, is an antioxidant and anti-inflammatory agent. Therefore, the potential protective activity of ferulic acid against b -amyloid peptide toxicity in vivo was examined. Mice were allowed free access to drinking water (control) or water containing ferulic acid (0.006%). After 4 weeks, Ab1-42 (410 pmol) was administered via intracerebroventricular (i.c.v) injection. Injection of control mice with Ab1-42 impaired performance on the passive avoidance test, the Y-maze test, and the water maze test. In contrast, mice treated with ferulic acid prior to Ab1-42 administration were protected from these changes. Ab1-42 decreased acetylcholine level in the cortex, which was tended to be ameliorated by ferulic acid. In addition, Ab1-42 increased immunoreactivities of the interleukin (IL)-6, interferon-g, endothelial nitric oxide synthase, and 3-nitrotyrosine in the hippocampus, effects also suppressed by pretreatment with ferulic acid. These results suggest that pretreatment with ferulic acid induces resistance of the brain to the toxicity of Ab1-42, and that ferulic acid may be useful as a potential chemopreventive agent against AD.
ANTIPLASMODIAL ACTIVITY OF SEMISYNTHETIC AURONES AGAINST Plasmodium falciparum STRAINS K1 and NF54
1Freie Universität Berlin, Institut für Pharmazie, Berlin, Germany
2Robert Koch-Institut, Berlin, Germany
3Schweizerisches Tropeninstitut, Basel, Switzerland
A series of naturally occurring aurones was synthesized and tested for the ability to inhibit erythrocytic stages of Plasmodium falciparum strains in vitro. To validate the antiplasmodial potential of the investigated aurones and auronols, they were tested in vitro both against a drug-sensitive (NF54) and a chloroquine/ pyrimethamine resistant (K1) strain of P. falciparum. Of the twelve compounds tested, seven aurones/ auronols showed appreciable antiplasmodial activities (IC50 £ 0.3 µM), but none was as effective as chloroquine or artemisinin (quinghaosu) used as references (IC50 for K1 0.07 µM and 0.001 µM, respectively). The most active compound against P. falciparum strain K1 was 4,6,4’-Triacetyl-3’,5’-dimethoxy-2-[phenylhydroxy-methylene]-3(2H)-benzofuranone followed by 2’,6-Dihydroxy-2-[phenylmethylene]-3(2H)-benzofuranone and 4,6,4’-Trihydroxy-3’-methoxy-2-[phenylhydroxy-methylene]-3(2H)-benzofuranone with IC50 values of 0.007, 0.03 and 0.03 µM, respectively. Notably, the multi drug-resistant P. falciparum strain K1 was more sensitive to tested aurones than the drug-susceptible strain NF54 against which most compounds were either inactive or only moderately active. All tested compounds exhibited similar, moderate cytotoxic activity against human squamous carcinoma (KB) and melanoma (SK-Mel) cells (IC50 £ 3.0 µM).
Antiplasmodial testing was conducted using a modification of the [3H]-hypoxanthine incorporation assay by Desjardins for determing intra-erythrocytic inhibition of parasite growth. Briefly, P. falciparum-infected human red blood cells in hypoxanthine-deficient culture medium were exposed to serial drug dilutions in microtiter plates for 48 hours at 37 °C in a 4 % CO2 -enriched humidified atmosphere before [3H]-hypoxanthine was added for another 24 h and its incorporation by viable parasites assessed.
The drug resistant strain K1 was more sensitive to most of the aurones than strain NF54, e.g. IC50 values of 0.007 µM (K1) vs. 0.180 µM (NF54) for bracteatintriacetat. One possible explanation might lie in an altered energy metabolism as it is often observed in drug resistant mutants of protozoa. Possibly drug resistant strains must divert a large part of their energy from reproduction to serving mechanisms that neutralize the effects of the antibiotic. This could also lead to the resistant strain being more restricted in its metabolic resources that can be invested in new cellular defense strategies, rendering is more susceptible to new antiparasitics.
To summarize, we present certain aurones as new antiplasmodial agents with a first preliminary indication for their structure/ activity-relationships. Further, we observed, that the drug-resistant P. falciparum strain K1 was orders of magnitude more sensitive for these aurones than strain NF54 which is sensitive to current antimalarials. The results may contribute to find even more potent antimalarial aurones and chalcones from natural sources and will be discussed intensively.
1Department of Analytical Toxicology Gülhane Military Medical School, Ankara, Turkey
2Ankara University, Faculty of Pharmacy, Department of Pharmacognosy, Turkey
3 Ankara University, Faculty of Veterinary, Department of Pathology, Turkey
4Department of Pharmacology, Gülhane Military Medical School, Ankara, Turkey
5Marmara University, Department of Pharmacology, School of Medicine, Istanbul, Turkey
Aqueous extract from root of Inula heterolepsis Boiss. was prepared and tested for its ability to treat alcoholic hepatic injury in rats. Alcoholic hepatic injury was experimentally induced by administering ethanol in liquid diets for 28 days. Alcoholic rats were divided into two groups. First group of rats were given 200 mg/kg/day plant extract in 1% methyl cellulose solution via 2 mm diameter orogastric tube. Related doses of extract preparations were given 12 h intervals for 10 days. Differences between the recovery of tissue injury, with and without Inula plant extract, were evaluated. Second group of rats were given vehicle. Liver, testis and kidney injuries due to the chronic alcohol consumption were proven biochemically and histopathologically. At the end of treatment each group of rats were sacrificed, blood samples were obtained and serum SGOT, SGPT, alkaline phosphatase, and albumin levels were measured. SGOT, SGPT and alkaline phosphatase levels were significantly higher in alcoholics rats due to the tissue damage comparing with intact, vehicle and Inula treated gropus of rats ( p<0.05). On the other hand, biochemical test results were found similar in Inula and vehicle administered alcoholic rats. Liver, testis, kidneys, stomach, intestine, heart, lungs and bladder were examined histopathologically. Histological changes were scored numerically. Lungs, stomach, intestine, heart and bladder were found in their normal histopathological shape in the Inula and vehicle groups of rats. According to our study, root extract of Inula heterolepsis Boiss. has slight therapeutic effect on alcoholic liver, kidney and testis damages in rats. Comparing the therapeutic effects among these organs, liver seemed to be effected meaningfully.
1 Department of Food Chemistry, Public Health Institute – Subotica, Zmaj Jovina 30, 24000, Subotica, Yugoslavia
2 Institute of Chemistry, Faculty of Natural Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21000, Novi Sad, Yugoslavia
3 Department of Pharmacy, Division of Pharmacognosy, University of Athens, Panepistimioupolis Zografou, 157 71 Athens, Greece
4 National Center for Poisoning Control, Military Medical Academy, Crnotravska 17, 11000, Belgrade, Yugosalvia
Angelica archangelica L. (Archangelica officinalis Moench or Hoffm.) is a tall, striking plant belonging to the Apiaceae family. Mainly the roots with rhizomes are used (Angelica radix) in loss of appetite, peptic disorders, as a spasmolytic and antimicrobially active carminative [1, 2]. While the chemistry is well documented, there is limited documented pharmacological information available for A. archangelica, to justify its use [3].
The roots of Angelica were extracted with 80% aq. ethanol solution (AI), and after evaporation of the solvent, the slurry water extract was submitted to liquid-liquid repartition in petrol ether (AII), chloroform (AIII), ethyl acetate (AIV) and n-butanol (AV). Compounds present in the extracts were identified using thin layer chromatography on silica gel and cellulose as stationary phase and reversed phased HPLC-DAD. The extracts were investigated on their radical scavenger capacity (RSC) measuring spectrophotometrically the disappearance of DPPH* at 515 nm according to the procedure described by Soler-Rivas C. et al. (2000) [4]. To determin which compounds in the extracts are responsible for the RSC the dot-blot test on siliga gel TLC layers after staining with DPPH* solution were employed. The Angelica extracts were analysed for potential antibacterial activity against different Gram-positive and Gram-negative bacteria (Bacillus subtilis, Escherichia coli, Micrococcus luteus, M. flavus, Pseudomonas aeruginosa, Staphylococcus epidermidis, S. aureus and Streptococcus faecalis) by the disc-diffusion methode [5].
According to TLC analysis the phenolic compounds identified are mainly phenolic acids and coumarins. The chloroformic extract (AIII) of Angelica was shown to possess the greatest radical scavenger capacity as well as highest antibacterial activity of all extracts tested. The most resistant bacteria were Pseudomonas aeruginosa and Streptococcus faecalis.
Wichtl M. (1994). Scientific Publishers, Stuttgart.
Blumenthal M. (ed.) (1998). American Botanical Council, Austin, Texas.
Newall A. C., Anderson A. L., Phillipson J. D. (1996). The Pharmaceutical Press, London.
Soler-Rivas C., Espin J. C., Wichers H. J. (2000). Phytochemical Analysis, 11, pp.330-338.
Dey P. M., Harborne J. B. (series ed.) (1991). Academic Press, Hartcourt Brace Jovanovich - Publishers, London, San Diego, New York, Boston, Sydney, Tokyo, Toronto, pp. 47-58.
1 Department of Pharmacognosy, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Yugoslavia
2 Laboratory for Microbiology, Faculty of Biology, University of Belgrade, Studentski trg 3, 11000 Belgrade, Yugoslavia
Aerial parts of Galium verum L. are traditionally used as sedative, tonic, stomachic, for healing wounds and skin diseases [1]. Phytochemical studies showed the presence of flavonoids, alizarin type of anthraquinones, coumarins, iridoids and tannins. In this work, the antimicrobial activity of petrolether and methanol extracts, as well as the antimutagenic effect of methanol extract of G. verum herb have been screened.
Plant material was collected in the vicinity of town Sombor, Serbia, in June 2000. Dried and grounded herb was macerated with light petroleum (1:10) for two days followed by extraction with 70% methanol using the same procedure. The solvents were evaporated under reduced pressure. Petrolether extract, dissolved in petrolether (1:3), and methanol extract dissolved in sterile water (1:5) were tested against several microorganisms: Staphylococcus aureus ATCC25923, Staphylococcus aureus ATCC29213, Staphylococcus epidermidis ATCC12228, Pseudomonas aeruginosa ATCC27853, Escherichia coli ATCC25922, Escherichia coli SY252, Escherichia coli SY252 uvrArfa, Sarcina lutea ATCC9341, Saccharomyces cerevisiae ATCC9763, using the disc diffusion method [2]. No inhibition zones were observed with petrolether and methanol extract. However, weak bacteriostatic activity against Pseudomonas aeruginosa with both extracts was found. The potential antimutagenic effect was studied using E. coli K12 reversion assay (argE3®Arg+) (Test A) designed for detection of bioantimutagens preventing spontaneous and UV induced mutagenesis by modulation of DNA repair and replication [3]. Inhibition of UV induced mutagenesis was about 43 % with 5 ml/plate of the extract in the repair proficient strain E. coli SY252. Results showed no effect of the extract on spontaneous mutation rate in the same strain. Further investigation of the therapeutic potential of G. verum herb is in progress.
Tucakov J., Phytotherapy, “Rad”, Beograd, 1995
Mahon, C.R., Manuselis, G., Textbook of Diagnostic Microbiology, W.B. Sounders Company, Philadelphia, 1995
Simic, D., Vukovic-Gacic, B., Knezevic-Vukcevic, J., Detection of natural bioantimutagens and their mechanisms of action with bacterial assay-system, Mut. Res., 402, 51-57, 1998
Department of Biochemistry, College of Natural Science, Dongguk University, Kyongju 780-714, Korea
* College of Pharmacy, Pusan National University, Pusan 609-735, Korea
Antimutagenic compounds act by either inactivating mutagens or interfering in the process of mutagenesis, which lead to their antimutagenicity by reducing the frequency or rate of spontaneous or induced mutation.
Antimutagenic agents may prevent cancer because they can either destroy mutagens in or out of body cells or block mutagens which damage DNA and cause mutations in cells.
As a part of our continuing search for anticancer agents from natural sources, we have investigated the antimutagenic activities of the total extract of whole plant of Rumex acetosa L. (Polygonaceae), which has been used in Korean folk medicine as analgesic, antipyretic and anticancer drug, and its fractions by Ames test using NPD as a mutagen for S. typhimurium TA98 and NaN3 for S. typhimurium TA100.
Antimutagenic activity of emodin, which revealed the most active cytotoxicity, was found to be 26.2% against NPD and 85.9% against NaN3 at a dose of 1.0 mg per plate.
The most active fraction was a methylene chloride fraction, showing 6% antimutagenic activity against NPD and 61.6% activity against NaN3 at a concentration of 1.0 mg per plate. Cytotoxicity of the methanol extract and its fractions against five cultured human tumor cell lines, A549 (non small cell lung), SK-OV-3 (ovary), SK-MEL-2 (melanoma), XF498 (central nervous system) and HCT-15 (colon) was examined in vitro. Among the tested samples, the methylene chloride fraction was most effective, exhibiting IC50 values of 13.17, 13.46, 18.73, 18.35 and 17.62 mg/ml against above cell lines, respectively. These results suggest that the methylene chloride fraction possesses potent antimutagenic and anticancer constituents. We have isolated four components from the methylene chloride fraction and identified their structures as chrysophanol, physcion, emodin and emodin-8-O-b-D-glucopyranoside.
Institut für Pharmazie, Humboldt-Universität zu Berlin, Goethestr. 54, D-13086 Berlin, Germany
* Research & Development, Flachsmann AG, Rütiwisstr., CH-8820 Waedenswil, Switzerland
Green tea is widely consumed but in China it has been used medicinally for 5000 years. It was suggested that green tea extracts possess anti-inflammatory as well as anticarcinogenic properties [1]. We examined the effects of a triple-standardized green tea extract (EFLA 085942) and some catechin derivatives on enzymes related with inflammation and cancer. The serin proteinase elastase, the metallopeptidase collagenase, and the metallopeptidase neutral endopeptidase seem to play an important role in the degradation of connective tissue (collagenase, elastase) and in inflammation (elastase, neutral endopeptidase). Uncontrolled high levels of the activity of these enzymes in the human body are expected to be one reason for various diseases such as rheumatoid arthritis, gastric mucosal injury [2] or tumor metastasis.
We determined the activity of the leukocyte elastase (HLE, EC 3.4.21.37) spectrophotometrically according to Melzig et al. [3]. The activity of the Clostridium histolyticum collagenase (EC 3.4.24.3) was measured fluorimetrically according to the procedure of Twining [4]. The activity of neutral endopeptidase (NEP, EC 2.4.24.11) was determined fluorimetically according to the procedure of Melzig et al. [5].
We found out that elastase and neutral endopeptidase were more inhibited by green tea extracts than collagenase (IC50collagenase: 200µg/ml, IC50elastase: 2.5µg/ml, IC50NEP: 40µg/ml).
(-)-Epigallocatechingallate was the most potent inhibitor of all three enzymes (IC50collagenase: 250µM, IC50elastase: 5µM, IC50NEP: 30µM). Collagenase was not inhibited by the three other tested catechin derivatives ((+/-)-catechin, (-)-epicatechin, (-)-epigallocatechin) in concentrations of 300µM. For elastase we found a weak inhibition by (-)-epigallocatechin (300µM: about 20%), while there was no effect by (+/-)-catechin and (-)-epicatechin on the enzyme activity (300µM). (+/-)-Catechin, (-)-epicatechin, and (-)-epigallocatechin inhibited the activity of NEP weakly about 20%.
As there is a potent inhibitory activity by (-)-epigallocatechingallate but only a weak or no inhibition by the other catechin derivatives on collagenase, elastase, and neutral endopeptidase, the gallate or a phenyl residue in position 3 of the catechin seems to be important for the interaction with the enzymes.
The enzyme mostly influenced by the green tea extract and (-)-epigallocatechingallate was elastase. We compared the inhibition rate of the green tea extract (standardized on 15% (-)-epigallocatechingallate, polyphenols, purinalcaloides, and glutamic acid-ethylamide) with the inhibition rate of pure (-)-epigallocatechingallate. We found out that the inhibition by the extract was 6 times stronger than the inhibition by (-)-epigallocatechingallate. It might be useful to standardize green tea extracts on epigallocatechingallate because it seems to be one effective component. But it has been shown above it is obviously not the only effective one.
Katiyar SK et al. (1996) Int J Oncol 8: 221-226
Harada N et al. (2000) Dig Dis Sci 45: 1210-1216
Melzig MF et al. (1999) Pharmazie 54: 712
Twining SS (1984) Anal.Biochem.143: 30-34
Melzig MF et al. (1996) Pharmazie 51: 501-503
1Department of Pharmaceutical Biology, Philipps-University of Marburg, Germany
2 Bionorica Arzneimittel GmbH Neumarkt, Germany
Interactions between ligands and the GABAA receptor have been investigated mainly by radioreceptor assays which require the use of radioactively labeled ligands and include several washing steps. Especially interactions of ligands with adverse binding kinetics are often not detectable because the half-life of the receptor-ligand complex is shorter or analogous to the time required for the separation process. Using Fluorescense Correlation Spectroscopy (FCS) receptor binding studies can be carried out directly in solution and on the membrane surface of living cells without any separation procedures.
With this work we describe a GABAA receptor binding assay using a fluorescently labeled muscimol and rat hippocampal neurons.
After labeling muscimol with the fluorophore Alexa Fluor® 532, the specific binding constants of the dye-labeled ligand to the GABAA binding site were determined. We found a high specific binding affinity of the dye-labeled ligand to its receptor site with a KD-value in the low nanomolar range. Displacement studies of 5 nM Alexa® 532–muscimol with a 1000-fold excess of non-labeled muscimol show an non-specific binding of about 11%. Therefore the binding behavior of the fluorescently labeled muscimol is not essentially influenced by the bulky dye. Additionally, a concentration-dependent increase of the specific Alexa® 532–muscimol binding was demonstrated by the positive cooperative activity of co-incubated midazolam.
With this study, we demonstrate that FCS will be an even more valuable tool for future studies of ligand-receptor interactions, particularly when investigating low molecular weight ligands with adverse binding kinetics. In this respect, the synthesis of dye-labeled ligands for various receptor sites and the development of FCS receptor-binding assays will be very promising for pharmacological research.
Acknowledgement: This study was financially supported by Bionorica Arzneimittel GmbH Neumarkt, Germany.
1 Institute for Medicinal Plants Research, Tadeusa Koscuska 1, 11000 Belgrade, Yugoslavia
2 Faculty of Medicine, Dr. Subotica 8, 11000 Belgrade, Yugoslavia
The aerial parts of Gentiana lutea have not attracted much therapeutic interest till now. On the other hand, recent studies pointed out an interesting chemical composition of the aerial parts. In our previous study of activity of G. lutea extracts against Mycobacterium bovis ethanolic extract D, obtained from flowers, showed inhibition with minimum inhibitory concentration (MJC) of 1000 µg/mL. As the extract D consisted of considerable proportion of isogentisin that compound was isolated and was subjected to antituberculosis tests. MJC for isogentisin was found to be 500 µg/mL.
Antimicrobial activity of G. lutea extracts obtained from leaves and flowers was now studied. Plant material was harvested on its natural habitat, on mountain Suvobor (800 m). Samples (leaves and flowers) were air-dried, and extracted in a Soxhlet apparatus with MeOH and CHC13 for 24h. Solvents were evaporated under vacuum and the samples obtained were: GLL 1 (methanolic extract of G. lutea leaves), GLL2 (chloroformic extract of G. lutea leaves), GLF1 (methanolic extract of G. lutea flowers) and GLF2 (chloroformic extract of G. lutea flowers). Samples were dissolved in MeOH and analysed by HPLC (instrument: Hewlett Packard HPLC model 1090; DAD detection, HP 1040 A; column Lichrospher RP18, 5 µm, 250 x 4 mm I. D., Merck; mobile phase: A - acetonitrile, B- HPLC water with 1 % 0.1N H3PO4 ; flow rate 0.8 mL/min, eluation by linear gradient).
Antimicrobial activity against Staphylococcus aureus ATCC 25923 (Gram positive), Bacillus IP 5832 (spore-forming), Escherichia coli ATCC 25922 (Gram negative), Candida albicans ATCC 24433 and Aspergilus niger was determined by agar well diffusion. Stationary-phase culture was suspended in saline solution in concentration of approximately 105 cfu/mL and 200 mL and the suspension was spread over the surface of Mueller-Hinton (for bacteria) and Sabouroud (for yeast) agar plate. Test samples at dilution of 1 mg/100 mL were added in each well, and after incubation of 24 h zones of inhibition were measured.
By agar well diffusion test no activity was found against Gram positive, gram negative, spore forming bacteria and C. albicans. Activity against A. niger was detected in samples GLL2, GLF 1 and GLF2 with zone of inhibition of 2 mm for GLL2, 2 mm for GLF 1 and 3 mm for GLF2. Gentisin was the dominant compound in GLL2, while isogentisin was dominant in GLF1 and GLF2.
aInstitute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH) Zurich, Winterthurerstr. 190, 8057 Zürich, Switzerland
bSwiss Federal Institute for Water Research and Water Pollution Control (EAWAG), 8600 Dübendorf, Switzerland
Cyanobacteria are known as a rich source of secondary metabolites exhibiting a wide variety of biological activities, for example toxic, antibacterial, antifungal and antineoplastic effects [1,2].
We have investigated a total of 44 lypophilic and hydrophilic extracts obtained from 22 samples of cultured freshwater and terrestrial cyanobacteria for their biological activities. 9 % of all extracts showed antifungal activity against Candida albicans and 54.5 % demonstrated to be active against at least one gram-positive bacterium (bioautographic assay, zone of inhibition >2 mm at 500 µg). None of the extracts showed activity against gram-negative bacteria. 4.5 % of the extracts exhibited a significant lethal effect (>60 %) against brine shrimp (Artemia salina) at 500 ppm and 38.6 % cytotoxic activity against KB-cells at 50 ppm.
One antibacterial active strain, Scytonema spirulinoides Born. (EAWAG, 161a), was selected for further detailed fractionation. The cyanobacterium was mass cultured and the bioactive inorganic medium separated from the cells. After that, the medium was subjected to solid phase extraction on Amberlite XAD-2 resin and the resin subsequently eluted with methanol. The antibacterial active methanol extract was fractionated by reversed-phase VLC. The final purification was performed by reversed-phase HPLC (RP 18) and yielded one pure compound. The isolate was identified by NMR spectroscopy (1D and 2D experiments) and high resolution mass spectrometry (HR-MALDI) as 1,10-Bis-(2,6-dihydroxy-3-methyl-phenyl)-1,10-dihydroxy-decan-4,7-dione (1) a new diaryldecanoide derivative. Characterisation of its biological activity is in progress.
(1)R.J.P. Cannell, Pestic. Sci. 39 (1993) 147.
(2)M.A. Borowitzka, J. Appl. Phycol. 7 (1995) 3.
1Institute of Chemistry, Faculty of Natural Sciences, University of Novi Sad, Trg D.Obradovica 3, 21000 Novi Sad,Yugoslavia
2Department of Pharmacognosy, Faculty of Pharmacy, Vojvode Stepe 450, Belgrade, Yugoslavia
Excessive production of free radicals is known to induce oxidative damage in cells and causes several serious diseases. This is why in recent time a great attention is devoted to the research of new effective antiradical and antioxidative substances. Due to the negative effects of some synthetic antioxidants (BHA, BHT), there is an increased tendency for their replacement with natural ones.
The genus Hieracium comprises over 1000 species, some of them with very limited distribution. In the present study we investigated radical scavenger capacity (RSC) of methylene chloride (CH2Cl2) and methanol (MeOH) extracts of seven Hieracium sp. (subgenus Hieracium) from Montenegro (Mt. Durmitor): H. gymnocephalum Griseb. ex Pant., H. suborientii (Zahn) Sell et West., H. blecicii Niketic, H. naegelianum Pancic, H. coloriscapum Rohlena and Zahn, H. guentheri-beckii Zahn, H. rotundatum Kit. ex Schultes. With the exception of H. rotundatum, all the analyzed species are Balkan endemites, H. suborienii and H. blecicii are SE Dinaric Alps local endemites. In the previous study we found that these species are rich in flavonoids and phenolic acids (1).
The RSC of examined plant extracts was investigated to the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH.) and OH. radicals. DPPH. scavenging effects were recorded spectrophotometrically, monitoring the transformation of DPPH. stable radical into the reduced form (DPPH-H) (2). OH. antiradical activity was recorded, following degradation of 2-deoxyribose into TBA-reactive substances, induced by the OH. radicals, generated in Fenton`s reaction (3).
The results showed that MeOH extracts of all tested Hieracium spp. act as powerful scavengers of DPPH. radicals. The highest scavenging affect was obtained with MeOH extracts of H. naeglianum and H. rotundatum (IC50= 1.5 mg/ml), and the lowest one with H. suborienii (IC50= 2.8 mg/ml). CH2Cl2 extracts exhibited lower RSC. The highest activity was seen with the extract of H. rotundatum (IC50=27 mg/ml), and the lowest with H. coloriscarpum and H. guentheri-beckii (IC50=100 mg/ml). All examined extracts inhibited OH. radicals, generated in the Fenton`s reaction. Highest inhibition (51%) was obtained with MeOH extracts (5% w/V) of H. naegelianum. OH. radical scavenging activity of CH2Cl2 extracts were significantly lower. In CH2Cl2 extracts the triterpenes a- and b-amyrine and 21-a-hydroxy-taraxasterol were identified, whereas in MeOH extracts flavonoids and phenolic acids (1,4). Our results indicate high antiradical properties of Hieracium species which are mostly due to the presence of phenolic compounds in MeOH extracts.
Petrovic S.D., Loescher, R., Gorunovic, M.S., Merfort, I. (1999) Biochem. Syst. Ecol. 27, 651-656.
Soler-Rivas, C., Espin H.C., Wicher H.J. (2000) Phytochem. Anal., 11, 330-338
Cheesman, K.H., Beavis, A., Esterbauer, H. (1988) Biochem.J. 252, 649-653
Petrovic S.D., Gorunovic, M.S., Wray, V., Merfort, I. (1999) Phytochemistry, 50, 293-296.
ANTIMICROBIAL ACTIVITY OF VARIOUS EXTRACTS PREPARED FROM Sesbania grandiflora BARK
Department of Pharmacy, FMIPA, Institut Teknologi Bandung, Bandung, Indonesia
Antimicrobial activity of n-hexane, ethyl acetate and ethanol extracts of the bark of „turi“ [Sesbania grandiflora (L.) Pers., Leguminosae] had been carried out. The antimicrobial activity was tested against several skin and gastrointestinal tract infecting-rnicrobes. Result showed that only the ethyl acetate extract was active against in Salmonella typhi, Shigella flexneri, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Pseudomonas pyroceaneae, and Microsporum gypseum,. and a bioautographic test showed that the active compound was supposed to be flavonoids. All extracts had no activity against Candida albicans
1. Denyer, S.P. et al. (1990), Guide to Microbiological Control in Pharmaceutical, Elis Horwood. London, 11-28.
2. Hugo, W. B. et al. (1977) Pharmaceutical Biology. Blackwell Sci. Publ. London, 1 16-136.
State Chemical-Pharmaceutical Academy, 197376 Saint-Petersburg, Russia
Caragana spinosa (L.) Vahl ex Hornem. is a shrub growing in western Sibiria and used as an anti-rheumatic and anti-arthritic in folk medicine. Different Caragana species such as Caragana sinica (Buchoz.) Rehd. and Caragana chamlagu Lamarck are reported to contain oligomeric stilbenes which were shown to inhibit the activity of proteinkinase C (PKC). Caragana spinosa contains quercetin (3,5,7,3’,4’-pentahydroxyflavone) that inhibits PKC activity. PKC is thought to regulate the activity of lymphocytes, mast cells, polymorphonuclear leukocytes (PMNL) and to play a critical role in autoimmune diseases progression. Caragana spinosa dry spirit shoot extract (CSE) was previously shown to depress the activity of macrophages, T- and B-lymphocytes at doses 50 and 100 mg/kg (1/50 and 1/25 LD50, respectively). Thus, we studied the immunodepressive effects of CSE in animal autoimmune disease model and its antiallergic properties.
Adjuvant arthritis (AA) was induced in rats by intradermal injection at base of the tail with 0.1 ml complete Freund’s adjuvant (CFA). 21 days after that the blood level of circulate immune complexes (CICs) and the activity of PMNL in nitroblue tetrazolium test were measured. Antiallergic properties of CSE were studied in common anaphylaxis reaction currently used for this purpose in Russia. Guinea-pigs were subdermally (s.d.) sensibilized with 0.1 ml rat serum (RS). Two weeks later the anaphylaxis shock was induced by intraheart injection of 0.2 ml RS.
The AA progression was accompanied by elevation of CISs and reactive oxygen species production in PMNL. CSE (50 mg/kg, administrated i.p. at days 5-11 after immunization) significantly reduced (82%, P<0.001) the level of CICs and PMNL activity (67%, P<0.001) in rats with AA. Inoculation with CSE (50 mg/kg, administrated s.d. during 4 days after sensibilization) either completely averted (in 75% of guinea-pigs) or strongly reduced the strength of anaphylaxis reaction to RS.
These findings suggest the CSE ability to prevent hyperproduction of CICs and reactive oxygen species that cause tissue injury typical for autoimmune diseases. Significant immunodepressive and antiallergic activity of Caragana spinosa might be useful in treating such diseases as rheumatoid arthritis and different hypersensitivity reactions.
1Department of Clinical Pharmacy and Biopharmacy,
2Department of Pharmacognosy, School of Pharmacy, College of Medicine of the University of Lagos. P.M.B. 12003 Idi-Araba, Lagos-Nigeria
The clinical efficacy of many herbal remedies is not in doubt. However, not much is known about the safety and tolerability of most herbal products used in therapy. National and International efforts regarding herbal medicine should be directed towards toxicity and efficacy research as this would facilitate the incorporation of herbal medicines into national primary health care to achieve health for all especially in developing countries.
Jubi formular® is marketed as a herbal formulation in Nigeria. It is available as 250 mg rusty brown fine powder with a lot of cellulose fibres in red transparent gelatin capsules. It is claimed to be a blood normalizer and booster and is used to treat diabetes and hypertension. Jubi formular® is claimed to contain Sorghum bicolor stem, Harungana madagascariensis bark and Parquetina nigrescens leaves. Though both S. bicolor and H. madagascariensis are used in the treatment of anemia in Nigerian Traditional Medicine, there are no reports in literature, except for the use of S. bicolor stem as a natural colorant because of the presence of high level of flavonoid apigenin. H. madagascariensis contains anthraquinone glycosides (1) and P. nigrescens contains cardiac glycosides (2).
Qualitative TLC/HPLC analysis confirmed the presence of only S. bicolor as a constituent of the herbal formulation. Acute toxicity testing was carried out with various aqueous concentrations of the Jubi formular® and S. bicolor extracts administered orally to the different groups of mice, while control group received distilled water. Percentage mortality was determined and LD50 calculated by probit analysis within 95% confidence limits to obtain dosages used for short-term toxicity on a new batch of healthy mice. General health Status was observed during the testing, they were sacrificed on the 8th day, the vital organs (heart, kidneys, livers and lungs) were removed, and processed for histopathological evaluations.
LD50 for Jubi formula® and S. bicolor was 202.18 mg/kg and 349.68 mg/kg body weight, respectively, indicating the formula to be more toxic than S. bicolor. The short-term toxicity study showed the animals with loss of appetite, inactivity, dehydration and impaired vision. Histopathological results showed no remarkable pathological changes in the heart, varying degrees of congestion and oedema in the lungs, liver and kidneys suggestive of disturbances in the blood circulation, pointing towards a left sided failure that may be due to high blood pressure or valve disorder.
In conclusion, anyone with cardiovascular problem must be counselled to use this herbal preparation with caution.
(1) Berthold, R., Wehih, W. and Rencheteun, 1. (1965) Helv. Chim. Acta 48 (7), 1634-1658.
(2) Marks, W. H., Fong, H. H. S. and Farnsworth, N. R. (1975) J. Pharm. Sci. 64 (10), 1674-1676.
Department of Pharmacology, College of Medicine, University of Lagos, Lagos, Nigeria
The effects of Crinum glaucum aqueous extract on immediate hypersensitivity reactions were investigated on the rat passive cutaneous anaphylactic reaction (1), rat peritoneal mast cell degranulation (2) and also allergic bronchoconstriction in the guinea pig (3).
The extract showed a significant (P<0.05) reduction in area of dye leakage. The values obtained were 40.34 ± 3.80, 21.37 ± 3.73, 13.57 ± 1.49 and 6.39 ± 0.84 mm2 for control, 100, 200 and 400 mg/kg extract administration, respectively.
The extract (20 – 80 mg/kg body wt./day) administered for five days inhibited mast cell degranulation of normal and passively sensitized rats induced by dextran and antigen, respectively (Figure).
Morphological examination also indicated that the extract protected the degranulation of the rat peritoneal mast cells. Allergic bronchoconstriction in actively sensitized guinea pigs was inhibited by the extract (200 – 400 mg/kg).
Results obtained indicated that the aqueous extract demonstrated dose-dependent and significant inhibitory effects on passive cutaneous anaphylaxis and mast cell degranulation in rats. It also inhibited bronchoconstriction in sensitized guinea pigs. These effects observed were comparable to those of sodium cromoglycate.
These results substantiate the efficacy of the extract in the treatment of asthma, in traditional medicine.
1. Gautam, A. M., Ali, A. A., Gupta, P. P. and Kar, K. (1989) Indian J Allergy Appl Immunol. 3: 13-19
2. Loeffler, L. J., Lovenberg, W. and Sjoerdsma, A. (1971) Biochem Pharmacol. 20: 2278-97
3. Barua, C. C., Gupta, P. P., Patriak, G. K., Kulshrestha, D. K. and Dhawan, B. N. (1997) Current Sci. 72:397-399
1 Institut für Pharmazeutische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany
2 The School of Pharmacy, University of Bradford, West Yorkshire, BD7 1DP, U.K.
3 Facultad Ciencias Quimicas y Farmacia, Universidad de San Carlos, Guatemala Ciudad, Guatemala
4 Facultad de Agronomia, Universidad de San Carlos, Guatemala Ciudad, Guatemala
The Guatemalan Asteraceae Eupatorium semialatum is used against malaria and other diseases in Central America (1). Recently, we reported the isolation and structure elucidation of sesquiterpene lactones of the eudesmanolide type as main components in the leaves of this plant (2,3). Since sesquiterpene lactones have been found to be active against Plasmodium parasites (4,5), we now have tested the main compounds on their activities against a chloroquine resistant strain (K1) of P. falciparum in vitro.
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Seven eudesmanolide sesquiterpene lactones derived from reynosin (1-4), balchanin (5-6), and magnolialide (7), and the new guaianolide 8 were found to be active. Their IC50 ± S.E. values ranged between 8.9 ± 1.8 and 31.7 ± 4.0 µM.
1. Cáceres, A. (1996) Plantas de Uso Medicinal en Guatemala. In: Girón, L.M. und Cáceres, A. (Eds.): Colección Monografías. Bd. 1. Guatemala : Editorial Universitaria, 89-90.
2. Lang, G., Passreiter, C.M., Medinilla, B., Castillo, J.-J. (2000) Z. Naturforsch. 55c, 511-515.
3. Lang, G., Passreiter, C.M., Medinilla, B., Castillo, J.-J. (2001) Pharmaceutical Biology, in press.
4. Francois, G., Passreiter, C.M., Woerdenbag, H.J., Voan Looveren, M. (1996) Planta Med. 62, 126-129.
5. Woerdenbag, H.J., Pras, N. Van Uden, W., Wallaart, T.E., Beekman, A.C., Lugt, C.B. (1994) Pharm. World Sci. 16, 169-180.
Institute of Medical Chemistry and Biochemistry
1 Institute of Pharmacology
2 Institute of Histology and Embryology
Medical Faculty, Palacký University, Hnìvotíská 3, 775 15 Olomouc, Czech Republic
3 Institute of Medical Chemistry and Biochemistry, 2nd Medical Faculty
Charles University, Plzeòská 221/130, 150 00 Prague 5, Czech Republic
Anthracycline antibiotics (AA), such as doxorubicin (DOX), are anti-neoplastic agents whose clinical effectiveness is limited by severe side effects, including cardiotoxicity. In addition, nephrotoxicity has been observed in rats. To prevent these adverse effects, free radical scavengers and/or transition metal chelators are used in clinical practice; dexrasoxane (DEX) being the most effective agent in this respect. In the present study the effects of silymarin, a known antioxidant extracted from Silybum marianum, on DOX-induced oxidative stress was evaluated.
Wistar rats (210±20 g) were randomly divided into 5 groups (n=6). Group 1 was fed with standard laboratory diet enriched with silymarin (1%, w/w, MADAUS Lot No. 65550 silymarin complex 66.1% [silibinin 30.1%, isosilibinin 9.1%, silydianin 12.0%, silychristin 14.9%, w/w], group 2 was fed the mixture of silymarin-lecithin (1%-1%, w/w). Groups 3-5 were fed with standard laboratory diet. Groups 1-4 were treated with DOX (1.5 mg/kg, i.v.) injected 1x weekly for 8 weeks. Group 3 was additionally treated with DEX (30 mg/kg, i.v.) injected 15 min before administration of DOX. Saline-treated animals (group 5) served as a control. The body weight of the animals was measured weekly. Weight loss was observed beginning at the sixth week of experiment in all groups treated with DOX.
Eight weeks after the final drug administration the animals were anaesthetised, the animals’ ECG were taken and blood, urine, heart, kidney, spleen, liver, lung, and thymus were collected. Groups 1, 2 and 3 had the same kidney and spleen mass as control. Neither silymarin nor DEX had any effect on the DOX-induced increases of the heart/body weight ratio. Statistically significant decreases in plasma urea and creatinine levels were observed in the groups 1-3 compared to the group 4. These data suggest a nephroprotective effect of silymarin and DEX, which was confirmed morphologically. Triacylglycerols and total cholesterol were similar to control in group 3 only, while groups 1 and 2 values were the same as in group 4. The protein profile-contractile, collagenous, and metabolic fractions of the right and left ventricle in groups 1-4 was altered with respect to the control. On this basis, we assume that neither of the studied compounds, silymarin or dexrasoxan, had a complete myocard-protective effect. However the ECG analysis in the groups 1-3 exhibited fewer abnormalities then did group 4. We found a statistically significant protective effect in groups 1-3 on the parameter of lipid peroxidation damage (TBARS) and GSH level in liver and lung. In the serum of the groups 1-4, the total antioxidant capacity (evaluated by cyclic voltammetry) was not changed compared to the control.
Conclusion: From the parameters followed in this study, no major differences in the protective effects silymarin vs. dexrasoxan were found. To evaluate whether silymarin has a nephroprotective effect and if it could be potentially used as an adjuvant in the anthracycline therapy, a series of more detailed in vivo experiments is required.
Acknowledgements: This work was supported by the Grant Agency (303/97/P081) and The Ministry of Education, Youth and Sports of the Czech Republic (MSM 151100003).
School of Pharmacy, Mashhad University of Medical Sciences, P.O. Box 91775-1365, Mashhad, Iran.
Antinociceptive effects of aqueous and ethanolic extracts of Zataria multiflora have been reported previously (1). These findings prompted us to investigate the possible isolation of the active component(s) of Z . multiflora responsible for antinociceptive activity.
Antinociceptive activity was evaluated using hot-plate and writhing tests. Morphine (10 mg/kg, i. p.) and diclofenac (10 mg/kg, i. p.) were used as positive controls. Analysis of variance followed by the multiple comparison test of Tukey-Kramer were used for comparison of data. Differences with p> 0.05 were considered significant.
The methanol-chloroform (1:1) extract of the aerial parts of the plants was prepared and partitioned between methanol-water (9:1) and n-hexane. The more active hydroalcoholic fraction was concentrated and further partitioned between methanol water (3:2) and chloroform. The hydroalcoholic layer showed a dose-dependent and significant activity. For all fractions the activity was observed at 100 mg/kg. Further fractionation of the hydroalcoholic fraction is underway to isolate the active component(s).
1. Hosseinzadeh, H.; Ramezani, M.; Salmani, G. J. Ethnopharmacol., 73, 379-385 (2000).
1Department of Pharmacognosy, Faculty of Pharmacy, University of Belgrade, Yugoslavia
2National Poison Control Centre, Military Medical Academy, Belgrade, Yugoslavia
3Institute of Pharmaceutical Biology, University of Freiburg, Germany
Tanacetum larvatum (Griseb.) Kanitz (Asteraceae) is a local endemic species, which is distributed on rocky places in Yugoslavia (Serbia-Kosovo, Montenegro) and Albania [1]. Recently, it was shown that the leaves contain the sesquiterpene lactone parthenolide at a concentration of 1.1% [2]. We have studied the chloroform extract prepared from the aerial blooming parts of the plant for anti-inflammatory and anti-ulcer activity.
Carrageenan-induced rat paw oedema test has been used as an experimental model for screening the anti-inflammatory activity according to the modified method of Oyanagni and Sato [3]. The extract was administrated p.o. in doses of 25, 50, 100 and 200 mg/kg to rats and compared with indomethacin, which was used as a reference at a dose of 8 mg/kg p.o. The results show that the extract slightly reduced the oedema in a dose dependent manner to 8.6, 32.8, 37.0 and 49.5% at doses of 25, 50, 100 and 200 mg/kg, respectively. Indomethacin has strong anti-inflammatory effects. It reduced inflammatory response to 26.62% at a dose of 8 mg/kg, but caused large gastric lesions. When indomethacin was concomitantly given with the extract (200 mg/kg), the ulcerogenic action was significantly reduced (see Table).
The anti-inflammatory activity may be partly due to the inhibition of the transcription factor NF-kB. This protein is a pivotal mediator of the immune system by regulation the transcription of various inflammatory mediators [4]. The chloroform extract impaired NF-kB DNA binding in an electrophoretic mobility shift assay. It can be assumed that parthenolide, which inhibits NF-kB at a 20 mM concentration [5], is responsible for this effect.
Table. Anti-inflammatory and ulcerogenic effect of indomethacin giving alone and concomitantly with the extract of T. larvatum
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Treatment (mg/kg p.o.) |
Inflammatory response (%) |
Lenght of gastric lesions (mm) |
Lesion area (mm2) |
Gastric damage score |
% of animals with lesions |
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Control (1 ml/kg p.o. DMSO) |
100 ± 13.73
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0 |
0 |
0 |
0 |
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Indomethacin (8mg/kg) |
26.62 ± 14.23
|
32.6 ± 24.60 |
5.98 ± 6.64 |
3.6 ± 2.40 |
100 |
|
Indomethacin (8mg/kg) + Extract (200mg/kg) |
18.39 ± 6.97
|
2.9 ± 3.54 |
0.195 ± 0.41 |
1.1 ± 1.64 |
80 |
1. Josifovic, M. (Ed.): The Flora of FR Serbia, Vol. VII. SANU, Belgrade, 1975
2. Vajs, V. et al. (2000) Arh. farm. 3-4: 252-253
3. Oyanagni, Y., Sato, S. (1991) Arzneim.-Forsch./Drug Res. 41(I): 5
4. Baeuerle, P., Henkel, T. (1994) Ann. Rev. Immun. 12: 141-79
5. Rüngeler, P. et al. (1999) Biorg. Med. Chem. 7: 2343-2352
aDepartment of Chemistry, University of Turku, FIN-20014 Turku, Finland
bInstitute of Bio-Medicine, Department of Anatomy, University of Turku, Kiinamyllynkatu 10, FIN-20014 Turku, Finland
A 70% methanol extract of Terminalia chebula fruit was studied in vitro for its antiproliferative activity on two breast cancer cell lines, MCF-7 (human breast cancer cells) and S115 (mouse breast cancer cells); two prostate cell lines, PC-3 (human prostate cancer cells) and PNT1A (human immobilized prostate gland cells) and HOS (human osteosarcoma cells) with cytotoxicity assays, ([3H]-thymidine incorporation, ATP determination, and Coulter Counter) and cell-death-mechanism assays, (Flow Cytometery and Hoechst DNA staining). In cytotoxicity assays the extract showed strong activity (ID50 in mg / ml range) on all cell lines included in the study. The flow cytometric results showed necrosis as the mechanism of cell death by the extract. According to the Hoechst DNA Staining assay, 400 mg / ml of the extract killed the cells apoptotically. ATP assay guided chromatographic fractionation of crude T. chebula fruit extract yielded chebulainic acid, and ellagic acid which were tested by ATP assay in comparison to five known phenolics of Terminalia (gallic acid, ehtyl gallate, tannic acid, 2.3.4-trihydroxy benzoic acid, and luteolin). Chebulinic acid (IC50 = 52.21 ± 0.16 mM) > Tannic acid (IC50 = 59.0 ± 0.19 mM) > and ellagic acid (IC50 = 78.5 ± 0.24 mM) were confirmed as the most active compounds in our study. In vitro antioxidant analyses of the extract showed 89% inhibition of lipid peroxidation (ID50 = 195.0 mg / ml) and 92% DPPH radical scavenging potential. This strong antiproliferative potential of the extract and its phenolics could be attributed to their remarkable antioxidant properties.
1 Institute for Medicinal Plants Research, Tadeusa Koscuska 1, 11000 Belgrade, Yugoslavia
2 Faculty of Medicine, Dr. Subotica 8, 11000 Belgrade, Yugoslavia.
Phytochemical investigation of Gentianella species, which grows in Serbia and Montenegro, mainly are not done. Our study on Gentiana, Centaurium and Swertia species resulted in the determination of simple and quick general chromatographic pattern which permitted identification and evaluation of the characteristic compounds in the crude drug, that are g-pyrones and secoiridoids. Preliminary study of Gentianella species included G. crispata, G. bulgarica, G. albanica and G. germanica. Similar chromatographic profiles were noticed. All species were characterised by the presence of bellidifolin and bellidifolin-8-O-glucoside. Among secoiridoids gentiopicrine dominated.
Here we investigated the antimicrobial activity of G. crispata and G. bulgarica extracts. Plant material was harvested on natural habitats. G. crispata was collected on Durmitor (2300 m) and G. bulgarica on Stara mountain (1700 m). Samples (aerial parts) were air-dried, and extracted in a Soxhlet apparatus with MeOH and CHC13 for 24 h. Solvents were evaporated under vacuum and the samples obtained were termed GC1 (methanolic extract of G. crispata aerial parts), GC2 (chlorophormic extract of G. crispata aerial parts), GB1 (methanolic extract of G. bulgarica aerial parts) and GB2 (chlorophormic extract of G. bulgarica aerial parts).
Antimicrobial activity against Staphylococcus aureus ATCC 25923 (Gram positive), Bacillus IP 5832 (spore-forming), Escherichia coli ATCC 25922 (Gram negative), Candida albicans ATCC 24433 and Aspergilus niger was determined by agar well diffusion. Stationary-phase culture was suspended in sahne solution in concentration of approximately 105 cfu/mL and 200 mL and the suspension was spread over the surface of Mueller-Hinton (for bacteria) and Sabouroud (for yeast) agar plate. Test samples at dilution of 1 mg/100 mL were added in each well, and after 24 hours of incubation the zones of inhibition were measured.
By agar well diffusion test no activity was found against Gram positive, gram negative and spore forming bacteria. Activity against C. albicans was detected in all samples with zone of inhibition of 3 mm for GC1, 4 mm for GB1, 4 mm for GC2 and 7 mm for GB2. Activity against A. niger was detected in the methanolic extracts GC1 and GB1 (2 mm).
*Central Laboratory for Drug & Food Analysis, Ministry of Health, P.O.Box 59082, Riyadh-11525, Saudi Arabia
**College of Pharmacy, King Saud University, P.O.Box 2457, Riyadh-11451, Saudi Arabia
Traditional claims about the medicinal properties of several natural drugs have now been verified on modern scientific grounds. Many international organisations including the World Health Organisation are encouraging more efforts in this direction1. A survey of the literature revealed that little attention has been paid to evaluate the toxicity of natural drugs. Such toxicity data may provide essential information regarding measure to be taken during preclinical testing of such drugs. Commiphora molmol (Oleo-gum-resin) has been proven to possess significant anti-inflammatory, antipyretic and antihistaminic effects in animal models. C. molmol treatment showed a dose-dependent mitodepressant effect in the femoral cells of mice and reduction of RNA levels in hepatic cells as compared to the control. However, pretreatment with C. molmol could neither alter the biochemical and cytological effects of cyclophosphamide nor showed any additive effect of both treatments. The cyctoxicity of C. molmol was also confirmed in Ehrlich solid tumor cells2. Teucrium polium showed anti-ulcer3 and hypoglycemic activities4. Trigonella foenum-graecum was found to possess hypoglycemic activity5.
In the present study Commiphora molmol, Teucrium polium and Trigonella foenum-graecum were subjected to acute toxicity studies in mice for 24 h and chronic toxicity evaluation for 3 months. External morphological changes, visceral toxicity, haematological changes, spermatogenic dysfunction and effect on body weight and vital organs weight were recorded. C. molmol showed no signs of toxicity. In Teucrium polium treated animals during the first month two male mice were observed to be anemic. During the second month of treatment, one of these male mice showed abnormal movement while the other developed alopecia. Two other male mice suffered from genital degeneration and putrification of sex organs during the third month of treatment with Teucrium polium. Increased mortality, increase in weight of liver, kidney and decrease in weight of testis was observed in the Teucrium polium group. In the group treated with Trigonella foenum-graecum one male mouse developed a fore limb tumor after 30 days of treatment. During the second month two other mice were found to have tumor in the fore limb and inflammation in the hind limb. Teucrium polium and Trigonella foenum-graecum treatment caused significant sperm damage.
Akerele, O. (1992). W.H.O. guidelines for the assessment of herbal medicine. Fitoterapia LXIII: 99-110.
Al-Harbi, M.M., Qureshi, S., Raza, M., Ahmed, M.M., Giangreco, A.B. and Shah, A.H. (1994). Anticarcinogenic effect of Commiphora molmol on solid tumors induced by Ehrlich carcinoma cells in mice. Expermental Chemotherapy 40: 337-347.
Twaij, H.A., Albader, A.A. and Abul-Khail, A. (1987). Anti-ulcer activity of Teucrium polium. Int. J. Crude Drug Res. 25: 125-128.
Gharaibesh, M.N., Elayan, H.H. and Salhab, A.S. (1988). Hypoglycemic effects of Teucrium polium. J. Ethnopharmacology 24: 93-99.
Sharma, R.D., Raghuram, T.C. and Rao, N.S. (1990). Effect of fenugreek seeds on blood glucose and serum lipids in type I diabetes. Eur. J. Clin. Nutr. 44: 301-306.
1 Institut für Pharmazie, Pharmazeutische Biologie, Freie Universität Berlin, Königin-Luise-Str. 2+4, D-14195 Berlin, Germany
2 Institut für Pharmazie, Pharmazeutische Technologie und Biotechnologie, Kelchstr. 31, D-12169 Berlin, Germany
3 Robert Koch-Institut, Abt. Infektionskrankheiten, Nordufer 20, D-133353 Berlin, Germany
Leishmania are obligate intracellular parasites of the monocyte-macrophage system. Even for chemotherapy, cure of leishmaniasis depends on the activation of microbicidal mechanisms of host cells. Interferon-gamma (IFN-g), produced by natural killer- (NK) or T-cells, is the major macrophage-activating cytokine. Immunomodulation can also be achieved by external signals, such as certain plant metabolites. Expanding on our recent studies on polyphenols (1), we tested 4 plant polyphenols for cytokine induction in Leishmania-parasitised macrophages.
Macrophage-like RAW 264.7 cells were infected in vitro with L. donovani promastigotes and exposed to 50 µg/ mL of gallic acid (1), 3,5-digalloyl shikimic acid (2), epigallocatechin 3-gallate (3) or a proanthocyanidin hexamer for 4 h. IFN-g plus bacterial endotoxin (LPS) served as positive control for immunological macrophage activation. Total RNA was extracted and semi-quantitative RT-PCR performed with primers for interleukin (IL)-1, IL-10, IL-18, tumor necrosis factor-alpha (TNF-a), inducible nitric oxide synthase (iNOS) and hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) as internal standard.
IL-1, iNOS and TNF-a were expressed at transcription level in all Leishmania-infected cells treated with the polyphenols or IFN-g +LPS. IL-18 was also induced by IFN+LPS or the test compounds. Interestingly, IL-10 was marginally expressed in infected, non-treated cells, but strongly only in epigallocatechin 3-gallate-treated RAW cells. Corresponding to the known down-regulating activity of IL-10, expression of both iNOS and IL-1 appeared much weaker in its presence.
In conclusion, gallic acid (1), 3,5-digalloyl shikimic acid (2), epigallocatechin 3-gallate (3), and a proanthocyanidin hexamer activated macrophage functions in terms of cytokine gene expression. However, the spectra of induced cytokine mRNAs at 4 h differed significantly. These data show that plant polyphenols possess immunomodulatory properties with positive implications for their use as novel antileishmanial drugs.
1. Kolodziej, H., Kayser, O., Latté, K.P. & Kiderlen, A.F. (1999) Plant Polyphenols 2 (Gross, G.G., Hemingway, R.W., Yoshida, T., eds.) Kluwer Academic, New York, 575-594.
Institut für Pharmakologie und Toxikologie der WWU, Domagkstr. 12, D-48149 Münster, Germany
Clinical studies demonstrate a comparable antidepressant efficacy of Hypericum perforatum (St. John`s wort) extract with tricyclic antidepressants such as imipramine. Onset of efficacy of imipramine and Hypericum extract occurs typically after several weeks of treatment, so it is believed that the medication causes delayed central nervous system adaptations. Since changes to the binding properties of beta-adrenergic receptors have been reported as potential indicators of antidepressant activity we used quantitative radioligand receptor-binding-studies to examine in rats the effects of short-term (2 weeks) and long-term (8 weeks) oral administration of imipramine (15 mg/kg p.o.), Hypericum extract (500 mg/kg p.o.), hypericin (0.2 mg/kg p.o.) and miquelianin (0.6 mg/kg p.o.) on the regulation of beta-adrenergic receptors in rat frontal cortex. Hypericin and miquelianin have been chosen as both substances proved to be remarkably active in the forced swimming test (1,2).
[125I]CYP binding to b-adrenergic receptors was found to be decreased after short as well as after long-term treatment with imipramine. Interestingly, short-term treatment with St. John´s wort had no effect on b-adrenoceptor regulation, while long-term treatment with Hypericum extract elicited a marked increase in b-binding in the frontal cortex. A similar effect on b-receptor density was observed for fluoxetine (3), a selective serotonin reuptake-inhibitor (SSRI). Therefore it can be speculated that serotonergic neurotransmission may play an important role in mediating the antidepressant effects of St. John’s wort, a finding which is confirmed by our recent results (submitted for publication). The increase in b-binding may further be explained by either effects of so-far untested compounds in the extract or a synergistic action of substances in combination.
However, hypericin or miquelianin alone had no effect on b-receptor density, neither after 2 nor after 8 weeks. Our data clearly show that long-term, but not short-term administration of St. John´s wort, modifies binding properties of b-adrenergic receptors in the rat frontal cortex. Because St. John´s wort, hypericin and miquelianin had different effects on adrenergic neurotransmission, they might have subtly different actions. The extent to which other constituents of St. John’s wort contribute to its activity on b-adrenergic receptors is under current investigation.
Butterweck V., Korte B., and Winterhoff H. (2001a) Pharmacological and endocrine effects of Hypericum perforatum and hypericin after repeated treatment. Pharmacospychiatry (in press)
Butterweck V., Petereit F., Winterhoff H., and Nahrstedt A. (1998) Solubilized hypericin and pseudohypericin from Hypericum perforatum exert antidepressant activity in the forced swimming test. Planta Med. 64, 291-294
Palvimaki E.P., Laakso A., Kuoppamaki M., Sylvalathi E., Hietala J. (1994), Up-regulation of b 1-adrenergic receptors in rat brain after chronic citalopram and fluoxetine treatments. Psychopharm. 115(4), 543-6
1Institut für Biochemie
2Institut für Anatomie
Medizinische Fakultät, Universität Leipzig, Leipzig, Germany
Matrix-Metalloproteinases (MMP) form a family of structurally and functionally related zinc endopeptidases that play an important role in the degradation of extracellular matrix proteins and are thus implicated in the reconstruction of the extracellular matrix and of connective tissue. Therefore, these proteinases are involved in the pathogenesis of several disorders such as rheumatoid arthritis, atherosclerosis, tumor growth, metastasis, and fibrosis. MMPs are secreted by a number of different cell types, each of which can secrete a different set of MMPs that may be altered in response to the disease. In atherosclerosis and metastasis, particularly MMPs produced by vascular endothelial cells are involved in the remodelling of the vessel wall as part of the pathogenesis. Since garlic is believed to have anti-atherogenic properties, we were interested in whether garlic-derived organosulphur compounds might affect the secretion and activity of MMPs liberated from vascular endothelial cells.
Human coronary artery endothelial cells (HCAEC) were maintained in Microvascular Endothelial Growth Medium-2 (Clonetics Cell Systems) which is supplemented with various hormones and growth factors. Experiments concerning the expression of MMPs were performed during the growth phase of the HCAEC between two and six days after subcultivation with changes of the culture medium every second day. At 9 or 24 h after a medium change, culture medium was taken and analysed for secreted MMP-1,-2,-3,-8,-9, and -13 using specific ELISA calibrated by external and internal standards. During this incubation period, cells were also exposed to several garlic-derived organosulphur compounds, diallyl disufide (DADS), allyl mercaptan (AM), and S-allylcysteine (SAC) in concentrations from 0.5 to 500 µM. No signs of cytotoxic effects were seen, even at the highest concentrations.
The HCAEC cells secreted mainly MMP-1 and -2, while MMP-8 and -13 were hardly detectable. MMP-9 was also very low and, in contrast to HUVEC (human umbelical vein endothelial cells), was not stimulated by a mixture of TNF-a, forskolin and PMA in the HCAEC. During the 9 h incubation period, MMP-2 secretion was blocked by DADS with an EC50-value of approximately 30 µM and a maximal effect at 500 µM (about 75% reduction). AM was much less effective, while SAC apparently did not show any effect. In the case of MMP-1, DADS was similarly effective, whereas AM and SAC appeared to act slightly stronger than with MMP-2.
These results indicate that DADS, which is a common product of garlic-derived organosulphur compounds and is easily detectable in blood after garlic consumption, has some potency to reduce the production of MMPs by HCAEC cells. Whether this effect observed during short-term incubations is enforced after long-term exposure to DADS remains to be established. Nonetheless, even a slight decrease of MMP secretion may be of significance for pathogenetic processes, e.g. in atherosclerosis, because the balance between the MMPs and their inhibitors, the tissue-inhibitors of matrix metalloproteinases (TIMPs), which is the main determinant of the matrix degrading activity, may be changed. The question of whether secretion of TIMPs by endothelial cells is also affected by garlic-derived compounds will be addressed in future studies.
a Fundação Ezequiel Dias, Rua Conde Pereira Carneiro 80, 30510-010 Belo Horizonte, Minas Gerais, Brazil
b School of Biosciences, University of Nottingham, University Park Campus, Nottingham NG7 2RD, U.K.
Studies of P. hexandrum as axenic shoot cultures and/or dedifferentiated tissues are limited, partially attributed to a short seasonal availability for plants of this species. Additionally, some studies have revealed that adventitious rooting of shoots was difficult. In view of the importance of establishing in vitro propagation systems for P. hexandrum, a rare and threatened species, studies were undertaken to investigate the effects of the Rhizobial lipo-oligosaccharide NGR234 (a nod-factor) on the stimulation of rooting of somatic embryo-derived plantlets. It was speculated that this or related compounds may be capable of inducing and promoting rooting of such recalcitrant species. Somatic embryos were produced via embryogenic cell suspensions cultures established from root-derived callus, cultured in liquid UM medium in the dark. Differentiation of somatic embryos and subsequent shoot formation was achieved when embryos were transferred to full-strength MS medium with 0.45 mg l-1BAP. Somatic embryo-derived shoots (with no evidence of a root system) were individually cultured on full-strength MS medium supplemented with 10-5 M NGR234 in the dark for 7 d at 22° ± 1°C. Shoots were thereafter transferred to half-strength MS medium lacking growth regulators. Plantlets were maintained under a 16 h photoperiod (42 mmol m-2 s-1) at 22° ± 1°C for 4 weeks. Rooting (50%) of such plantlets was obtained within 35 d of transfer. This is the first report describing the recovery of fully developed somatic embryo-derived plants of P. hexandrum following promotion of rooting in response to a lipo-oligosaccharide such as NGR234. The supplementation of culture medium with lipo-oligosaccharides opens new approaches for the rooting of somatic embryo-derived plantlets of P. hexandrum, which, in turn, provides a realistic basis for its routine micropropagation.
Institute of Pharmaceutical Biology, University of Basel, Benkenstrasse 254, CH-4108 Witterswil, Switzerland
Hydroalcoholic extracts of Vitex agnus-castus have been shown to exert dopaminergic actions via subtype D2 receptors characterized by D2 antagonists 125I-sulpride and 3H-spiperone (1, 2). Further evidence for dopamine D2 agonistic action is based on inhibition of acetylcholine release in rat striatal slices (2). A reduced hypersecretion of prolactin mediated by such dopaminergic actions of Vitex agnus-castus extracts may prevent premenstrual symptoms (PMS).
Here, we studied binding characteristics of 3H-spiperone to both calf striatum and human dopamine D2-CHO cells in the presence of various Vitex agnus-castus extracts. Free and receptor bound radioactivity was separated by filtration. Ethanolic extracts derived from fruits were prepared by maceration. 3H-spiperone displacement experiments in the presence of various Vitex extracts revealed sigmoid competition curves with IC50 values in the range of 10 to 50 microgram native extract per ml. Thereby, specific binding was calculated by the difference between total binding (TB) and non-specific binding (NSB; (+)-butaclamol) in the absence or presence of corresponding extract concentrations. Concentrations >1 microgram extract/ml resulted in an increased NSB: at 5 microgram extract/ml NSB reached the level of TB, while it even increased >500% of TB at 100 microgram/ml. At such high NSB levels, the determination of specific binding becomes inaccurate. Fractionation of the dry extract by stepwise extraction with either hexan or ether followed by MeOH also failed to provide reasonable NSB. Binding experiments with 3H-haloperidol revealed even higher NSB than with 3H-spiperone. The described phenomenon is unique with respect to such high levels of NSB compared to other radioligand bindings, where NSB is usually at a reasonable low level in the presence of corresponding extract concentrations.
In conclusion, competition binding experiments with Vitex agnus-castus extracts to D2 receptors failed to provide reliable results due to high non-specific binding of the radioligand to the filter. Other separation techniques like centrifugation or the use of additional radioligands may overcome this technical problem and may result in reliable competition binding data confirming the known dopaminergic effect of Vitex agnus-castus .
Jarry et al. (1994) Exp. Clin. Endocrinol. 102: 448-454.
Meier et al., (2000) Phytomedicine 7: 373-381.
T.J. Schmidta, J. Heilmannb
aInstitut für Pharmazeutische Biologie der Heinrich-Heine-Universität, Düsseldorf, Germany
bInstitut für Pharmazeutische Wissenschaften, ETH Zürich, Switzerland.
Due to their high degree of cytotoxicity, many sesquiterpene lactones (STLs) have been widely recognized as potential lead structures for the development of anti-tumoral drugs.1,2 Apart from the fact that cytotoxicity is mediated by potentially alkylating structure elements such as a,b-unsaturated carbonyl groups, capable of undergoing Michael-type addition to biological macromolecules, little is known about the structural determinants of cytotoxicity. Detailed knowledge on the contributions of different structural features would be highly desirable as a first step in the direction of lead structure optimisation. In continuation of a previous QSAR study on cytotoxicity of helenanolide type STLs3, we have now investigated a set of 35 sesquiterpene lactones representing five different structural groups (1 germacranolide, 3 guaianolides, 23 pseudoguaianolides, 7 eudesmanolides and 1 carabranolide; figure 1 shows an overlay of all 35 structures with the g-lactone rings superposed) in order to obtain a more general description of the factors responsible for cytotoxic activity and to allow for prediction of cytotoxicity for a wider range of compounds. Cytotoxic IC50 values towards the KB nasopharynx carcinoma cell line were experimentally determined for each compound. The range of biological activity in this data set varies over more than two orders of magnitude (-log IC50 (M) between 3.9 and 6.2). QSAR analysis was carried out by analysis of 30 descriptors of molecular and physicochemical properties obtained by molecular modeling techniques. Selection of significant descriptors was carried out by genetic algorithm/partial least squares (GA-PLS) statistics.4 The best model obtained by this procedure so far contains significant contributions from 9 of the 30 descriptors (two significant PLS components), namely, three electronic parameters derived from AM1 bond orders in the respective a,b-unsaturated structure elements (33), the solvent-accessible surface of activated double bonds (20) and accessible non-polar surface (8*), energies of second and third lowest unoccupied MOs (20*), dipole moment (7), and deviation from a spheric molecular shape (11*). (In brackets: relative contributions to the model in %; parameters marked with * show a negative regression coefficient). Statistical parameters: r2 = 0.83, s=0.22, q2=0.76, SDEP=0.26. Fig. 2 shows a plot of experimental vs. calculated pIC50 values.
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Figure 1 |
Figure 2 |
The model provides insight into the factors that determine STL cytotoxicity and allows for prediction of the cytotoxic activity of untested compounds with almost 80% certainty (100*q2). Further QSAR models obtained with different approaches and their application to literature data will be discussed.
1. Schmidt, TJ 1999 Current Org. Chem. 3, 577-605.
2. Picman, AK 1986 Biochem. Syst. Ecol. 14, 255-281.
3. Schmidt TJ 1999 Pharm. Pharmacol. Lett. 9, 9-13.
4. UNC QSAR server: http://mmlin1.pha.unc.edu/~jin/QSAR/
aInstitut für Pharmazeutische Biologie der Heinrich-Heine-Universität, Düsseldorf, Germany bFaculty of Pharmacy, Philadelphia University, Amman, Jordan
cSwiss Tropical Insitute, Basel, Switzerland
Sesquiterpene lactones (STLs) such as helenalin 1 and many others are known to possess a wide spectrum of biological activities, which are mediated in most cases through the presence of a,b-unsaturated carbonyl groups capable of alkylation of biological nucleophiles.1,2 Among the many reports on antimicrobial effects of STLs, also anti-protozoal activity has been reported.1,2
Trypanosomiasis caused by Trypanosoma brucei rhodesiense (East African Sleeping Sickness) and T. cruzi (Chagas‘ disease) are among the the most serious infectious diseases in the tropical regions. Search for new pharmacological agents to control these infections is therefore an important goal. We have now investigated in-vitro the potential acitivity of six sesquiterpene lactones isolated from Arnica (1-5) and Inula species (6) (Asteraceae) against trypomastigotes of both Trypanosoma species.
The resulting IC50 values show that bifunctional alkylants (1 and 2) as in many other biological assays, are the most potent agents in this group. T. b. rhodesiense was found to be generally more sensitive to STLs than T. cruzi.
With an IC50 of 0.027 µM against T. b. rhodesiense, helenalin 1 appears to be a promising candidate for further evaluation. Cytotoxic IC50 values of 1 towards mammalian cells are generally about an order of magnitude higher.3,4 It appears noteworthy, that mexicanin I (2), a diastereomer of 1, is less active by a factor of almost 16, so that obviously stereochemistry plays an important role which will have to be investigated in detail. Both, 1 and 2, possess two potential alkylant centers, the cyclopentenone (CP) and the a-methylene-g-lactone (ML) group. Compounds 3 and 4 possessing only the former or the latter, respectively, differ in activity, 3 being more active by a factor of 5.5 which indicates that the CP structure may be more influential on anti-trypanosomal activity. Further studies on the mechanism of action and on structure-activity relationships are in progress.
1. Picman, AK 1986 Biochem. Syst. Ecol. 14, 255-281.
2. Schmidt, TJ 1999 Current Org. Chem. 3, 577-605.
3. Kupchan, SM et al. 1971 J. Med. Chem. 14, 1147-1152.
4. Schmidt TJ 1999 Pharm. Pharmacol. Lett. 9, 9-13.
BIOLOGIGCAL ACTIVITY OF BIFLAVONOIDS FROM Ochna macrocalyx AND A FURTHER CONSITUENT
1 Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, U.K.
2 Institut für Pharmazeutische Biologie, Albert-Ludwigs Universität, Stephan-Meier-Str. 19, 79104 Freiburg, Germany
Ochna macrocalyx Oliv. (Ochnaceae) is a medicinal tree from the Western Usambara Mountains of Tanzania. Its powdered yellow bark is used for female disorders and gastrointestinal problems, and its ethnobotanical importance led to a biochemical and phytochemical investigation (1).
A crude extract of the bark was obtained by reflux with ethanol. 200 mg/ml of the crude extract was found to have NF-kB inhibitory activity with no visible cytotoxicity. Fractionation of the ethyl acetate extract led to the isolation of four polyphenolic compounds via sephadex and preparative thin layer chromatography. Biflavonoids calodenin B (3) and trans-dihydro calodenin B (2), cordigol, and a cordigone like compound 1. Structures were elucidated using 1D and 2D NMR (1H and 13C) experiments and FAB mass spectroscopy.
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Antimicrobial investigations have been carried out on calodenin B, shown to have activity against three strains of methicillin resistant Staphylococcus aureus (MRSA). The compound was serially diluted (2 fold) into 10 wells of a 96 well microtitre plate and incubated with inoculum at 36 °C for 18 h. Preliminary minimum inhibitory concentrations obtained on MRSA strains RN 4220, 1199-B and XU212 were 32, 32 and 16 mg/ml, respectively. MTT reduction assays using MCF-7 breast cancer cells were used to determine the degree of cytotoxicity of the compounds. With calodenin B and 1, cytotoxicity was observed at concentrations above 30mg/ml. |
Calodenin B was observed to be cytotoxic to HeLa cells at concentrations above 80ng/ml and at lower concentrations it had no NF-kB inhibitory activity. Further work is being carried out to establish the source of the NF-kB inhibition observed for the crude extract.
1. Schlage, C., Mabula, C., Mahunnah, R.L.A., Heinrich, M. (2000) Medicinal Plants of the Washambaa (Tanzania): Documentation and Ethnopharmacological Evaluation. Plant Biology 2: 83 – 92.
2. Marston. A., Zagorski, M.G., Hostettmann, K. (1988) Antifungal Polyphenols from Cordia goetzei. Helvetica Chimica Acta 71:1210-1219
3. Tang, S., Schlage, T., Gibbons, S., Heinrich, M. (2000) Biflavonoids from Ochna macrocalyx - a Medicinal Plant from the Usambara Mountains (Tanzania). Poster presented at the ISE Conference in Zurich, Switzerland
1Department of Plant Physiology, Institute for Biological Research, 29 Novembra 142, 11 000 Belgrade, Yugoslavia
2Department of Pharmacy, Division of Pharmacognosy, University of Athens, Panepistimiopolis Zografou, 157 71 Athens, Greece
Essential oils of Origanum onites, Satureja thymbra, Salvia fruticosa (Greek sage), and S. pomifera subsp. calycina plants growing wild in Greece and their components; carvacrol camphor, and 1,8-cineole, were assayed for antifungal activity against 13 fungal species. Among the fungi tested were food poisoning, plant, animals and human pathogenic species.
In order to determine minimum inhibitory and fungicide concentrations (MIC and MFC) microdilution test was used. The oils showed inhibition against all investigated fungi. The highest and broadest activity was shown by the carvacrol containing oils (O. onites and S. thymbra), with MIC of 0.05-0.1 ml/ml and MFC of 0.05-0.2 ml/ml. The essential oil of sage was the least effective, with MIC and MFC of 5.0-20.0 ml/ml and 5.0-25.0 ml/ml, respectively.
Carvacrol possessed the highest and 1,8-cineole possessed the lowest level of antifungal activity among the components tested. The effect produced by the essential oils was compared with a standard antifungal agent bifobnazol. The essential oils of O. onites and S. thymbra and all the investigated components investigated possessed greater antifungal activities that this commercial fungicide.
Adams, R. P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured Publishing Co Illinois, USA (1995).
Daouk, K. D., Dagher, M. S., Sattout, J. E. Antifungal activity of the essential oil of Origanum syriacum L. Journal of Food Protection, 58, 1147-1149 (1995).
Hannel, H., Raether, W. A more sophisticated method of determining the fungicidal effect of water-insoluble preparations with a cell harvester, using miconazole as an example. Mycoses 31, 148-154 (1988).
Department of Pharmacology & Toxicology, Zayed Complex for Herbal Research & Traditional Medicine, Post Box 29300, Ministry of Health, Abu Dhabi, United Arab Emirates
Many animal models of morphine dependence are available, varying in the dose, route and duration of administration of morphine. We used a model of morphine dependence developed by Itoh et al. (1998) in our laboratory. In this model, morphine dependence is induced by administering morphine (5 mg/kg., s.c.) twice daily for 5 days and withdrawal symptoms are precipitated using naloxone (5 mg/kg., i.p.). Withdrawal symptoms such as diarrhoea and body weight reduction were observed in the animals, following this protocol. However most of the animals failed to exhibit jumping, which is one of the most obvious symptoms of withdrawal induced by naloxone in morphine-dependent mice. Therefore, we decided to modify this model by changing the morphine administration regime. Instead of administering the same dose of 5 mg/kg of morphine twice a day to the mice for 5 day, as described in Itoh’s method, we followed a gradually increasing dose schedule for morphine administration. Following doses were administered s.c. to the mice twice a day, one in the morning and one in the evening : 2.5 mg/kg on day 1, 5 mg/kg on day 2, 10 mg/kg on day 3, 20 mg/kg on day 4 and day 5. Drinking water was substituted with an aqueous solution of morphine (0.1 mg/ml on day 1 and day 2; 0.2 mg/ml on day 3, day 4 and day 5). Naloxone (20 mg/kg., i.p.) was used to precipitate withdrawal symptoms, 1 h after the last dose of morphine on day 5. We used only female mice in the study as in pilot studies, female mice were found to exhibit better withdrawal symptoms compared to male mice. In this modified model, 90% of the mice studied exhibited withdrawal symptoms viz. jumping, diarrhoea and body weight loss.
A proprietary Chinese herbal drug (FPK) used to treat opiate addiction was evaluated in our laboratory using the modified model. Morphine dependence was induced in four groups of animals consisting of 10 female mice each by following the above protocol. Groups I and II were co-administered with FKP at the doses of 1.25 g/kg and 2.5 g/kg, p.o. respectively, for 5 days, starting from day 1 of morphine administration. Group III and IV were administered with the vehicle orally for 5 days. On day 5, Groups I, II and III were given a single dose of naloxone (20 mg/kg, i.p.) to precipitate withdrawal symptoms and Group IV was given saline (i.p.) only. Jumping, body weight reduction after 1 h of naloxone administration and diarrhoea were quantitatively measured for each animal in the four groups. Withdrawal symptoms viz. jumping, diarrhoea and body weight reduction were observed in Groups I, II and III, but they were absent in Group IV. In Group I and II, diarrhoea and body weight reduction were significantly less, compared to in Group III. However there was no significant difference in the number of jumps elicited by the animals in Group I and II compared to Group III.
The modified model of morphine dependence was found to be a convenient method for screening anti-addiction drugs in a short period. In our laboratory, the model developed by Itoh et al. (1998) did not exhibit jumping, probably due to the strain difference of the mice or the observation cages used. The Chinese herbal formula was found to alleviate some of the withdrawal symptoms of morphine dependence in mice which supports the claims of the manufacturer.
1. Itoh A, Noda Y, Mamiya T, Hasegawa T, Nabeshima T. Methods. 1998, Find Exp Clin Pharmacol 20(7) 619-625.
Department of Pharmacology and Toxicology, Zayed Complex for Herbal Research & Traditional Medicine, Post Box 29300, Ministry of Health, Abu Dhabi, U.A.E.
Alpinia galanga (Fam. Zingiberaceae) locally known as Kholengan, is a small shrub distributed throughout Arabia. The local traditional healers consider this plant to be a good remedy for impotence and nervous debility (Ghazanfar, l994). However, these claims have not been verified scientifically. The present study was undertaken to investigate the effect of 10% ethanolic extract of A. galanga (rhizomes) on intracavernous pressure (ICP) and copulatory behaviour in male rats. ICP in male rats was measured using an in vivo technique (Chen et. al., l992). Male Sprague-Dawley rats were anaesthetized with pentobarbital sodium and the prepuce was de-gloved to expose the proximal shaft of the penis. A 26-gauge needle filled with normal saline was inserted into the cavernous tissue and ICP was recorded. Administration of 5 mg of the extract in 0.1 ml saline into the cavernous tissue produced a significant increase in ICP (28.2 ± 4.7) compared to the control ( 15.6 ± 3.1) and positive control-sodium nitroprusside ( 50.7 ± 7.8), values being expressed as a % of basal ICP.
The effect of extract of A. galanga was tested on copulatory behaviour in albino mice (Islam et al., l99l) following the acute oral treatment at the dose of 400 and 800 mg/kg. Receptive female mice were brought into oestrous with a single subcutaneous dose of 1 mg of estradiol benzoate, and 600 µg of progesterone, 72 h and 4 h before testing, respectively. The treated male was introduced into the cage with receptive female and copulatory behaviour was observed for 30 min. The parameters studied include mount latency (ML), intermission latency (IL), ejaculation latency (EL), mount frequency (MF), intermission frequency (IF), ejaculation frequency (EF) and mean intermission interval (MII). The results from the copulatory behaviour study show that acute treatment with A. galanga produced a significant increase in ejaculation frequency and mean intromission interval at the dose of 800 mg/kg as compared to the control (Table 1).
Table 1. Effect of A. galanga on copulatory activity in male rats
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Treatment
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ML |
IL |
EL |
MF |
IF |
EF |
MII |
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Control
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169.78 ± 39.30 |
248.50 ± 46.03 |
653.35 ± 65.11 |
6.43 ± 0.78 |
26.79 ± 2.16 |
2.93 ± 0.16 |
97.32 ± 13.11 |
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A. galanga 400 mg/kg |
253.90 ± 61.28 |
266.30 ± 60.05 |
660.40 ± 8.64 |
3.86 ± 1.08 |
21.90 ± 2.67 |
3.20 ± 0.13 |
107.00 ± 16.89 |
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A. galanga 800 mg/kg |
124.00 ± 35.01 |
157.43 ± 45.99 |
528.71 ± 48.17 |
5.57 ± 1.20 |
30.71 ± 25.87 |
3.57* ± 0.20 |
56.22 ± 6.51* |
*Significantly different from control * < 0.05 Student t-test
The results of the present study indicate that 10% ethanol extract of Alpinia galanga enhances the sexual activity in male animals supporting its traditional use as a sexual stimulant.
1. Ghazanfar, D.A. (l994) Handbook of Arabian Medicinal Plants, CRC Press.
2. Chen K. K, et.al. (1992) Neurosci. Lett. 141(2) 218-22.
3. Islam M.W. et al. (l991) Phytotherapy Research 33, 67-72
1 Institut für Pharmakologie und Toxikologie der Westfälischen Wilhelms-Universität, Domagkstr.12, D-48149Münster, Germany
2 Dr. Willmar Schwabe GmbH &Co, Willmar-Schwabestr.4, 76227 Karlsruhe, Germany
3 National Public Health Institute, PL719, 00101 Helsinki, Finland
Up to 30% of the German population drink large amounts of alcohol regularly. This is the reason for an increased risk of liver damage and neurological complaints. Moreover, the danger of becoming dependent on alcohol is heavily heightened by this chronic misuse. Therefore it would be interesting to find substances reducing the ethanol intake. As the risk of developing alcohol addiction in man is defined genetically, a suitable test animal is the genetically determined alcohol-dependent rat (1).
As experimental animals alcohol-preferring female AA- rats (National Public Health Institute, Helsinki) were used, which were bred and selected over many generations for high alcohol consumption (more than 5g/kg/d).The two bottle choice paradigm was used, with the choice to drink ethanol 10% (V/V) or water during the dark period (18.00 to 6.00).
The animals were treated with kava extract ID 501632080, which was suspended in 0,2% agar and administered orally by gavage shortly before the dark phase. At first, the reproducibility of ethanol intake (g/kg) and ethanol preference (%) of the test animals had to be investigated. A fairly constant amount of ethanol was consumed daily, the preference amounted more than 80%.
To exclude unspecific (toxic) effects food intake and total fluid intake were recorded additionally every day. Kava was administered in a dose range from 50 to 600mg/kg b.w.. Even 50mg/kg b.w. caused a slight reduction of ethanol intake and ethanol preference.
Following administration of 100mg/kg b.w., a decrease of 10% was observed. After 200mg/kg b.w., ethanol intake and ethanol preference were lowered significantly in all experiments. By further increasing the kava dose, the reduction of ethanol intake and ethanol preference raised dose-dependently. Following 600mg/kg, ethanol intake- and preference amounted only 40% of the pretreatment values, while food intake (g/kg) and total fluid intake (g/kg) were not affected at all even by the highest dose.
After repeated treatment with 100 or 200mg/kg b.w., the effect was not lessened , ethanol intake and ethanol preference were uniformly diminished by 13 resp. 26%.
As neither food intake nor total fluid intake were affected and as the effect remained unaffected after repeated administration this effect of the kava preparation on ethanol preference seems to be a specific one. This effect of kava is clearly superior to that of flupenthixol (2), which in higher doses reduces ethanol intake and total fluid intake simultaneously.
Hints on the mode of action shall be obtained by interference with substances with known central activity.
Kiianmaa K., Hyytiä P., Sinclair J., (1992) Development of an Animal Model of Ethanol Abuse. Neuromethods, Vol.24: Animal Models of Drug Addiction. Eds: A.Boulton, G.Baker, and P.H. Wu
Soyka M., de Vry J., (2000) Flupenthixol as a potential pharmacotreatment of alcohol and cocaine abuse/dependance. European Neuropsychopharmacology 10, 325-332
1Institut für Pharmazeutische Biologie, Albert-Ludwigs-Universität Freiburg, Stephan-Meier-Str. 19, 79104 Freiburg, Germany
2Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, 29-39 Brunswick Sq., London WC1N 1AX, U.K.
Olinia rochetiana A.Juss is a medicinal plant collected during ethnobotanical fieldwork in the western Usambara-Mountains, Tanzania(1). It is used traditionally as an infusion to treat toothache and inflammation of the gum. The Oliniaceae are a small monogeneric family, consisting of shrubs and small trees originating from Africa. Very little has been published about the phytochemistry of this family except the occurrence of cyanogenic glucosides in four species(2) and the caffeic acid derivative olinoside and related compounds from O. usambarensis Gilg(3) which is considered by some authors to be a synonym of O. rochetiana while others consider it a separate species (Sebola, personal communication).
Continuing our search for plant-derived inhibitors of the transcription factor NF-kB we screened selected medicinal plants from the Usambara-Mountains. An ethanolic extract of the leaves was active at 400mg/ml using an electrophoretic mobility shift assay (EMSA). Bioassay guided fractionation using successive liquid partitioning between petrolether, ethylacetate and water indicated that the ethylacetate fraction was the only active one. The fraction was active down to similar concentrations as the crude extract although the yield was only 15%. Sephadex LH 20 and MCI gel CHP20P fractionation led to the isolation of 5 compounds: the caffeic acid derivatives olinoside, for which we propose a revised stucture, 4-O-glucosyl-caffeic-acid, 6-O-caffeoyl-umbelactone and the ellagitannins tellimagrandin II and pedunculagin. The structures were elucidated by NMR (1H-, 13C-NMR, COSY, HMQC, HMBC, NOESY), ESI-MS and by comparison with published data. Only the first two compounds have previously been reported from this species. The isolated compounds were tested for their effect on NF-kB activation using the luciferase assay with stably transfected HeLa cells. Some activity was observed with Olinoside (120mg/ml), 6-O-caffeoyl-umbelactone (30mg/ml) and Tellimagrandin II (50mg/ml). It is doubtful, whether the effects observed at these concentrations are of therapeutic relevance.
The activity of the ethanolic extract cannot be explained by the activity of the single components. There is a significant loss in activity during the isolation procedure. Possible reasons for this loss will be discussed in the poster.
C. Schlage, C. Mabula, R.L.A. Mahunnah, M. Heinrich, Plant Biology 2 (2000) 83-92.
A. Nahrstedt and J. Rockenbach, Phytochemistry 34 (1993) 433-436
E. Nyandat, E. Rwekika, C. Galeffi, G. Palazzino and M. Nicoletti, Phytochemistry 33 (1993) 1493-1496
Centre For Post-Graduate Studies & Research In Botany, G.T.P.College Campus,
Nandurbar-425412, Maharashtra, India
Leaf extracts of eight angiosperm plants viz. Aristolochia bracteata Lam (Aristolochiaceae), Argemone mexicana Linn. (Papavaraceae), Boerhhavia difusa Linn. (Nyctaginaceae), Butea monosperma (Lam.) Taub. (Fabaceae), Cassia tora Linn. (Caesalpinaceae), Enicostemma littorale Bl. (Gentianaceae), Nyctanthes arbortristis Linn. (Oleaceae) and Xanthium strumarium Linn. (Compositae), have been tested for biocidal properties against four pathogenic fungi namely: Alternaria brassicola, Colletotrichum capsici, Fusarium oxysporum and Rhizoctinia solani. These plants are being widely used in India as folk medicines for controlling different skin diseases of humans as well as cattle. All plants chosen are sumptuously available in the forests and wastelands; many of them are predominant weeds in the crop fields throughout the country causing economic loss to the farmers.
Fresh leaves (100 g) of each plant were cleaned with tap water, crushed in a mortar and transferred to a conical flask. After adding 500 ml-distilled water, the extract was boiled for 10 min, cooled and filtered. The extract was then tested (individually and in combinations) for anti-fungal activity. The pathogenic fungi were multiplied on Potato-Dextrose-Agar (PDA) medium. Mycelial disks (6 mm in diameter) from 7-day old cultures were inoculated in the petri dish containing PDA. In the center of each Petri dish a well of 10 mm diameter was made with a flamed cork borer. Each well was filled with 10 ml of plant extract. The plates were kept in the refrigerator for one hour for diffusion and then incubated for 5-days at 30±°C. The zone of inhibition was measured in mm diameter, after the incubation period.
Except for Cassia tora and Xanthium strumarium, all plants showed biocidal activity. However, individually, Aristolochia bracteata, Butea monosperma, Enicostemma littorale and Nyctanthes arbortristis were more efficient than the rest of the plants while a combination of Butea and Nyctanthes was proved to be the best in controlling the fungal growth. The ethanolic extract of each plant (1000µg/ml) supplemented in the medium also showed similar results. Field experiments with a mixture of Butea and Nyctanthes leaf extracts showed significant control of leaf spot disease caused by A. brassicola on cabbage.
1Dipartimento di Economia e Merceologia, Università di Trieste, Italia
2Faculte de Medicine et Pharmacie, Université Mohammed V, Rabat, Morocco
3Dipartimento di Scienze Farmaceutiche, Università di Salerno, Italia
4Faculte des Sciences, Université Mohammed V, Rabat, Morocco
Thymus willdenowii Boiss, trivial name “z’itra”, is a species of the Lamiaceae family endemic to Morocco and Tunisia, where its leaves are currently employed by the traditional medicine as a remedy against bronchitis, colitis, rheumatisms and similar inflammatory illnesses [1, 2].
The biological activities of some species of the genus Thymus are already described, such as the spasmolytic and the antioxidant ones, both ascribable to the polyphenolic compounds, while thyme essential oil is reported to possess antimicrobial properties [3-5]. However, T. willdenowii was never submitted to phytochemical or to pharmacological investigations. To verify the therapeutic value of the plant, we submitted the leaves to a bioassay-oriented fractionation procedure for anti-inflammatory activity, using the Croton oil ear test in mice as assay.
The first step of fractionation yielded four extracts and, among them, the most active was the chloroform one. Its potency was similar to that of indomethacin, the non steroidal anti-inflammatory drug used as reference, being the relevant ID50 values (dose giving 50 % oedema inhibition) 83 and 93 µg/cm2, respectively. The subsequent fractionation of the chloroform extract revealed ursolic acid and oleanolic acid as the active principles responsible for the observed anti-inflammatory activity. Ursolic acid was about two times more active than indomethacin (ID50 = 0.12 and 0.26 µMoles/cm2, respectively), whereas the potency of oleanolic acid (ID50 = 0.29 µMoles/cm2) was similar to that of the reference drug. Fractionation of the methanol extract led to the isolation of two flavonoids new for the genus Thymus, luteolin-3’-O-glucuronide and eriodictyol-7-O-glucoside. The role of these results in the evaluation of the traditional use of the plant is discussed.
1. Bellakhadar J., Claisse R., Fleurentin J., Younos C. - J. Ethnopharmacol. 35: 123-143 (1991).
2. Thiri B. - These de Doctorat de 3° Cycle, Université Mohamed V, Faculté des Sciences, Rabat, Moroc, N. 1441.
3. Vand Den Broucke C.O., Lemli J.A., Lamy J. - Plantes Medicinales et Phytotherapie XVI: 310-317 (1982).
4. Miura K., Nakatami N. - Agric. Boil. Chem. 53: 3043-3045.
5. Lattaoui N., Tantaoui-Elaraki A. - J. Ess. Oil Res. 6: 165-171.
Department of Pharmacology and Toxicology, Zayed Complex for Herbal Research & Traditional Medicine, Post Box 29300, Ministry of Health, Abu Dhabi, U.A.E.
Crotalaria aegyptica (Family: Fabaceae) is a perenniel herb commonly found in Arabia. The leaves of another species of Crotalaria viz. C. retusa, are used to treat diarrhoea in traditional medicine (Warrier et al., 1995). The present study was designed to evaluate the anti-diarrhoeal activity of Crotalaria aegyptica.
A standardised aqueous extract of the aerial parts of C. aegyptica was used in the study. In vitro effects of the extract on gastro-intestinal tract were studied on isolated rabbit jejunum and in vivo studies were carried out in rats.
Rabbit jejunum was isolated, cut into 1 inch pieces and set up in an organ bath (Aziba et al., 1999). After 30 min of stabilization period, the extract at concentrations 0 (control), 0.3 and 1.0 mg/ml were added to the bath in different sets of experiments and the responses were recorded using a transducer - amplifier - chart recorder assembly.
Anti-diarrhoeal effect of the extract was studied using the castor oil-induced diarrhoea model in rats (Awouters et al., 1975). Wistar rats were administered the extract at the dose of 1 g/kg., p.o., followed by 1 ml of castor oil after 1 h. The numbers of droppings of the stool were counted at 1, 2 and 4 h after castor oil administration and the weight of the total stool output in 24 h were recorded in both groups.
The extract produced about 90% reduction in amplitude of spontaneous contractions in rabbit jejunum at the concentration of 1.0 mg/ml. Frequency of contractions was not affected by the extract. The inhibitory effect of Crotalaria extract were prevented, although not completely, with the pre-treatment of either phentolamine or propranolol. In rats treated with Crotalaria extract, the stool output in 24 h was significantly reduced compared to the control group, indicating anti-diarrhoeal effect. However the numbers of stool in treated group at 1,2 and 4 h were not significantly different from control group (Tab. 1).
Table 1. Effect of C. aegyptica extract on stool output in rats
|
Group |
Body weight (g) |
Number of stool after castor oil administration |
Weight of total stool output in 24 h (g) |
||
|
1 h |
2 h |
4 h |
|||
|
Control |
232.50±12.56 |
0.33±0.26 |
1.33±0.55 |
1.67±0.45 |
8.46±0.46 |
|
Crotalaria |
225.83±16.86 |
0.50±0.26 |
0.50±0.26 |
1.00±0.40 |
6.07±0.45* |
* Significantly different from control, P<0.05, Student’s t-test.
The results indicate anti-diarrhoeal effects of C. aegyptica extract. Reduction in the amplitude of intestinal motility as observed in the rabbit jejunum could be one of the mechanisms behind this effect. As both the alpha- and beta-specific antagonists reduced the inhibitory effects of extract on the amplitude of jejunal contractions, it may be assumed that the effect is at least in part mediated through adrenergic receptors in the gut. Plant may be further studied to confirm the anti-diarrhoeal effect using other models.
Warrier PK, Nambiar VPK and Ramankutty C (eds.) (1995) Indian Medicinal Plants, Orient Longman Ltd., Madras. Vol. 2, p 218
Department of Pharmacology and Toxicology, Zayed Complex for Herbal Research & Traditional Medicine, Post Box 29300, Ministry of Health, Abu Dhabi, U.A.E.
Fagonia indica DC. Pord. (Polygonaceae) is a shrub distributed throughout Arabia, growing in sandy deserts. The decoction of dried leaves or fresh juice of whole plant are used by the local healers as anti-inflammatory and in stomach ailments (Ghazanfar, 1994). The aim of the present investigation is to evaluate the possible anti-inflammatory activity of F. indica.
F. indica were collected from Sharjah in UAE, dried in shade, powdered and extracted with 10% ethanol. Two models were used for screening anti-inflammatory activity of the extract. Acute inflammation was induced in the hind paw of SD rats by injecting carrageenan into the subplantar tissue. The extract was administered orally at two dose levels (0.5 & 1.0 g/kg) in two different groups of animals, 30 min before carrageenan injection. The hind paw volume was measured before and after the carrageenan challenge plethysmometrically (Burch et. al, 1990) at 1 h, 2 h, 4 h, 6 h and 24 h. Sub-acute anti-inflammatory activity of the extract was assessed using cotton pellet granuloma method in rats (Pelzer et al., 1998) at the dose of 1.0 g/kg administered orally for 7 days.
Table 1 The anti-inflammatory effect of F. arabica extract on rat oedema induced by carrageenan
|
Group |
Initial paw volume (ml) |
Percentage of initial paw volume after carrageenan at: |
||||
|
1 h |
2 h |
4 h |
6 h |
24 h |
||
|
Control
Fagonia 500 mg/kg
Fagonia 1000 mg/kg
|
1.30±0.10
1.38±0.06
1.31±0.07 |
134.5±3.9
119.9±2.5*
14.4 ±2.1** |
183.4±6.7
152.4±5.1*
156.5±8.0* |
195.4±7.8
167.4±6.1*
177.1±4.0 |
186.4±7.3
169.0±3.1*
167.2±1.7* |
141.5±5.4
129.2±1.6
132.3±4.5 |
*P<0.05, **P<0.01 Vs Control group, Student’s t-test
Our results showed that the extract of F. indica at the doses 0.5g/kg and 1.0 g/kg, significantly reduced the increase in hind paw volume induced by carrageenan. The effect started 1 h after extract was given orally, and lasted for 6 h (Table 1). Subacute treatment in cotton pellet granuloma method at the dose of 1.0 g/kg, p.o., also showed significant anti-inflammatory activity in rats. The weight of cotton pellet in control group was 245.40 ± 17.80 mg, while it was reduced to 202.75 ± 9.65 mg in the treated group.
The results clearly demonstrate anti-inflammatory activity of 10% ethanolic extract of F. indica in the doses studied, supporting the traditional use of F. indica. Further studies are suggested to elucidate the exact mode of action and its therapeutic value in the treatment of inflammation.
Ghazanfar, S.A. (1994) Handbook of Arabian Medicinal Plants, CRC Press Inc.
Burch,R.M. & Christopher,D.(1990) Arch. Pharmacol. 342,189- 193 .
Pelzer, LE. et al. (1998) Farmaco. 53(6), 421-4.
Department of Pharmacology and Toxicology, Zayed Complex for Herbal Research &
Traditional Medicine, Post Box 29300, Ministry of Health, Abu Dhabi, U.A.E.
Cleome africana (Fam. Cleomaceae) is a native of Caribbean region mainly distributed in tropics and temperate zone (Migahid, 1978). It is considered to have a function of relieving inflammation and pain and has been used traditionally to treat rheumatism. This study was designed to evaluate the analgesic activity of the plant, through the writhing test in mice and the tail flick test in rats. The aqueous extract of the dried whole plant was prepared and used for the experiments.
The writhing test used normal albino mice of body weight 25-30 g (Emele, 1963). The animals were administered orally with the aqueous extract at 800 mg/kg, while the control ones were treated with distilled water. One hour later, 0.6% acetic acid was injected intraperitoneally at 10 ml/kg to induce writhing. The onset time of writhing was observed and the writhing number was counted in 25 min.
The tail flick test was performed using Tail Flick Unit-Ugo Basile. One rat was kept on the platform with its tail covering the small window of a beam of infrared light. When the rat felt pain, it flicked it’s tail and the counter of the instrument automatically stopped and gave the reaction time. Seven Dawley rats were administered orally with the extract of C. africana at 800 mg/kg, while the control ones were given distilled water. The tail flick was tested at 0 h, 1 h, 2 h, 4 h and 6 h after the administration.
In the writhing test, the mice in the control group and the extract treated group started to writhe (onset) at 178 ± 16 s and 527 ± 88 s separately, and the writhing number in 25 min was 47 ± 6 and 11.5 ± 4 separately. The result showed that water extract of C. africana significantly delayed the onset time of writhing (p=0.0037), as well as reduced the writhing number caused by acetic acid (p=0.0001). In the tail flick test, the reaction time was calculated as the percent of the initial value. In the control group, the reaction times, at 1 h, 2 h, 4h and 6h after the administration, were 96 ±15, 158± 23, 172±29 and 170 ±26 separately; and were 119 ± 13, 192 ±30, 260± 34 and 181± 25 in the extract treated group. This indicated that the water extract of C. africana might prolong the rats’ reaction times to pain, though the differences were not statistically significant.
The present study proves that the aqueous extract of C. africana has analgesic activity in animals and this supports the traditional medicinal value of this plant.
Migahid. A. M. (1978) Flora of Saudi Arabia, Riyadh University Publication, Riyadh, UA, p. 59
Emele J.F., et al. (1963) Fed Proc, 22:248
1Mahidol University, Department of Microbiology, Faculty of Pharmacy, Bangkok 10400, Thailand
2Mahidol University, Department of Pharmaceutical Botany, Faculty of Pharmacy, Bangkok 10400, Thailand
The use of stem bark, wood and fruit of Alangium salviifolium (L. f.) Wang. for treatment of symptoms and diseases was found describing in Thai traditional remedy. This plant, also known as Proo, Ploo and ma kue ka, is widely cultivated in many parts of Thailand. In screening for microbial inhibitory spectrum of some local plant, ground wood of A. salviifolium was macerated with ethanol and water. By evaporating ethanol and lyophilizing aqueous extract, 3.65 and 4.59% yields of ethanolic part and lyophilized powder were obtained, respectively. Inhibitory spectrum of extracts was tested against 85 isolates of different organisms, i.e. 36 of filamentous fungus (dermatophytes) and 18 of unicellular fungus (Candida albicans), 4 representative species of Gram positive, 6 Gram negative and 21 isolates of anaerobic bacteria (Propionibacterium spp.). Inhibitory properties were considered as zone diameters in agar disc diffusion test.
According to average inhibitory diameter, inhibitory effectiveness of ethanolic and aqueous extract against dermatophytes (27.39 and 32.34 mm) was not significantly different from each other and was statistically similar to ketoconazole’s (33.88 mm) (p > 0.01). On the contrary, both extracts exerted significantly different effect on Candida albicans (8.61 and 12.69 mm) and differed from ketoconazole’s (28.71mm) (p < 0.01).
Inhibitory zones from activities of ethanolic and lyophilized extract against Propionibacterium spp. were not significantly different (29.85 and 30.72 mm) either with the reference drug, ampicillin (36.5mm)(p > 0.01). While Gram positive and Gram negative bacteria expressed various susceptibilities to both extracts.
This study exhibited the promising tendency to access the active ingredient in response to microbial inhibitory effects of this plant. Therefore, this finding leads to a further contact dermatitis test in order to develop A. salviifolium (L. f.) extract for external herbal products.
Department of Pharmacology and Toxicology, Zayed Complex for Herbal Research and Traditional Medicine, P.O. Box 29300, Ministry of Health, Abu Dhabi, U.A.E.
A large number of plant drugs are used traditionally to treat various ailments in the Gulf region. In the present study, the plants used for the treatment of chronic diseases (Ghazanfar, 1994), where long term treatment is required, namely, Zizyphus spina-christi L. (Rhamnaceae), and Teucrium stocksianum Boiss. (Lamiaceae) were selected for safety evaluation. The safety evaluation of the these plants included the evaluation of acute, sub-acute and sub-chronic toxicity in albino mice. Tests for clastogenic activity in mice and teratogenicity in rats were also carried out. For acute study, plant extracts were administered p.o. at the dose of 400 and 800 mg/kg, once only and were compared with control. The mice were examined for the toxic signs and symptoms, with a battery of tests including locomotor activity, somatic responses, reflex action, sensitivity tests, tremors/convulsions, and were compared with controls. For sub-acute study (15 days) and sub-chronic study (90 days), the plant extracts were administered orally, at a daily dose of 400 mg/kg. During treatment, the animals were observed for toxic signs and symptoms and body weight changes were recorded. At the end of the treatment, the blood was withdrawn and the animals were sacrificed. The body organs including brain, kidney, liver, spleen, heart, lung, testes, ovary and uterus were examined and their individual weights were recorded. The blood was analysed for WBC, RBC, Hgb, PCV, MCV, MCHC and platelets and the serum was analysed for total protein, albumin, globulin, SGOT, SGPT, LDH, CPK, BUN, creatinine, iron, chloride, calcium, magnesium, sodium and potassium. The clastogenic activity was studied in mouse bone marrow cells to quantitate micronucleus formation. Plant extracts-treated groups at the doses 3.2 g/kg, 1.6 g/kg and 0.8 g/kg, (p.o.) were assessed for micronuclei, at 30 h post dose and compared with positive control and vehicle control. For teratogenicity studies plant extracts were administered by gavage from 6-15 days of gestation, at a daily dose of 800 mg/kg. The dams were sacrificed on day 21 of gestation and maternal and fetal parameters were evaluated.
Our results for acute study showed an increased rate of respiration and restricted movement, at both dose level tested with all the plant extracts studied. Sub-acute treatment for 90 days with the plant extracts produced no significant change in body weight. However, Teucrium groups gained weight in the first week of treatment only. In the Teucrium group, two animals showed swelling in the testes and penis on day 18 of treatment. No significant difference in the organ weights and haematological parameters studied were observed with any of the plant extracts tested. A significant increase in SGOT and decrease in CPK value were observed in the Teucrium group. The Zizyphus group on the other hand showed no significant changes in the biochemical parameters studied. The frequency of micronuclei did not increase and both the plants were found devoid of clastogenic activity. Teratogenicity data revealed no teratogenic or fetotoxic effects with any of the plants studied. Oral LD50 values were determined for Zizyphus spina–christi ( > 6400 mg/kg) and Teucrium stocksianum (>6400 mg/kg). Data from the present investigation provide some indications on the toxicity profile of the plants which may be helpful in assessing their suitability for clinical use.
Ghazanfar, D.A (l994) Handbook of Arabian Medicinal Plants, CRC Press.
1 Institute of Pharmaceutical Biology, University Freiburg, 79104 Freiburg, Germany
2 Escuela de Quimica and CIPRONA, Universidad de Costa Rica, San José, Costa Rica
3 Institute of Physical Chemistry, University Freiburg, 79104 Freiburg, Germany
*these both authors contributed equally to the work
Human neutrophil elastase (HNE) is a major granule proteinase of human neutrophils. Its primary role appears to be in the intracellular degradation of foreign proteins during phagocytosis by neutrophils. A second role is likely to be in aiding the movement of these cells through connective tissue matrices while it is heading towards a target. During these processes, HNE is released and controlled by endogenous proteinase inhibitors. However, intense neutrophil infiltration results in an imbalance between the amount of HNE and endogenous inhibitors. Accumulating HNE can then cause abnormal degradation of healthy tissue resulting in the development of diseases such as pulmonary emphysema, rheumatoid arthritis or cystic fibrosis (1, 2). Therefore, natural or synthetic inhibitors of HNE could be of considerable interest in the treatment of these inflammatory diseases.
Recently, we have demonstrated that the caffeate, ferulate and coumarate of borneol, but not the free acids inhibited HNE in an in vitro assay. Bornyl caffeate was the most active compound with an IC50 value of 1.6 mmol/ml (3). Using the X-ray structure of HNE (4) and the program FlexX (5) we here present docking experiments by which the different activities of the bornyl derivatives as well as the free acids may be explained.
These calculations reveal that the presence of the two o-dihydroxy groups in bornyl caffeate are especially favorable for an interaction within the active site of elastase. They enable a simultaneous anchoring of this inhibitor via hydrogen bonds in the oxyanion hole and at the sidechains of His57 and Ser195 which form the catalytic triad together with Asp102. In case of bornyl coumarate and ferulate no placements of the inhibitor within the binding site were found. Similar results were obtained for the corresponding free acid compounds.
1. Bernstein, P.R., Edwards, P.D. and Williams, J.C., (1994) Progress in Medicinal Chemistry. 31, 59-120
2. Döring, G., (1994) Am. J. Respir. Crit. Care Med. 150, S114-S117
3. Melzig, M.F., Löser, B., Lobitz, G.O., Tamayo-Castillo, G., Merfort, I. (1999) Pharmazie. 54, 9
4. Navia, M.A., McKeever, B.M., Springer, J.P., Lin, T., Williams, H.R., Fluder, E.M., Dorn, S.C.P. and Hoogsteen, K., (1989) Proc. Natl. Acad. Sci. USA. 86, 7-11
5. Rarey, M., Kramer, B., Lengauer, T., Klebe, G. (1996) J. Mol. Biol.. 261, 470-489
School of Pharmacy, Tokyo University of Pharmacy and Life Science, 192-0392 Tokyo, Japan
Effects of some plant-derived essential oils and psychotropic drugs on plasma ACTH level of menopausal model rats under restriction stress were studied. Ovariectomized female rats were used as an experimental menopausal model. Restriction stress experiments were carried out as described by Yamada et a1.1.We found that inhaling of chamomile oil (Anthemis nobilis) or lemon oil vapour decreased the increase of plasma ACTH level in ovariectomized rats induced by restriction stress. The plasma ACTH level was not influenced by chlorpromazine, haloperidol and anti-depressants. On the other hand, the increase was inhibited by administration of diazepam. The plasma ACTH level decreased further when diazepam or bromazepam was administered along with inhalation of chamomile oil or lemon oil vapour. Flumazenile (an anti-benzodiazepine) blocked the decrease in plasma ACTH level induced by inhaled chamomile oil or lemon oil vapour. These results suggest that essential oils may have a strengthening effct on GABA nergic activity in central neurons2.
Table: Effect of Inhalation of Essential Oil Vapour and Diazepam on Restriction Stress-induced Plasma ACTH Level in Menopausal Model Rats
OVX
:Ovariectomy, OVX-rat used as menopausal model. Data are means ±
S. E. (n=5)
a) p<0.01 vs OVX, b) p<0.01 vs Stress, c) p<0.01 vs Diazepam and Chamomile Oil
1) K.Yamada, T. Miura, Y.Mimaki and Y. Sashida, Biol. Pharm. Bull. 19, 1244 (1996)
2) K.Yamada, T. Miura, Y.Mimaki and Y.Sashida, Aroma Research 1, 24 (2000)
SESQUITERPENE LACTONES AND HUMAN NEUTROPHIL ELASTASE
1 Institute of Pharmacy, Humboldt-University Berlin, Germany
2 Institute of Pharmaceutical Biology, University Freiburg, 79104 Freiburg, Germany
3 Escuela de Quimica and CIPRONA, Universidad de Costa Rica, San José, Costa Rica
4 Institute of Pharmaceutical Biology, University Saarbrücken, Germany
5 Institute of Physical Chemistry, University Freiburg, 79104 Freiburg, Germany
Human neutrophil elastase (HNE) is a major granule serine proteinase of neutrophils. It is directed to the degradation of type IV procollagen thus contributing to the destruction of basement membranes during inflammation (1). It was shown that this enzyme is involved in diseases such as pulmonary emphysema, rheumatoid arthritis or cystic fibrosis (1). Looking for natural compounds which inhibit this proteinase 15 structurally different sesquiterpene lactones (SLs) of the germacranolide, eudesmanolide, guaianolide and pseudoguainolide type were investigated in an in vitro assay using isolated human neutrophil elastase.
Whereas four SLs exhibited no effect up to a concentration of 200 mM, seven showed a moderate inhibitory activity with IC50 values between 69 and 102 mM. The remaining three were more active. Podachaenin, isolated from Podachaenium eminens (2), induced a 50 % inhibition (IC50) of elastase at a 4.7 mM, whereas the other two germacranolides possessed IC50 values of 15 and 28.6 mM. Further studies, such as cysteine reactivation assay and inhibitor dialysis experiments were undertaken to find out whether inhibition of elastase was due to a covalent binding within the active site. In contrast to 2-methyl-4H-3,1-benzoxazin-4-on for which acylation within the active site can be assumed (3) and which was used as a positive control, SLs do not react in a similar way. The inhibitory activity of all tested SLs was abolished by subsequent addition of cysteine to the mixture of HNE and SLs.
Using the X-ray structure of HNE (4) and the program FlexX (5) we undertook docking experiments to explain the different activities of the SLs. From these studies it can also be deduced that SLs may not inhibit elastase by acylating an amino acid of the catalytic triad. However, it is not possible to explain the activity of the SLs with a uniform molecular inhibition mechanism. Additionally, it should be taken into account, that Sls could also indirectly influence elastase by inhibition of the cytokine IL-8 (6).
Bernstein, P.R., Edwards, P.D. and Williams, J.C., (1994) Progress in Medicinal Chemistry. 31, 59-120
Castro, V., Murillo, R., Klaas, C.A., Meunier, C., Mora, G., Pahl, H.L., Merfort, I.(2000) Planta Med. 66, 591 – 595
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1Department of Toxicology
2Department of Pharmacognosy K. Marcinkowski University of Medical Sciences, 10 Sieroca, 61-771 Poznan, Poland
3Faculty of Chemistry, A. Mickiewicz University, 6 Grunwaldzka, 60-780 Poznan, Poland
The leaves and stems of A. vulgaris contain flavonoid C- and O-glycosides, of which apigenin 4'-methyl ether 6-C-glucoside (isocytisoside) is a predominant compound (1). The studies described here concern flavonoids present in the Aquilegia exudate. The fresh leaves of A. vulgaris were extracted by shaking with AC2O at room temp. for 1 min. TLC of the extract showed the presence of compounds I and II (15% HOAc: Rf = 0.53 and 0.58; H2O: Rf = 0.20 and 0.85, respectively). Compound II was identified as isocytisoside. In addition to the isocytisoside signals the 1H NMR spectrum of I showed a signal at 3.15 ppm, corresponding to a malonyl CH2. Moreover, non-equivalence of protons at C-6" (H-6" A at 4.50 and H-6" B at 3.95 ppm) indicated that the malonyl residue was connected to this position. Comparison of 13C NMR spectra of I with isocytisoside showed three additional carbon resonances at d 173.0; 42.0 and 171.6 ppm as expected for the methylene and carbonyl moieties. The site of malonylation is defined as the C-6 of glucose by the 3.0 ppm downfield shift of C-6 glucose relative to isocytisoside. I and II were also investigated by liquid secondary ion mass spectrometry (LSIMS) both in positive and negative mode. Standard LSI mass spectra allowed to determine their molecular weights. High-resolution data confirmed their molecular formulae. On the basis of WE linked scan mass spectra the fragmentation pathways of the studied compounds was deduced. Thus, I was characterized as 4'-methoxyapigenin 6-C-(6"-O-malonyl) glucopyranoside. The above-mentioned flavonoid C-glycoside malonate I has been isolated for the first time from a plant source.
Antioxidant properties of an ethanolic extract of the leaves of A. vulgaris and isocytisoside were evaluated. Free radical scavenging ability was tested with the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assay. Effect of preparations tested on microsomal lipid peroxidation was assayed in three systems: stimulated by Fe3+ NADPH/ADP (enzymatic), stimulated by Fe2+/ascorbate (nonenzymatic) and non-stimulated (endogenous). Additionally the ability of chelating ferrousion was examined. a-Tocopherol was used as a positive control. The results indicate that the compounds tested are potential antioxidants. However it can be suggested that they work rather like iron chelators not chain breaking antioxidants.
The effect of an ethanolic extract of the leaves of A. vulgaris and isocytisoside on the activities of SDH, AspAT, ALAT, GGTP and cholesterol concentration were examined in blood plasma of Wistar rats intoxicated with CCl4. As a result, significant decrease of enzyme activities, previously elevated by CCl4 was observed after administration of an ethanolic extract of A. vulgaris. The effect was similar to that caused by acetylcysteine. Isocytisoside decreased enzymes activity to a much less extent. The substances tested did not change the level of cholesterol significantly.
Pathomorphological changes in the liver of rats treated with CCl4 were reduced both by ethanolic extract of the leaves of A. vulgaris and isocytisoside.
Acknowledgement: the work was supported by a grant (KBN Nr 6 P05F 001 20)
I.Bylka W., I. Matlawska (1997) Acta Polon.Pharm. 54,331.
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