Comparison of hormonal activity (estrogen, androgen and progestin) of standardized plant extracts for large scale use in hormone replacement therapy.

Extracts from red clover (Trifolium pratense), soybean (Glycine max.) and black cohosh (Cimicifuga racemosa) are frequently used as alternative compounds for hormone replacement therapy (HRT) to treat menopausal disorders. Fifteen commercially available products made either from red clover, soybean or black cohosh were tested in in vitro assays in this study. The main polycyclic phenolic compounds of soy and red clover products were biochanin A, genistein, daidzein, formononetin, and glycitein. In red clover products glycitein was not abundant. All the compounds showed clear estrogenic activity through estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) and affinity to progesterone receptor (PR) and androgen receptor (AR), whereas the compounds from black cohosh did not. This was corroborated by synthetic isoflavones such as biochanin A, daidzein, genistein and formononetin. They exerted affinity to PR and AR in the range of 0.39-110 mM. Statistical analysis applying principal component analysis (PCA) revealed that all red clover and soy products are grouped in different clusters. Red clover products showed a higher affinity to AR and PR than soy products, which is explained by the higher amount of isoflavones present. In vitro assays and chemical analysis showed that theoretical estrogenic activity expressed as equivalent E2 concentration is in the same range as recommended for synthetic estrogens. Broader spectrum of action and hypothesized lower side effects by action through ERbeta make them suitable for alternative hormone replacement therapy.

Highly hydroxylated guaianolides of Achillea asiatica and Middle European Achillea species.

From flower heads of Achillea asiatica (L.) Serg., three new guaianolides were isolated by repeated column chromatography and HPLC. The constitution and the stereochemistry of these new, labile compounds were determined by MS, one ((1)H, (13)C, selective (1)H-TOCSY and (1)H-NOESY) and two-dimensional NMR experiments ((1)H, (1)H-COSY, (1)H, (13)C-HSQC, (1)H, (13)C-HMBC). The substances were identified as 8 alpha-angeloxy-2 alpha, 4 alpha,10 beta-trihydroxy-6 beta H,7 alpha H, 11 beta H-1(5)-guaien-12,6 alpha-olide (1), 8 alpha-angeloxy-1 beta,2 beta:4 beta,5 beta-diepoxy-10 beta-hydroxy-6 beta H, 7 alpha H, 11 beta H-12,6 alpha-guaianolide (2) and 8 alpha-angeloxy-4 alpha,10 beta-dihydroxy-2-oxo-6 beta H,7 alpha H, 11 beta H-1(5)-guaien-12,6 alpha-olide (3). They were also detected in Middle European species (Achillea collina, Achillea ceretanica (2x and 4x), Achillea roseoalba, Achillea asplenifolia) by HPLC, TLC and off line MS and have not been described before. The possibility that these compounds might be products of an oxidation process is discussed.

Estrogenic activity of two standardized red clover extracts (Menoflavon) intended for large scale use in hormone replacement therapy.

Isoflavones are the most potent estrogenic compounds in red clover extracts. Standardized extracts have been discussed as an alternative for hormone replacement therapy. Variation due to extraction procedure and natural seasonal variation and variations originating from agricultural conditions have prevented the large scale use of such phytochemicals. An improved extraction procedure and careful analysis of the raw material yielded in a highly standardized preparation (Menoflavon) with an average isoflavone content of approximately 9% (dry weight) determined by HPLC. The estrogenic activity has been further evaluated by a yeast two plasmid system using estrogen receptor alpha (ER alpha) and estrogen receptor beta (ER beta). An estrogenic activity corresponding to a transactivational capacity of ca. 18 microg 17 beta-estradiol per g red clover extract for ER alpha and ca. 78 microg 17 beta-estradiol per g red clover for ER beta was obtained. The difference is explained by the higher affinity of ER beta to isoflavones than that observed for ER alpha. Calculation of potency from isoflavone content measured by HPLC yielded a comparable potency to that experimentally determined by the bioassay. The high content of isoflavones as well as the higher transactivational potency for ER beta than ER alpha make these extracts interesting candidates for HRT.

Antiprotozoal activity of Neurolaena lobata.

Extracts, fractions and sesquiterpene lactones from Neurolaena lobata (L.) R. Br. (Asteraceae), a traditional medicinal plant from Guatemala, were tested in vitro against Leishmania spp. promastigotes, Trypanosoma cruzi trypomastigotes and epimastigotes and Trichomonas vaginalis trophozoites. The ethanol extract inhibited the parasite growth of L. mexicana, T. cruzi and T. vaginalis significantly. The pure germacranolides 1 and a mixture of 2 and 3, isolated from the ethonal extract, were highly active against L. mexicana and T. cruzi.

Qualitative and quantitative determination of sesquiterpenoids in Achillea species by reversed-phase high-performance liquid chromatography, mass-spectrometry and thin-layer chromatography.

A reversed-phase high-performance liquid chromatographic method was developed as a universal analysis system in order to determine and quantify antiphlogistic sesquiterpenoids in different Achillea species. Identification was performed by HPLC and diode array detection as well as by monitoring the HPLC fractions by TLC and MS. Using santonin as internal standard, HPLC separations were achieved with a methanol-water gradient system using RP 8 LiChrospher 100 (5 microm) as stationary phase. For validation, sample analyses were performed, using the two tetraploid species A. collina and A. pratensis. The method allows the identification and quantification of the main compounds achillicin, 8alpha-tigloxy-artabsin, 8alpha-angeloxy-artabsin, arglanin and santamarin with variation coefficients between 3.4 and 4.7% (total content) using santonin as internal standard. For the different compounds recovery was found between 81 and 107% performing multiple analyses of A. collina and A. pratensis.

New 12a-hydroxyrotenoids from gliricidia sepium bark

The investigation of a methanolic extract of Gliricidia sepium bark afforded, in addition to vestitol and 2'-O-methylvestitol, three new 12a-hydroxyrotenoids, gliricidol (1), 2-methoxygliricidol (2), and gliricidin (3). The structures of 1-3 were elucidated by analysis of their spectroscopic data. Compounds 1-3 exhibited activity against Artemia salina larvae.

Plants used in Guatemala for the treatment of protozoal infections: II. Activity of extracts and fractions of five Guatemalan plants against Trypanosoma cruzi.

The activities of crude plant extracts of five plants popularly used in Guatemala against bacterial and protozoal infections and some of their fractions have been evaluated against the trypomastigote and epimastigote forms of Trypanosoma cruzi in vitro. The most active fraction of Neurolaena lobata has also been screened in vivo. Main in vitro activities against trypomastigotes have been observed for the hexane and ethanol extracts of N. lobata (Asteraceae). Both extracts were also active against epimastigotes, whereas all other extracts tested had no effect on epimastigotes. For the hexane extracts of Petiveria alliacea (Phytolaccaceae) and Tridax procumbens (Asteraceae) a marked inhibition of trypomastigotes has been found. Also the ethanol extracts of Byrsonima crassifolia (Malpighiaceae) leafs and Gliricidia sepium (Papilionaceae) bark showed some trypanocidal activity. Fraction 2 of the ethanol extract of N. lobata was highly active against T. cruzi as well in vitro as in vivo. The chloroforme fraction of P. alliacea showed a high inhibition of trypomastigotes in vitro. Also three fractions of the active extract of B. crassifolia inhibited T. cruzi trypomastigotes. No fraction of G. sepium bark extract showed a marked trypanocidal activity.

Guaianolide-endoperoxide and monoterpene-hydroperoxides from Achillea nobilis.

From flower heads of Achillea nobilis two chrysanthemol derivatives (1, 2), tanaparthin-beta-peroxide (3), and 5-hydroxy-3,6,7,4'-tetramethoxyflavone (4) were isolated by repeated column chromatography and HPLC. Structure elucidation has been performed by means of MS, 1H-NMR, 13C-NMR, and 2D-NMR experiments.

About the bufadienolide complex of "red" squill.

Red coloured bulbs of plants of the URGINEA MARITIMA aggregate from the northern and southern Mediterranean area were shown to exhibit different bufadienolide compositions. Therefore, the bufadienolide complex of "red squill" samples from Tunesia and from Sardegna was investigated. Twelve bufadienolides were isolated from bulbs of tetraploid squill from Tunesia, sixteen from tetraploid samples from Sardegna. The Compounds were identified by means of FAB-MS, (1)H-NMR, and (13)C-NMR studies or comparison with authentic substances. Both of the samples contained scilliroside ( 14), scillarenin 3- O-beta- D-glucoside ( 2), proscillaridin A (1), scilliphaeosidin 3- O-beta- D-glucoside ( 5), scilliglaucoside ( 17), scilliphaeoside ( 4), and 12- EPI-scilliphaeoside ( 7); the bulbs from Tunesia contained additionally glucoscilliphaeoside ( 6) and the four new bufadienolides 12- EPI-glucoscilliphaeoside ( 10), 12beta-hydroxyscilliglaucosidin 3- O-beta- D-glucoside ( 21), 12- EPI-scilliphaeosidin 3- O-beta- D-glucoside ( 8), and 12- EPI-scilliphaeosidin 3- O-alpha- L-rhamnosido-alpha- L-rhamnoside ( 9). In the bulbs from Sardegna scülaren A ( 3), gamabufotalin 3- O-alpha- L-rhamnoside ( 20), scilliglaucoside ( 17), scillirubrosidin 3- O-alpha- L-rhamnoside ( 11), scillirubroside ( 12), 12beta-hydroxyscilliroside ( 15), 5alpha-4,5-dihydroscillirosidin 3- O-alpha- L-thevetosido-beta- D-glucoside ( 19), and deacetyl-scilliroside ( 8) besides the new Compounds 10-carb-oxy-5beta,14beta-dihydroxybufa-3,20,22-trienolide 5- O-beta- D-glucoside ( 18) and scilliglaucogenin ( 16) were found in addition. Not only quantitative but even qualitative differences in the bufadienolide pattern between the samples of the northern and the southern Mediterranean area were proved. By now it is not clear whether the investigated samples belong to different chemodemes of URGINEA NUMIDICA or are two different species of the URGINEA MARITIMA aggregate.

In vitro propagation of Achillea asplenifolia VENT. through multiple shoot regeneration.

A method for the micropropagation of Achillea asplenifolia VENT. (Asteraceae) is described. Axillary shoots regenerated from nodal explants of adult plants could be stimulated to multiple shoot formation on the basal medium of Murashige and Skoog (1962), supplemented with different cytokinins. The best proliferation (5.33 shoots per culture), together with a healthy appearance of the cultures, was achieved with N-benzyl-9-(2-tetrahydropyranyl)adenine at a concentration of 1 mg/l. Shoots could be easily rooted on hormone-free MS basal medium and subsequently acclimatized to greenhouse and field conditions with 100% survival rate. DNA-fingerprinting, chromosome counts, the proazulene pattern as well as the essential oil analysis proved the true to type character of the micropropagated plants.

Cardenolides from Adonis aestivalis.

Four cardenolides were isolated for the first time from the aerial parts of Adonis aestivalis. The compounds were identified by spectrometry and for 3-epi-periplogenin, helveticoside also by comparison with authentic substances. Two new cardenolides were structurally elucidated: strophanthidin-3-O-beta-D-digitoxosido-alpha-L-cymarosido-be ta-D-glucoside and strophanthidin-3-O-beta-D-digitoxosido-beta-D-digoxoside-bet a-D-diginosido-beta-D-glucoside.

[Isolation and structure elucidation of further new saponins from Solidago canadensis]. / Isolierung und Struktur weiterer neuer Saponine aus Solidago canadensis.

Four new main saponins (canadensis-saponins 5-8) (compounds 5-8) were isolated from Solidago canadensis L. (Asteraceae). Using GC/MS, FAB-MS, and mainly 2D-NMR techniques their structures were identified as 3-O-[beta-D-glucopyranosyl(1----3)-beta-D- glucopyranosyl]-28-O-[beta-D-galactopyranosyl(1----2)-alpha-L- rhamnopyranosyl-(1----3)-beta-D-xylopyranosyl-(1----4)-[beta-D- xylopyranosyl-(1----3)]-alpha-L-rhamnopyranosyl-(1----2)-[beta-D-apio -D- furanosyl-(1----3)]-beta-D-6-deoxyglucopyranosyl-(1----)]-bayog enin(5),3-O- [beta-D-glucopyranosyl-(1----3)-beta-D-glucopyranosyl]-28-O-[beta-D- galactopyranosyl-(1----2)-alpha-L-rhamnopyranosyl-(1----3)-beta-D- xylopyranosyl-(1----4)-[beta-D-xylopyranosyl-(1----3)]-alpha-L- rhamnopyranosyl-(1----2)-[beta-D-apio-D-furanosyl-(1----3)]- arabinopyranosyl-(1----)]bayogenin(6),3-O-[beta-D-glucopy ran osyl-(1----3)- beta-D-glucopyranosyl]-28-O-[beta-D-galactopyranosyl-(1----2)- alpha-L-rhamnopyranosyl-(1----3)-beta-D-xylopyranosyl-(1----4)-[beta-D- xylopyranosyl-(1----3)]-alpha-L-rhamnopyranosyl-(1----2)-[alpha-L- rhamnopyranosyl-(1----3)]-beta-D-6-deoxyglucopyranosyl-(1----)]-++ +bayogenin (7), and 3-O-[beta-D-glucopyranosyl-(1----3)-beta-D-glucopyranosyl]-28-[O- beta-D-galactopyranosyl-(1----2)-alpha-L-rhamnopyranosyl-(1----3)-beta-D - xylopyranosyl-(1----4)-[beta-D-xylopyranosyl-(1----3)]-alpha-L- rhamnopyranosyl-(1----2)-[alpha-L-rhamnopyranosyl-(1----3)]arabinopyr anosyl - (1----)[-bayogenin (8).

[Sesquiterpenelactones of Achillea setacea with antiphlogistic activity]. / Entzündungshemmende Sesquiterpenlactone von Achillea setacea. Sesquiterpenelactones of Achillea setacea with Antiphlogistic activity.

From the aerial parts of Achillea setacea W. & K. (Asteraceae) the main sesquiterpenes were isolated. Their structures were determined by means of 2D-NMR and MS as 11,13-dehydrodeacetylmatricarin (1) (= 14-deoxylactucin), rupicolin A (2), and rupicolin B (3). These are the first compounds with an alpha-methylene-gamma-lactone structure isolated from a species belonging to the Achillea millefolium aggregate. Achillicin, achillin, 8-hydroxyachillin, 8-acetoxyachillin, and matricin could not be detected in A. setacea. For both rupicolin B and 11,13-dehydrodeacetylmatricarin an anti-inflammatory activity was found in the croton oil ear test.

Four major saponins from Solidago canadensis.

Four new bisdesmosidic saponins each containing eight carbohydrate units were isolated from Solidago canadensis. GC, GC-MS, FABMS analysis and mainly the use of 2D NMR techniques allowed their identification as bayogeninglycosides (canadensissaponins 1-4) 3-O- [beta-D-glucopyranosyl-(1----3)-beta-D-glucopyranosyl]-28-O-[alpha-L- rhamnopyranosyl-(1----3)-beta-D-xylopyranosyl-(1----4)-[beta-D- xylopyranosyl-(1----3)]-alpha-L-rhamnopyranosyl-(1----2)-[beta-D- apio-D-furanosyl-(1----3)]-beta-D-6-deoxyglucopyranosyl- (1----]-bayogenin; -(1----2)-[beta-D-apio-D-furanosyl-(1----3)]-ara- binopyranosyl-(1----]-bayogenin; -[alpha-L-rhamnopyranosyl-(1----3)]-beta- D-6-deoxyglucopyranosyl-(1----]-bayogenin and - [alpha-L-rhamnopyranosyl- (1----3)]-arabinopyranosyl-(1----]-bayogenin.

Bufadienolides from Urginea pancration.

From bulbs of URGINEA PANCRATION (Steinh.) G. de Philippe (Hyacinthaceae) 5 bufadienolides were isolated by a combination of column and droplet counter-current chromatography; their structures were elucidated mainly by FAB-MS and (1)H- and (13)C-NMR spectroscopy. Besides the already known scilliglaucoside ( 1), scillirubroside ( 2), and scilliroside ( 3), two new bufadienolides were identified: 5alpha-4,5-dihydroscillirosidin- 3beta- O-alpha- L-thevetosido-4'-beta- D-glucopyranoside and arenobufagin-3beta- O- L-rhamnopyranosido-4'-beta- D-glucopyranoside.

Cardenolides from Ornithogalum boucheanum.

From leaves and bulbs of ORNITHOGALUM BOUCHEANUM (Kunth) Aschers. et Graebn. (Liliaceae), cardenolides were isolated by a combination of column and droplet counter-current chromatography. Their structure elucidation was performed mainly by means of (1)H-NMR, (13)C-NMR, EI-MS, and LD-MS studies as well as by acid and/or enzymatic hydrolysis of the glycosides followed by identification of genins and sugar moieties (TLC, GC). Eight cardenolides were identified as sarmentogenin-3- O-6'-deoxy-beta- D-allosido-beta- D-xylosido-alpha-L-rhamnoside, sarmentogenin-3- O-6'-deoxy-beta- D-allosido-beta- D-xylosido-beta- D-apioside, 15beta, 16alpha-dihydroxyuzarigenin-3- O-beta- D-digitoxosido-beta- D-xylosido-alpha- L-rhamnoside, sarmentogenin-3- O-beta- D-digitoxosido-beta- D-xylosido-beta- D-apioside, syriogenin-3- O-beta- D-digitoxosido-beta- D-xylosido-alpha- L-rhamnoside, sarmentogenin-3- O-alpha- L-rhamnosido-beta- D-apioside, uzarigenin-3- O-beta- D-digitoxosido-beta- D-xylosido-alpha- L-rhamnoside, and digitoxigenin-3- O-6'-deoxy-beta- D-allosido-beta- D-xyloside. This is the first report of the occurrence of 15beta, 16alpha-dihydroxyuzarigenin. The presence of three genins - syriogenin, uzarigenin, and digitoxigenin - in the genus ORNITHOGALUM is described for the first time. For cardenolides, not only the combination of three different monosaccharides in one glycoside is unusual, but, especially, the occurrence of apiose, which was not known for cardiac glycosides until now.

Structure of Triterpene-Sapogenins of Aesculus glabra1.

After extraction of coarsely ground seeds of AESCULUS GLABRA Willd. with 80% methanol the saponin fraction was submitted to enzymatic and acid hydrolysis, giving qualitatively identical TLC-spectra of the sapogenins. For separation of the obtained sapogenins silica gel-columns with chloroform-methanol-water mixtures as mobile phases were employed. Five main components (genins G-A to G-E), identified as 21,22-diangeloyl-barringtogenol C, 21,22-diangeloyl-R (1)-barrigenol, 21-angeloyl-barringtogenol C, 22-angeloyl-R (1)-barrigenol and 21-angeloyl-R (1) barrigenol have been isolated. Structure elucidation was accomplished by (1)H-NMR and mass spectrometry of the genins, their acetates and several isopropyliden derivatives. The acid free sapogenins barringtogenol C and R (1) barrigenol (G-F and G-G) were found and identified by thin layer chromatography.

[Physcion-8-O-beta-D-gentiobioside, a new anthraquinone glycoside from rhubarb roots]. / Physcion-8-O-ß-D-gentiobiosid, ein neues Hauptglykosid aus Radix Rhei1.

A new anthraquinone glycoside - physcion-8-O-beta-D-gentiobioside - has been isolated from rhubarb roots in high yield. The structure was confirmed by H-NMR and MS-data as well as by direct comparison with the synthesized compound. The new substance was the main anthraquinone glycoside in some of the investigated samples of rhubarb roots and extracts.

[New cardenolides from Convallaria majalis]. / Neue Cardenolide aus Convallaria majalis.

From the leaves of Convallaria majalis L. eight glycosides of strophanthidin, cannogenol, sarmentogenin and 19-hydroxysarmentogenin, resp., were isolated and structurally elucidated as strophanthidin-3-O-6'-deoxy-beta-D-allosido-alpha-L-rhamnoside, strophanthidin-3-O-6'-deoxy-beta-D-allosido-alpha-L-arabinoside, strophanthidin-3-O-alpha-L-rhamnosido-2'-beta-D-glucoside, cannogenol-3-O-6'-deoxy-beta-D-allosido-beta-D-glucoside, cannogenol-3-O-6'-deoxy-beta-D-allosido-alpha-L-rhamnoside, 19-hydroxy-sarmentogenin-3-O-alpha-L-rhamnoside, sarmentogenin-3-O-6'-deoxy-beta-D-allosido-alpha-L-rhamnoside and sarmentogenin-3-O-6'-deoxy-beta-D-guloside, mainly by means of their H-NMR-spectra, by EI-MS and LD-MS. This is the first report of the occurrence of 19-hydroxy-sarmentogenin in nature. The presence of arabinosido-6-deoxyallose within the sugar chain of a cardiac glycoside was detected for the first time, as well. Including the new glycosides, a total of 38 cardenolides, derived from nine aglycones, have now been found in Convallaria majalis.