The effect of pH and ATP on the transport of oleanolic acid monoglycosides into isolated vacuoles of Calendula officialis leaves.

The transport of oleanolic acid and its glycosides (3-O-monoglucuronide F and 3-O-monoglucoside I) into the vacuoles of C. officinalis leaves was studied. It was found that these monoglycosides are transported into the vacuolar space whereas free oleanolic acid only binds with the tonoplast. The transport of monoglycosides depends on pH of the medium, the optimum pH for monoglucoside I and monoglucuronide F being 6.0 and 7.0, respectively. Moreover, it was demonstrated that the transport of monoglucoside I, in contrast to that of monoglucuronide F, depends on ATP at 0.5-2.0 mM concentrations. The presented results indicate that different mechanisms underlie the transport of these two compounds into vacuoles.     

Chemical structure and antifungal activity of a number of triterpenoids

Antifungal activity was tested in 49 pentacyclic triterpenoids and their glycosides, of plant and semisynthetic origin. Several of these compounds inhibited the multiplication of the yeast Saccharomyces carlsbergensis. The highest antifungal activity was found in the triterpene glycosides oleanolic acid and hederagenin, which have a free carboxyl group at C 28(27). Triterpenes of the meristotropic acid, macedonic acid, and lupan types had no fungistatic activity at concentrations up to 100 microgram/ml.     

Zellsaftgewinnung,AFS (apparent free space) und Vakuolenkonzentration der osmotisch wichtigsten mineralischen Bestandteile einiger Helgoländer Meeresalgen

Samples of cell sap in amounts suitable for chemical analysis of mineral compounds were obtained by high speed centrifugation of several frost-killed (-20° C) Helgoland algae under paraffin oil. In order to calculate the vacuole concentration of the mineral salts determined in these liquids, the extravacuolar solving space of the algae was measured as Lithium apparent free space (Li-AFS) after rinsing the algae for several minutes in balanced LiCl solutions. The Li-AFS was calculated from the results gained by flamephotometric determination of the Li-concentrations in the sap and in the bathing fluid. To avoid errors caused by interaction of Donnan-effects, the Li-AFS ofChaetomorpha linum kept in diluted sea water of different concentrations was measured. By plotting the results against the medium concentration, evidence was obtained to show that in 100 % Li-solution (corresponding osmotically to natural sea water of about 30 ‰), the Donnan effect was negligible. In the more diluted solutions, however, higher Li-AFS values were obtained. This would indicate that the differences in the distribution of Li-cations between medium and AFS, which are effected by the negative charges of indiffusible anions in the cytoplasm and of fixed acidic groups (e. g. R-COO^?) of phycocolloids in the wall material, will cause considerable errors if Li-concentrations are too low. Taking these experiences into account, AFS-values of several algae from Helgoland were measured by the same technique and the vacuole concentrations of the analysed mineral compounds calculated.     

Isolation of Vacuoles from Immature Apple Fruit Flesh and Compartmentation of Sugars, Organic Acids, Phenolic Compounds and Amino Acids

Vacuoles of immature apple fruit (Malus pumila Mill. var. domesticaSchneid.) were obtained by purification using Ficoll densitygradient centrifugation after lysis of the protoplasts by bothmild osmotic shock and the addition of EDTA and BSA. The recoverywas about 35% of the protoplasts. The isolated vacuoles hada mean diameter of about 100 µm. The distribution of sugars, organic acids, phenolic compoundsand amino acids in the vacuole, the cytoplasm and the free spacewas determined. Almost all of the fructose and glucose, themajor sugars of the tissue, were found in the vacuole. Sorbitolwas mainly located in the free space and the vacuole, and sucrosein the free space and the cytoplasm. More than 90% of the malicacid, the main organic acid, was located in the vacuole. Almostall of the phenolic compounds were also deposited in the vacuole. The volumes of the vacuole, the cytoplasm and the free spacein the whole tissue were calculated from the cell numbers ofthe whole tissue, the volume of the isolated protoplasts, andthe volume of the vacuoles present in the protoplast. The soluteconcentration in each compartment was estimated: vacuoles, 888mM; cytoplasm, 37 mM; free space, 57 mM. How these compartmentationsof solutes affected the translocation of sugars into the fruitand the cell expansion is discussed. ¹This paper is contribution A-159 of the Fruit Tree ResearchStation. (Received July 7, 1983; Accepted November 14, 1983)     

An acetophenone and three naphthalides from turkish Rhamnus libanoticus

A new acetophenone glycoside and two new naphthalide glycosides have been isolated from the bark of Turkish Rhamnus libanoticus together with 7-hydroxy-5-methoxyphthalide 7-O-β-D-glucoside. The structures of the new compounds were elucidated by spectroscopic methods as 2,6-dihydroxy-4-methoxyacetophenone 2-O-β- rutinoside, 8,9-dihydroxy-6-methoxynaphthalide 8-O-β-rutinoside, 8,9-dihydroxy-6-methoxynaphthalide 8-O-/3b-D glucoside, respectively.     

Flavonoids of the Muhlenbergia montana complex

Specimens from natural populations of Muhlenbergia montana (Nutt.) Hitch. and related species were analysed for their flavonoid content. Twenty-four flavonoids from 14 species were separated and 22 of the compounds identified. Most were glycosylated derivatives of luteolin, apigenin and tricin. Two flavonols, quercetin 3-O-rutinoside and quercetin 3-O-glycoside, and two flavanones were also identified. Flavonoid patterns were distinct for all perennial species and identical for the two annual species examined. Phenetic analysis of the flavonoid characters does not support the inclusion of the annual species M. crispiseta and M. peruviana as part of the M. montana complex.     

Dynamics of Nitrogenous Assimilate Partitioning between Cytoplasmic and Vacuolar Fractions in Carrot Cell Suspension Cultures

Bulk vacuole isolation, gas chromatography-mass spectrometry,, and high-performance liquid chromatography have been used to investigate the accumulation and partitioning of assimilated nitrogen supplied as ¹⁵NH₄Cl between vacuolar and extravacuolar (cytoplasmic) fractions of protoplasts from suspension cultures of carrot (Daucus carota L. cv Chantenay). Glutamine was the most abundant amino acid in the vacuole of protoplasts from late-exponential phase cells, whereas alanine, glutamate, and γ-aminobutyric acid were located primarily in the cytoplasmic fraction. In ¹⁵N-feeding studies, newly synthesized glutamine partitioned strongly to the vacuole, whereas glutamate partitioned strongly to the cytoplasm, γ-aminobutyric acid was totally excluded from the vacuole, and alanine was distributed in both compartments. Comparison of the ¹⁵N-enrichment patterns suggests that initial assimilation to glutamine occurs within a subcompartment of the cytoplasmic fraction. The protoplast-feeding technique may be extended to investigate cytoplasmic compartmentation further.     

Content and vacuole/extravacuole distribution of neutral sugars, free amino acids, and anthocyanin in protoplasts

Neutral sugar, free amino acid, and anthocyanin levels and vacuole/extravacuole distribution were determined for Hippeastrum and Tulipa petal and Tulipa leaf protoplasts. Glucose and fructose, the predominant neutral monosaccharides observed, were primarily vacuolar in location. Glutamine, the predominant free amino acid found, was primarily extravacuolar. γ-Methyleneglutamate was identified as a major constituent of Tulipa protoplasts. Qualitative characterization of Hippeastrum petal and vacuole organic acids indicated the presence of oxalic, malic, citric, and isocitric acids. Data are presented which indicate that vacuoles obtained by gentle osmotic shock of protoplasts in dibasic phosphate have good purity and retain their contents.     

Identification of eupalitin, eupatolitin and patuletin glycosides in Ipomopsis aggregata

Eupalitin, eupatolitin and patuletin 3-O-galactosides as well as eupalitin and patuletin 3-O-diglucosides have been isolated and identified from Ipomopsis aggregata (Polemoniaceae). These 6-methoxylated flavonols are important taxonomic markers in the family, occurring as they do in nine of the eighteen known genera. Microscale methods of identifying these and other related highly substituted flavonols are discussed. Studies of the mass spectral fragmentation patterns have shown that quercetagetin derivatives methylated in the A-ring can be distinguished from similar compounds methylated in the B-ring and from the analogous gossypetin derivatives.     

Antibacterial activity of triterpene acids and semi-synthetic derivatives against oral pathogens

Triterpene acids (ursolic, oleanoic, gypsogenic, and sumaresinolic acids) isolated from Miconia species, along with a mixture of ursolic and oleanolic acids and a mixture of maslinic and 2-a-hydroxyursolic acids, as well as ursolic acid derivatives were evaluated against the following microorganisms: Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, Streptococcus salivarius, Streptococcus sobrinus, and Enterococcus faecalis, which are potentially responsible for the formation of dental caries in humans. The microdilution method was used for the determination of the minimum inhibitory concentration (MIC) during the evaluation of the antibacterial activity. All the isolated compounds, mixtures, and semi-synthetic derivatives displayed activity against all the tested bacteria, showing that they are promising antiplaque and anticaries agents. Ursolic and oleanolic acids displayed the most intense antibacterial effect, with MIC values ranging from 30 microg/mL to 80 microg/mL. The MIC values of ursolic acid derivatives, as well as those obtained for the mixture of ursolic and oleanolic acids showed that these compounds do not have higher antibacterial activity when compared with the activity observed with either ursolic acid or oleanolic acid alone. With regard to the structure-activity relationship of triterpene acids and derivatives, it is suggested that both hydroxy and carboxy groups present in the triterpenes are important for their antibacterial activity against oral pathogens.     

Flavonoids of Astilbe and Rodgersia compared to Aruncus

The flavonoid profiles of Astilbe (four taxa studied) and Rodgersia (two taxa studied) are based on simple flavonol glycosides. Astilbe has 3-O-mono-, 3-O-di-, and 3-O-triglycosides of kaempferol, quercetin, and myricetin, while Rodgersia has only mono- and diglycosides of kaempferol and quercetin. Astilbe×arendsii was also shown to accumulate dihydrochalcone glycosides. The flavonoid profile of Rodgersia is the simplest recorded so far in the herbaceous Saxifragaceae. The flavonoids of two species of Aruncus were shown to be based upon kaempferol and quercetin 3-O-mono- and 3-O-diglycosides. One of the species also exhibited an eriodictyol glycoside. The triglycoside differences were not considered important, but the differences in myricetin occurrences were taken as evidence against derivation of Saxifragaceae from an Aruncus-like ancestor. Should such an event be proposed, however, serious consideration would have to be given to the current pattern of myricetin occurrence in the two families.     

Synthesis and preliminary evaluation of new ursolic and oleanolic acids derivatives as antileishmanial agents

A series of new ursolic and oleanolic acids derivatives was synthesized via ursolic or oleanolic acids, previously extracted from South American Ilex species. These new compounds were tested for in vitro antiparasitic activity on Leishmania amazonensis and Leishmania infantum strains. Some of these compounds showed activity against the promastigote forms of L. amazonensis or L. infantum, with IC(50) ranging from 5 to 12 microM. As expected, most of the compounds showed a significant level of cytotoxicity against monocytes (IC(50) = 2-50 microM). From a structure-activity relationships point of view, these pharmacological results enlightened mainly the importance of an acetylation at position 3 of the oleanolic acid skeleton in the activity against the L. amazonensis strain, and of a bis-(3-aminopropyl)piperazine moiety on the carboxylic function of ursolic acid against the L. infantum strain.     

Distribution of oleanolic acid glycosides in vacuoles and cell walls isolated from protoplasts and cells of Calendula officinalis leaves

The contents of oleanolic acid and its 3-0-glucuronide derivatives as well as of 3-0-glucoside derivatives were determined in vacuoles prepared from protoplasts and cell walls obtained from cells of Calendula officinalis leaves. In both cell compartments studied 37% of total cellular oleanolic acid were accumulated, 0.6% occurring as free oleanolic acid (only in vacuoles). Glucuronides accounted for 31.1% (20.7% in vacuoles and 10.4% in cell walls), and glucosides for 5.3% (2.6% in vacuoles and 2.7% in cell walls).     

Dual Capacity for Nutrient Uptake in Tetrahymena. II. Role of the Two Systems in Vitamin Uptake

SYNOPSIS Previously, we found that a mutant strain of Tetrahymena pyriformis without food vacuoles failed to grow unless the nutrient media were richly supplemented with vitamins and trace metals. Here we show that calcium folinate alone can replace the extra vitamin supplementation. The mutant requires ∼ 90-fold higher concentrations of folinate than the wild-type cells to give similar growth responses in a chemically defined medium. We infer that the food vacuole is an important route of uptake for this vitamin in the wild-type cells. We found no difference between mutant and wild-type cells in their requirements for nicotinic acid, pantothenic acid, riboflavin-monophosphate, and pyridoxal. We infer that an extravacuolar route contributes importantly to uptake of these 4 compounds.     

Dual capacity for nutrient uptake in Tetrahymena. II. Role of the two systems in vitamin uptake.

Previously, we found that a mutant strain of Tetrahymena pyriformis without food vacuoles failed to grow unless the nutrient media were richly supplemented with vitamins and trace metals. Here we show that calcium folinate alone can replace the extra vitamin supplementation. The mutant requires approximately 90-fold higher concentration of folinate than the wild-type cells to give similar growth responses in a chemically defined medium. We infer that the food vacuole is an important route of uptake for this vitamin in the wild-type cells. We found no difference between mutant and wild-type cells in their requirements for nicotinic acid, pantothenic acid, riboflavin-monophosphate, and pyridoxal. We infer that an extravacuolar route contributes importantly to uptake of these 4 compounds.     

Rational design and synthesis of topoisomerase I and II inhibitors based on oleanolic acid moiety for new anti-cancer drugs

Semisynthetic reactions were conducted on oleanolic acid, a common plant-derived oleanane-type triterpene. Ten rationally designed derivatives of oleanolic acid were synthesized based on docking studies and tested for their topoisomerase I and IIα inhibitory activity. Semisynthetic reactions targeted C-3, C-12, C-13, and C-17. Nine of the synthesized compounds were identified as new compounds. The structures of these compounds were confirmed by spectroscopic methods (1D, 2D NMR and MS). Five oleanolic acid analogues (S2, S3, S5, S7 and S9) showed higher activity than camptothecin (CPT) in the topoisomerase I DNA relaxation assay. Four oleanolic acid analogues (S2, S3, S5 and S6) showed higher activity than etoposide in a topoisomerase II assay. The results indicated that the C12–C13 double bond of the oleanolic acid skeleton is important for the inhibitory activity against both types of topoisomerases, while insertion of a longer chain at either position 3 or 17 increases the activity against topoisomerases by various degrees. Some of the synthesized compounds act as dual inhibitors for both topoisomerase I and IIα.     

Intracellular distribution and origin of pentacyclic triterpenes in Calendula officinalis leaves

In Calendula officinalis leaves the cyclization of squalene to β-amyrin and its further oxidation to oleanolic acid as well as the biosynthesis of all derivatives of oleanolic acid 3-glucoside and some derivatives of oleanolic acid 3-glucuronoside occur in the microsomal fraction. The final metabolites of oleanolic acid 3-glucoside series i.e. pentaglycosides, are translocated from this fraction, one to the cell wall and plasmalemma fraction and the other to the cytosol. The derivatives of oleanolic acid 3-glucoronoside are synthesized partially in other fractions and accumulate in the different membraneous structures of the cell.     

Cycloartane triterpene glycosides from Astragalus trigonus

Three new cycloartane glycosides, trigonoside I, II and III, and the known astragalosides I and II were isolated from the roots of Astragalus trigonus. The structures of the new glycosides were totally elucidated by high field (600 MHz) NMR analyses as cycloastragenol-6-O-β-xylopyranoside, cycloastragenol-3-O-[-l-arabinopyranosyl(1 → 2)-β-d-xylopyranosyl]-6-O-β- d-xylopyranoside and cycloastragenol-3-O-[-l-arabinopyranosyl(1 → 2)-β-d-(3-O-acetyl)-xylopyranosyl]-6-O-β-d-xylopyranoside.     

Two rhamnetin digalactosides and an oleanolic acid digalactoside from the flowers of Cassia laevigata

From the flowers of Cassia laevigata, two new rhamnetin glycosides, the 3-galactosyl(1→4)- galactopyranoside and the related 3-galactosyl(1→6)galactopyranoside, and oleanolic acid 3-galactosyl(1→4)- galactopyranoside have been isolated. These three glycosides have not been isolated earlier from any plant source. The known compounds quercetin, docosyl alcohol, carnaubyl alcohol, ceryl alcohol and octacosanol have also been obtained.