Two validated HPLC methods for the quantification of alizarin and other anthraquinones in Rubia tinctorum cultivars

Direct and indirect HPLC-UV methods for the quantitative determination of anthraquinones in dried madder root have been developed, validated and compared. In the direct method, madder root was extracted twice with refluxing ethanol-water. This method allowed the determination of the two major native anthraquinone glycosides lucidin primeveroside and ruberythric acid. In the indirect extraction method, the anthraquinone glycosides were first converted into aglycones by endogenous enzymes and the aglycones were subsequently extracted with tetrahydrofuran-water and then analysed. In this case the anthraquinones alizarin, purpurin and nordamnacanthal may be determined. The content of nordamnacanthal is proportional to the amount of lucidin primeveroside originally present. The indirect extraction method is easier to apply. Different madder cultivars were screened for their anthraquinone content.     

Formation of genotoxic metabolites from anthraquinone glycosides, present in Rubia tinctorum L.

Rubia tinctorum L., a medicinal plant used for the treatment of kidney and bladder stones, contains a characteristic spectrum of 9,10-anthraquinone derivatives, which are substituted in only one of the aromatic benzo rings. The majority of the anthraquinones present in the plant itself or in plant extracts are glycosides. We investigated the metabolism of two such glycosides, alizarinprimeveroside (AlP) and lucidinprimeveroside (LuP). AlP given orally to rats was metabolized to alizarin (Al) and 1-hydroxyanthraquinone (1-HA). The reductive cleavage of AlP was also observed after treatment of this compound with rat liver enzymes (S9) and NADPH. 1-HA has been reported to induce unscheduled DNA synthesis (UDS) in primary rat hepatocytes (PRH) and intestinal and liver tumors in rats after chronic treatment. The in vitro genotoxicity of 1-HA was confirmed by our present investigations. We also observed that the glycoside AlP was active at inducing UDS in PRH, but the compound was inactive in the Salmonella/microsome assay. Oral administration of LuP to rats resulted in the excretion of lucidin and rubiadin. When LuP was treated with rat liver extract and NADPH, the compound was reduced to rubiadinprimeveroside (RuP), which was hydrolyzed to rubiadin. We have recently shown that lucidin is highly genotoxic in a battery of short-term tests. We now report that rubiadin is also highly genotoxic in Salmonella typhimurium. However, in contrast to lucidin, it requires metabolic activation. In the UDS assay in PRH, rubiadin was even more potent than lucidin and equal to the positive control DMBA. In addition, the glycoside LuP is active in the Salmonella/microsome assay as well as in the UDS assay. The present work demonstrates that the uptake of the anthraquinone glycosides AlP and LuP leads to the rodent carcinogen 1-HA, and to the highly genotoxic compounds lucidin and rubiadin. This extends our previous studies and supports our suggestion that the therapeutic use of Rubia tinctorum may involve a carcinogenic risk.     

Identification of a mutagenic substance, in Rubia tinctorum L. (madder) root, as lucidin

Mutagenicity of the extract from Rubia tinctorum L. (madder) root was demonstrated on Salmonella typhimurium TA100 and TA98. The active substance wa purified and characterized by TLC, UV spectrum, IR spectrum, mass spectrum and [1H]NMR spectrum. All the mutagenic activity of the extract from the root of Rubia tinctorum L. was due to lucidin (1,3-dihydroxy,2-hydroxymethyl-9, 10-anthraquinone).     

Ipolearoside: A new glycoside from Ipomoea leari with anti-cancer activity

Ipolearoside, a new glycoside with anticancer activity, has been isolated from Ipomoea leari Paxt. On acid hydrolysis in methanol, it gave the aglycone methyl ester, characterised as methyl 3,11-dihydroxyhexadecanoate. Ipolearoside is a complex glycoside of 3,11-dihydroxyhexadecanoic acid and glucose, rhamnose and fucose.     

Staining human lymphocytes and onion root cell nuclei with madder root

We performed staining experiments on cells using natural dyes and different mordants using techniques that are used for wool and silk dyeing. The natural dye sources were madder root, daisy, corn cockle and yellow weed. Ferrous sulfate, copper sulfate, potassium tartrate, urea, potassium aluminum sulfate and potassium dichromate were used as mordants. Distilled water, distilled water plus ethanol, heptane, and distilled water plus methanol were used as solvents. All dye-mordant-solvent combinations were studied at pH 2.4, 3.2 and 4.2. The generic staining procedure was to boil 5–10 onion roots or stimulated human lymphocyte (SHL) preparations in a dye bath on a hot plate. Cells were examined at every half hour. For multicolor staining, madder-dyed lymphocytes were decolorized, then stained with Giemsa. The AgNOR technique was performed following the decolorization of Giemsa stained lymphocytes. Good results were obtained for both onion root cells and lymphocytes that were boiled for 3 h in a dye bath that included 4 g madder root, 4 g ferrous sulfate as mordant in 50 ml of 1:1 (v/v) methanol:distilled water. The pH was adjusted to 4.2 with 6 ml acetic acid. We conclude that madder root has potential as an alternative dye for staining biological materials.     

Staining human lymphocytes and onion root cell nuclei with madder root.

We performed staining experiments on cells using natural dyes and different mordants using techniques that are used for wool and silk dyeing. The natural dye sources were madder root, daisy, corn cockle and yellow weed. Ferrous sulfate, copper sulfate, potassium tartrate, urea, potassium aluminum sulfate and potassium dichromate were used as mordants. Distilled water, distilled water plus ethanol, heptane, and distilled water plus methanol were used as solvents. All dye-mordant-solvent combinations were studied at pH 2.4, 3.2 and 4.2. The generic staining procedure was to boil 5-10 onion roots or stimulated human lymphocyte (SHL) preparations in a dye bath on a hot plate. Cells were examined at every half hour. For multicolor staining, madder-dyed lymphocytes were decolorized, then stained with Giemsa. The AgNOR technique was performed following the decolorization of Giemsa stained lymphocytes. Good results were obtained for both onion root cells and lymphocytes that were boiled for 3 h in a dye bath that included 4 g madder root, 4 g ferrous sulfate as mordant in 50 ml of 1:1 (v/v) methanol:distilled water. The pH was adjusted to 4.2 with 6 ml acetic acid. We conclude that madder root has potential as an alternative dye for staining biological materials.     

Residues affecting hydrolysis of soy isoflavone glycosides, stability and catalytic properties of Thermotoga maritima β-glucosidase

The thermostable β-glucosidase A (TmBglA) from Thermotoga maritima is a promising biocatalyst for production of isoflavone aglycones. Use of enzymes with high specificity for soy isoflavone conjugates is however essential for efficient hydrolysis. The effect of the amino acids located in the aglycone binding pocket with non-conserved residues between specificity groups in family 1 glycoside hydrolase (GH1) was studied using wild-type TmBglA and 3 exchange mutants (M1-TmBglA, M2-TmBglA, M1M2-TmBglA). Three mutants were expressed in Escherichia coli, purified and characterized. They had shifts in both optimum temperature and thermal stability, and their narrowing pH-activity curve caused by removing the ionized side chain in mutation. All mutants demonstrated the decreased catalytic efficiency more effectively revealed with natural glycoside, salicin, than with artificial substrate, p-nitrophenyl-β-D-glucopyranoside, suggesting that these amino acids are the key residues to determine aglycone specificity. A lower hydrolysis of genistin and daidzin for M2-TmBglA than M1-TmBglA indicated that L400, A407 and E408 being preferable to V170, A171, V173, G174 and H180 residues of Tm-BglA could be essential for soy isoflavone glycoside binding and catalysis.     

Crystallographic analysis shows substrate binding at the -3 to +1 active-site subsites and at the surface of glycoside hydrolase family 11 endo-1,4-beta-xylanases

GH 11 (glycoside hydrolase family 11) xylanases are predominant enzymes in the hydrolysis of heteroxylan, an abundant structural polysaccharide in the plant cell wall. To gain more insight into the protein-ligand interactions of the glycone as well as the aglycone subsites of these enzymes, catalytically incompetent mutants of the Bacillus subtilis and Aspergillus niger xylanases were crystallized, soaked with xylo-oligosaccharides and subjected to X-ray analysis. For both xylanases, there was clear density for xylose residues in the -1 and -2 subsites. In addition, for the B. subtilis xylanase, there was also density for xylose residues in the -3 and +1 subsite showing the spanning of the -1/+1 subsites. These results, together with the observation that some residues in the aglycone subsites clearly adopt a different conformation upon substrate binding, allowed us to identify the residues important for substrate binding in the aglycone subsites. In addition to substrate binding in the active site of the enzymes, the existence of an unproductive second ligand-binding site located on the surface of both the B. subtilis and A. niger xylanases was observed. This extra binding site may have a function similar to the separate carbohydrate-binding modules of other glycoside hydrolase families.     

The genotoxicity of lucidin,a natural component of Rubia tinctorum L., and lucidinethylether,a component of ethanolic Rubia extracts

The genotoxic activity of lucidin (1,3-dihydroxy-2-hydroxymethyl-9,10-anthraquinone), a natural component of Rubia tinctorum L., was tested in a battery of short-term tests. The compound was mutagenic in five Salmonella typhimurium strains without metabolic activation, but the mutagenicity was increased after addition of rat liver S9 mix. In V79 cells, lucidin was mutagenic at the hypoxanthine-guanine phosphoribosyl transferase gene locus and active at inducing DNA single-strand breaks and DNA protein cross-links as assayed by the alkaline elution method. Lucidin also induced DNA repair synthesis in primary rat hepatocytes and transformed C3HI M2-mouse fibroblasts in culture. We also investigated lucidinethylether, which is formed from lucidin by extraction of madder roots with boiling ethanol. This compound was also mutagenic in Salmonella, but only after addition of rat liver S9 mix. Lucidinethylether was weakly mutagenic to V79 cells which were cocultivated with rat hepatocytes. The compound did not induce DNA repair synthesis in hepatocytes from untreated rats, but positive results were obtained when hepatocytes from rats pretreated with phenobarbital were used. We conclude that lucidin and its derivatives are genotoxic.Abbreviations DMBA 7,12-dimethylbenz(a)anthracene - HA hydroxyanthraquinones - LUE lucidinethylether - PRH primary rat hepatocytes - UDS unscheduled DNA synthesis     

On the mechanism of mitochondrial permeability transition induction by glycyrrhetinic acid

Glycyrrhetinic acid (GE), the aglycone of glycyrrhizic acid, a triterpene glycoside which represents one of the main constituents of licorice root, induces an oxidative stress in liver mitochondria responsible for the induction of membrane permeability transition. In fact, GE, by interacting with the mitochondrial respiratory chain, generates hydrogen peroxide which in turn oxidizes critical thiol groups and endogenous pyridine nucleotides leading to the opening of the transition pore. Most likely the reactive group of GE is the carbonyl oxygen in C-11 which, by interacting mainly with a Fe/S centre of mitochondrial complex I, generates an oxygen-centered radical responsible for the pro-oxidant action.     

On the mechanism of mitochondrial permeability transition induction by glycyrrhetinic acid

Glycyrrhetinic acid (GE), the aglycone of glycyrrhizic acid, a triterpene glycoside which represents one of the main constituents of licorice root, induces an oxidative stress in liver mitochondria responsible for the induction of membrane permeability transition. In fact, GE, by interacting with the mitochondrial respiratory chain, generates hydrogen peroxide which in turn oxidizes critical thiol groups and endogenous pyridine nucleotides leading to the opening of the transition pore. Most likely the reactive group of GE is the carbonyl oxygen in C-11 which, by interacting mainly with a Fe/S centre of mitochondrial complex I, generates an oxygen-centered radical responsible for the pro-oxidant action.     

Hydration of vinyl ether groups by unsaturated glycoside hydrolases and their role in bacterial pathogenesis.

Many pathogenic microorganisms invade mammalian and/or plant cells by producing polysaccharide-degrading enzymes (lyases and hydrolases). Mammalian glycosaminoglycans and plant pectins that form part of the cell surface matrix are typical targets for these microbial enzymes. Unsaturated glycoside hydrolase catalyzes the hydrolytic release of an unsaturated uronic acid from oligosaccharides, which are produced through the reaction of matrix-degrading polysaccharide lyase. This enzymatic ability suggests that unsaturated glycoside hydrolases function as virulence factors in microbial infection. This review focuses on the molecular identification, bacterial distribution, and structure/function relationships of these enzymes. In contrast to general glycoside hydrolases, in which the catalytic mechanism involves the retention or inversion of an anomeric configuration, unsaturated glycoside hydrolases uniquely trigger the hydrolysis of vinyl ether groups in unsaturated saccharides but not of their glycosidic bonds.     

Investigation on the mechanisms for biotransformation of saponins to diosgenin

In order to improve the efficiency of biotransformation of saponins in Dioscorea zingiberensis to diosgenin, a new enzymatic model was developed to investigate the mechanism of the metabolic systems. Four main saponin hydrolases (E1, E2, E3 and E4) were purified from Trichoderma reesei. Using progracillin as substrate, the enzymatic hydrolysis experiments with E1, E2, E3 and E4 were carried out respectively. Saponin concentrations during each biotransformation reaction were constructed with a kinetic model consisting of a few Michaelis–Menten equations. During biotransformation, C-26 glycoside and C-3 terminal glycoside were cleaved sequentially from saponins by E1, E2, E3 and E4. Then C-3 terminal rhamnoside and C-3 glycoside were released from the aglycone stepwisely by E2 and E3, to yield diosgenin. E2 and E3 were the key enzymes in the system, and cleavage of the C-3 glycoside from saponins was the rate-limiting step in the biotransformation process. The proposed enzymatic model might be used to analyze the mechanism for biotransformation of saponins to diosgenin.     

The crystal structure of human cytosolic beta-glucosidase unravels the substrate aglycone specificity of a family 1 glycoside hydrolase

Human cytosolic beta-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. In this study, the substrate preference of this enzyme was investigated by mutational analysis, X-ray crystallography and homology modelling. The crystal structure of hCBG was solved by the molecular replacement method and refined at 2.7 A resolution. The main-chain fold of the enzyme belongs to the (beta/alpha)(8) barrel structure, which is common to family 1 glycoside hydrolases. The active site is located at the bottom of a pocket (about 16 A deep) formed by large surface loops, surrounding the C termini of the barrel of beta-strands. As for all the clan of GH-A enzymes, the two catalytic glutamate residues are located on strand 4 (the acid/base Glu165) and on strand 7 (the nucleophile Glu373). Although many features of hCBG were shown to be very similar to previously described enzymes from this family, crucial differences were observed in the surface loops surrounding the aglycone binding site, and these are likely to strongly influence the substrate specificity. The positioning of a substrate molecule (quercetin-4'-glucoside) by homology modelling revealed that hydrophobic interactions dominate the binding of the aglycone moiety. In particular, Val168, Trp345, Phe225, Phe179, Phe334 and Phe433 were identified as likely to be important in determining substrate specificity in hCBG, and site-directed mutagenesis supported a key role for some of these residues.     

Positive cooperativity in binding by albumin: The system bovine serum albumin and alizarin yellow G cobinding by salicylic acid

The binding of alizarin yellow G—an azo derivative of salicylic acid—by bovine serum albumin has been investigated using the method of equilibrium dialysis. Six strong and a number of additional, weak binding sites have been found to be present. The system is characterized by strong positive cooperativity between the first and second sites. Six binding constants have been determined on the basis of a simplified mathematical model. The results are ~2 × 10⁴m^?1 for the first binding site, 6 × 10⁵m^?1 for the second, and between 4 × 10⁴ and 10⁵m^?1 for the rest. The phenomenon is discussed in terms of the existence of various conformers or of the conformational adaptability of albumin. Cobinding by salicylic acid does not displace alizarin yellow G but induces a conformational change in the protein which affects the absorption spectrum of the bound dye. As expected for this kind of heterotropic interaction, the spectrum of the system albumin-salicylic acid is similarly affected by the cobinding of alizarin yellow G.     

Excretion of miserotoxin and detoxification of the aglycone by grasshoppers (Orthoptera: Acrididae).

Two species of grasshoppers (Melanoplus bivittatus and M. sanguinipes) tolerated high levels of miserotoxin (3-nitro-1-propyl-beta-D-glucopyranoside) in their diet. Miserotoxin is a causative agent in cattle poisoning when timber milkvetch (Astragalus miser) is consumed. Toxic effects were averted by grasshoppers in part by excretion of the intact glycoside. When the aglycone was administered, detoxification was achieved by two routes: by oxidation of the aglycone to 3-nitropropionic acid which was then conjugated with glycine, and by glucosylation of the aglycone to miserotoxin, in each case followed by excretion.     

水提取法分离大豆异黄酮苷和苷元

本文建立了一种仅用水作为溶剂分离大豆异黄酮苷和苷元的方法。采用水加热提取的方法分离大豆异黄酮苷和苷元,分别对所用溶剂,提取温度,提取时间和物料比进行优化。实验上清液干燥后的固形物中大豆异黄酮苷含量为32．24％,苷元含量仅为3．05％,沉淀中大豆异黄酮苷元含量为72．03％,苷含量仅为4．28％。该方法经济,简单,绿色无毒,适合工业生产。     

The History of Staining Logwood Dyes

Except for the cochineal derivatives, logwood extract was the first of the important modern stains to be employed in histology. Certain other natural dyes, such as madder and indigo, had been used earlier, but they are of little significance in discussing the history of staining, because none of them nor even alizarin, the derivative of madder, are of any appreciable significance in these days of synthetic dyes. Hematoxylin, on the other hand, still continues a very important stain, and it has played an interesting part in the history of staining.     

The History of Staining Logwood Dyes

Except for the cochineal derivatives, logwood extract was the first of the important modern stains to be employed in histology. Certain other natural dyes, such as madder and indigo, had been used earlier, but they are of little significance in discussing the history of staining, because none of them nor even alizarin, the derivative of madder, are of any appreciable significance in these days of synthetic dyes. Hematoxylin, on the other hand, still continues a very important stain, and it has played an interesting part in the history of staining.     

Antifeedant activity of an anthraquinone aldehyde in Galium aparine L. against Spodoptera litura F

The insect antifeedant anthraquinone aldehyde nordamnacanthal (1,3-dihydroxy-anthraquinone-2-al) was identified in Galium aparine L., and isolated from the root powder of akane (Rubia akane), a member of the Rubiaceae. Structure-activity relationship (SAR) studies using a series of anthraquinone analogues suggested that the aldehyde group on the anthraquinone was more important than the quinone moiety for antifeedant activity against the common cutworm (Spodoptera litura). High levels of nordamnacanthal were found in the seed leaf stage and in callus tissue induced from seedlings of G. aparine, but its concentration decreased with plant development. Since these compounds are natural pigments for dying textiles, we also evaluated the antifeedant activity against the carpet beetle (Attagenus japonicus ), a textile pest was also evaluated. While nordamnacanthal had strong antifeedant activity against the common cutworm, it did not show any antifeedant activity against the carpet beetle. The most effective antifeedant against the carpet beetle was the major constituent in the extract of R. trictorum, lucidin-3-O-primeveroside, a food pigment.