Chemical extraction of indigo from Indigofera tinctoria while attaining biological integrity

Indigofera tinctoria was permeabilized with 20% methanol (v/v) at 25 °C and released 8±2 g indigo g^–1 dry plant material (excluding roots). This is equivalent to 42±11% of the total indican within the cells. The plants began to recover after 2 weeks.     

Indigo production in hairy root cultures of Polygonum tinctorium Lour.

The transformed root culture of Polygonum tinctorium Lour. was established by infecting leaf explants with Agrobacterium rhizogenes A4. These cultures were examined for their growth and indigo content under various culture conditions. Among the four different culture media tested, SH medium showed the highest yield for root growth (28 mg dry wt/30 ml) and indigo production (152 g/dry wt). In SH medium, 30 g sucrose l^–1, 2500 mg KNO₃ l^–1, 300 mg NH₄H₂PO₄ l^–1 were the best conditions for indigo production at pH 5.7. The production of indigo in hairy roots slightly increased with the addition of 200 mg chitosan l^–1 (186 g/dry wt) and 20 U pectinase l^–1 (181 g/dry wt).     

Indigo and indirubin derivatives from indoles in Polygonum tinctorium tissue cultures

Substituted indoles (5-bromo-, 5-fluoro-, 5-chloro-, 5-hydroxy-, 4-methyl-, 5-methyl-, 6-methyl-, and 7-methylindole) were fed at 1 mM for 10 to 21 days into the tissue cultures of Polygonum tinctorium including cell suspension, root and shoot cultures. They were converted into the corresponding indirubin and indigo derivatives with the exception of 5-hydroxyindole. The yield ranged between 0.7–1.1 mg/g dried weight for indigos and 0.007–0.024 mg/g dried weight for indirubins. Presence of the corresponding indican derivatives in the shoot culture suggested that the pigment production proceeded through the indican formation. © Rapid Science Ltd. 1998     

Influence of light on natural indigo production from woad (Isatis tinctoria)

In woad (Isatis tinctoria), field observations indicated, that after periods of dry sunny weather, indigo yields increased significantly, suggesting that light intensity and quality affected indigo precursor production. Therefore, woad was grown under different light intensities and in the presence or absence of supplementary UV light. In general, plants supplied with more light produced more indigo than those given lesser light. When plants under greater light regimes were transferred to lesser light conditions, then indigo production declined. The opposite was also true, indicating that greater indigo production can be obtained from plants harvested after periods of increased sunlight.     

Indigogenic substrates for detection and localization of enzymes

Indoxyl esters and glycosides are useful chromogenic substrates for detecting enzyme activities in histochemistry, biochemistry and bacteriology. The chemical reactions exploited in the laboratory are similar to those that generate indigoid dyes from indoxyl-beta-d-glucoside and isatans (in certain plants), indoxyl sulfate (in urine), and 6-bromo-2-S-methylindoxyl sulfate (in certain molluscs). Pairs of indoxyl molecules released from these precursors react rapidly with oxygen to yield insoluble blue indigo (or purple 6,6'-dibromoindigo) and smaller amounts of other indigoid dyes. Our understanding of indigogenic substrates was developed from studies of the hydrolysis of variously substituted indoxyl acetates for use in enzyme histochemistry. The smallest dye particles, with least diffusion from the sites of hydrolysis, are obtained from 5-bromo-, 5-bromo-6-chloro- and 5-bromo-4-chloroindoxyl acetates, especially the last of these three. Oxidation of the diffusible indoxyls to insoluble indigoid dyes must occur rapidly. This is achieved with atmospheric oxygen and an equimolar mixture of K(3)Fe(CN)(6) and K(4)Fe(CN)(6), which has a catalytic function. H(2)O(2) is a by-product of the oxidation of indoxyl by oxygen. In the absence of a catalyst, the indoxyl diffuses and is oxidized by H(2)O(2) (catalyzed by peroxidase-like proteins) in sites different from those of the esterase activity. The concentration of K(3)Fe(CN)(6)/K(4)Fe(CN)(6) in a histochemical medium should be as low as possible because this mixture inhibits some enzymes and also promotes parallel formation from the indoxyl of soluble yellow oxidation products. The identities and positions of halogen substituents in the indoxyl moiety of a substrate determine the color and the physical properties of the resulting indigoid dye. The principles of indigogenic histochemistry learned from the study of esterases are applicable to methods for localization of other enzymes, because all indoxyl substrates release the same type of chromogenic product. Substrates are commercially available for a wide range of carboxylic esterases, phosphatases, phosphodiesterases, aryl sulfatase and several glycosidases. Indigogenic methods for carboxylic esterases have low substrate specificity and are used in conjunction with specific inhibitors of different enzymes of the group. Indigogenic methods for acid and alkaline phosphatases, phosphodiesterases and aryl sulfatase generally have been unsatisfactory; other histochemical techniques are preferred for these enzymes. Indigogenic methods are widely used, however, for glycosidases. The technique for beta-galactosidase activity, using 5-bromo-4-chloroindoxyl-beta-galactoside (X-gal) is applied to microbial cultures, cell cultures and tissues that contain the reporter gene lac-z derived from E. coli. This bacterial enzyme has a higher pH optimum than the lysosomal beta-galactosidase of animal cells. In plants, the preferred reporter gene is gus, which encodes beta-glucuronidase activity and is also demonstrable by indigogenic histochemistry. Indoxyl substrates also are used to localize enzyme activities in non-indigogenic techniques. In indoxyl-azo methods, the released indoxyl couples with a diazonium salt to form an azo dye. In indoxyl-tetrazolium methods, the oxidizing agent is a tetrazolium salt, which is reduced by the indoxyl to an insoluble coloured formazan. Indoxyl-tetrazolium methods operate only at high pH; the method for alkaline phosphatase is used extensively to detect this enzyme as a label in immunohistochemistry and in Western blots. The insolubility of indigoid dyes in water limits the use of indigogenic substrates in biochemical assays for enzymes, but the intermediate indoxyl and leucoindigo compounds are strongly fluorescent, and this property is exploited in a variety of sensitive assays for hydrolases. The most commonly used substrates for this purpose are glycosides and carboxylic and phosphate esters of N-methylindoxyl. Indigogenic enzyme substrates are among many chromogenic reagents used to facilitate the identification of cultured bacteria. An indoxyl substrate must be transported into the organisms by a permease to detect intracellular enzymes, as in the blue/white test for recognizing E. coli colonies that do or do not express the lac-z gene. Secreted enzymes are detected by substrate-impregnated disks or strips applied to the surfaces of cultures. Such devices often include several reagents, including indigogenic substrates for esterases, glycosidases and DNAse.     

Why is indigo blue?

Although the color of indigo is strongly dependent on its environment, it is blue in most commonly encountered situations. Indigo's absorption at such long wavelengths for such a small molecule is unique, and I provide here an overview of the concepts advanced to account for this feature. A traditional valence-bond approach may be used to provide a reasonable qualitative explanation. A more rigorous, quantitative explanation is provided by molecular orbital methods of varying degrees of sophistication and several explanations have been proposed based on these models. Commonly, it is suggested that the important structural unit in determining color is based on the cross-conjugated “H-chromophore” concept. A second closely related explanation describes it as two symmetrically coupled merocyanine chains. Another proposal suggests that the basic chromophore may be interpreted as the aza analogue of two coupled anti aromatic-cyclopentadienyl ions. PiSYSTEM, a commercially available quantum mechanics program, has been used to provide a successful quantitative account of the colors of indigo and indirubin, a red isomer.     

Population Dynamics of Plant Nematodes in Cultivated Soil: Effect of Summer Cover Crops in Old Agricultural Land

In a 6-year cover crop sequence study, nematode population densities varied with different cover crops. Millet favored rapid increase of Belonolaimus longicaudatus and supported relatively large numbers of Pratylenchus brachyurus. Beggarweed and ''Coastal'' bermudagrass favored rapid increase of B. Iongicaudatus and to a lesser extent P. brachyurus and Trichodorus christiei. Hairy indigo and Crotalaria supported relatively large numbers of P. brachyurus but suppressed B. longicaudatus. Hairy indigo also supported increases of T. christiei and Xiphinema americanum. Marigold did not favor development of any parasitic nematode species present. Tomato transplant yield was inversely related to nematode population, particularly to B. Iongicaudatus. Largest yields were obtained from plots with smallest numbers of B. longicaudatus and smallest yields were from plots with largest numbers of B. longicaudatus.     

板蓝根中靛玉红提取方法研究

采用薄层色谱分析比较了水煎法和渗漉法对板蓝根有效成分--靛玉红的提取效果,并对以前的提取方法做了改进,提高了靛玉红的提取效率。