Riboflavin preparation

Crude riboflavin, prepared as described above starting with 43.6 g of 2-(o-biphehylazo)-4,5-dimethyl-l-ribityl amino-benzene, is washed with 50 ml of cold ethyl acetate, slurried with 180 ml of methanol at 65°C for thirty minutes. The methanol slurry is cooled to 10°C for thirty minutes, filtered, and the filtered material washed with 40 ml of cold methanol. The methanol washed riboflavin is then slurried with 180 ml of water at 80°C for thirty minutes, the slurry is cooled to 70°C, filtered, and the filtered material is washed with 40 ml of hot (70°C) water. The hot water-washed riboflavin is... 

Stepanov, A.L et al. (1984) Riboflavin preparation. Patent FR2546907, Inst Genetiki. 

In 1933, R. Kuhn and his co-workers first isolated riboflavin from eggs in a pure, crystalline state (1), named it ovoflavin, and deterrnined its function as a vitamin (2). At the same time, impure crystalline preparations of riboflavin were isolated from whey and named lyochrome and, later, lactoflavin. Soon thereafter, P. Karrer and his co-workers isolated riboflavin from a wide variety of animal organs and vegetable sources and named it hepatoflavin (3). Ovoflavin from egg, lactoflavin from milk, and hepatoflavin from Hver were aU. subsequently identified as riboflavin. The discovery of the yeUow en2yme by Warburg and Christian in 1932 and their description of lumiflavin (4), a photochemical degradation product of riboflavin, were of great use for the elucidation of the chemical stmcture of riboflavin by Kuhn and his co-workers (5). The stmcture was confirmed in 1935 by the synthesis by Karrer and his co-workers (6), and Kuhn and his co-workers (7). 

Photochemical decomposition of riboflavin in neutral or acid solution gives lumichrome (3), 7,8-dimethyl all oxazine, which was synthesized and characterized by Karrer and his co-workers in 1934 (11). In alkaline solution, the irradiation product is lumiflavin (4), 7,8,10-trimethyhsoalloxazine its uv—vis absorption spectmm resembles that of riboflavin. It was prepared and characterized in 1933 (5). Another photodecomposition product of riboflavin is 7,8-dimethy1-10-foTmylmethy1isoa11oxazine (12). 

Later, a completely different and more convenient synthesis of riboflavin and analogues was developed (34). It consists of the nitrosative cyclization of 6-(A/-D-ribityl-3,4-xyhdino)uracil (18), obtained from the condensation of A/-D-ribityl-3,4-xyhdine (11) and 6-chlorouracil (19), with excess sodium nitrite in acetic acid, or the cyclization of (18) with potassium nitrate in acetic in the presence of sulfuric acid, to give riboflavin-5-oxide (20) in high yield. Reduction with sodium dithionite gives (1). In another synthesis, 5-nitro-6-(A/-D-ribityl-3,4-xyhdino) uracil (21), prepared in situ from the condensation of 6-chloro-5-nitrouracil (22) with A/-D-ribityl-3,4-xyhdine (11), was hydrogenated over palladium on charcoal in acetic acid. The filtrate included 5-amino-6-(A/-D-ribityl-3,4-xyhdino)uracil (23) and was maintained at room temperature to precipitate (1) by autoxidation (35). These two pathways are suitable for the preparation of riboflavin analogues possessing several substituents (Fig. 4). 

Fermentative Manufacture. Throughout the years, riboflavin yields obtained by fermentation have been improved to the point of commercial feasibiUty. Most of the riboflavin thus produced is consumed in the form of cmde concentrates for the enrichment of animal feeds. Riboflavin was first produced by fermentation in 1940 from the residue of butanol—acetone fermentation. Several methods were developed for large-scale production (41). A suitable carbohydrate-containing mash is prepared and sterilised, and the pH adjusted to 6—7. The mash is buffered with calcium carbonate, inoculated with Clostridium acetohutylicum and incubated at 37—40°C for 2—3 d. The yield is ca 70 mg riboflavin/L (42) (see Fermentation). 

Catalysis by flavoenzymes has been reviewed and various analogues of FAD have been prepared e.g. P -adenosine-P -riboflavin triphosphate and flavin-nicotinamide dinucleotide ) which show little enzymic activity. The kinetic constants of the interaction between nicotinamide-4-methyl-5-acetylimidazole dinucleotide (39) and lactic dehydrogenase suggest the presence of an anionic group near the adenine residue at the coenzyme binding site of the enzyme. ... 

The varions flavin phosphates and their acetyl derivatives were identified by pH titration, electrophoresis, and H-NMR, which permit direct analysis of crude reaction prodncts as well as rapid purity check of commercial flavin mononucleotide or riboflavin 5 -monophosphate (FMN or 5 -FMN) [7]. Riboflavin 4 -monophosphate was determined as the main by-product of commercial FMN by preparative TLC on cellulose with n-butanol/acetic add/water (5 2 3, v/v) as a solvent [7]. 

Incorporation of a flavin electron donor and a thymine dimer acceptor into DNA double strands was achieved as depicted in Scheme 5 using a complex phosphoramidite/H-phosphonate/phosphoramidite DNA synthesis protocol. For the preparation of a flavin-base, which fits well into a DNA double strand structure, riboflavin was reacted with benzaldehyde-dimethylacetale to rigidify the ribityl-chain as a part of a 1,3-dioxane substructure [49]. The benzacetal-protected flavin was finally converted into the 5 -dimethoxytri-tyl-protected-3 -H-phosphonate ready for the incorporation into DNA using machine assisted DNA synthesis (Scheme 5a). For the cyclobutane pyrimidine dimer acceptor, a formacetal-linked thymine dimer phosphoramidite was prepared, which was found to be accessible in large quantities [50]. Both the flavin base and the formacetal-linked thymidine dimer, were finally incorporated into DNA strands like 7-12 (Scheme 5c). As depicted in... 

The types of compounds that can be analyzed by fluorometry are rather limited. Benzene ring systems, such as the vitamins riboflavin (Figure 8.13) and thiamine, are especially highly fluorescent compounds and are analyzed in foods and pharmaceutical preparations by fluorometry. Metals can be analyzed by fluorometry if they are able to form complex ions by reaction with a ligand having a benzene ring system. 

Experiment 24 Fluorometric Analysis of a Prepared Sample for Riboflavin... 

Note Riboflavin solutions are fight sensitive. Store them in the dark as you prepare them. 

Your instructor has prepared a 50 ppm riboflavin stock solution in 5% (by volume) acetic acid. Prepare a series of calibration standard solutions that are 0.2, 0.4, 0.6, 0.8, and 1.0 ppm from the 50 ppm stock solution and again use 5% acetic acid for the dilution. Use 25-mL volumetric flasks for these standards and shake well. 

Coloring agents are used in pharmaceutical preparations for purposes of esthetics. A distinction should be made between agents that have inherent color and those that are employed as colorants. Certain agents such as sulfur (yellow), riboflavin (yellow), cupric sulfate (blue), ferrous sulfate (bluish green), and cyanocobalamin (red), have inherent color and are not thought of as pharmaceutical colorants in the usual sense of the term. 

Stock solution 4. 100 x stock solution of vitamins was prepared by dissolving biotin (20 mg), folic acid (20 mg), pyrodoxine hydrochloride (100 mg), riboflavin (50 mg), thiamine hydrochloride (50 mg), nicotinic acid (50 mg), pantothenic acid (50 mg), vitamin B12 (1 mg), 4-aminobenzoic acid (50 mg) and thioctic acid (50 mg) in deionized water. The volume was adjusted to 1.0 L. The solution was filtered, sterilized and stored as 10 mL aliquots at —20 °C. 

The ionisation state of molecules in the solution state appears to be an important variable in photodegradation mechanisms. A recent pubhcation on riboflavin oral liquid preparations shows that the formulation is most photostable at pHs between 5 and 6, where the non-ionised form predominates [78]. The rate of photolysis increase 80-fold at pH 10.0, owing to increased redox potential. Conversely, at pH 3.0, the increased photolysis is associated with the excited singlet state, in addition to the triplet state. 

Where patients are at risk of Wernicke s encephalopathy - for example, because of chronic alcohol abuse, hyperemesis gravidarum, or malnutrition - they should be given thiamine. In many countries no intravenous preparation of thiamine alone is available, and the compound preparations that are available are prone to cause anaphylactoid reactions, so they should be given by slow infusion, and with adequate facilities for resuscitation. A high potency preparation (Pabrinex ) that contains thiamine 250 mg in 10 ml with ascorbic acid, nicotinamide, pyridoxine and riboflavin, can be given by intravenous infusion over 10 min. 

FIGURE 5 Total peroxide (circles), catalase-resistant peroxides (squares), and H202 as the difference (triangles) were measured in three lots of oral multivitamins without riboflavin (Tri-Vi-Sol) and three lots of oral multivitamins with 0.6 mg of riboflavin (Poli-Vi-Sol) by time after the initial opening of the bottle. Compared with the preparation without riboflavin, the level of H202 was initially higher (P < 0.01) in (Poli-Vi-Sol), and that level dropped over time (P < 0.05). In both multivitamin preparations, the levels of catalase-resistant and total peroxide rose (P < 0.05) until day 8. Data represent the mean + standard error of the mean. Variations too small relative to the symbol are not shown [30]. 

Babior, who has studied this enzyme at several stages of its purification, found in lysates of PMNs which were activated with zymosan that of eight potential biological reductants only reduced pyridine nucleotides supported the formation of O ". The K , for NADPH was less than the K , for NADH and the activity was decreased in preparations from three patients with chronic granulomatous disease. In accord with predictions based on reaction 7, 0.55 molecule of O7 was measured per molecule of NADPH oxidized under conditions of saturating concentrations of cytochrome c The enzyme which was extracted with Triton X-100 from a granule-rich fraction from activated PMNs, required an external source of FAD for the formation of O from NADPH . Riboflavin and FMN would not substitute. Flavin adenine dinucleotide was proposed as a necessary cofactor, which was probably lost when the enzyme was treated with the detergent. 

The procedure to phosphorylate riboflavin derivatives on a preparative scale has recently been improved . These preparations, and also commercial FMN, contain a considerable amount of riboflavin phosphate isomers, which are difficult to separate by column chromatography. This problem is emphasized in the chemical synthesis of FAD where the yield is rather low (20-25 %). In this context, it is surprising that a modification of the synthesis of FAD from FMN published by Cramer and Neuhoeffer has not been noticed by workers in the flavin field. According to Cramer and Neuhoeffer, the yield of the chemical synthesis of FAD is drastically improved ( 70 % pure FAD). The procedure was successfully applied in the author s own laboratory (yield 60-70%). It is expected that the improved procedure of the FAD synthesis will stimulate the active-site directed studies on flavoproteins because the problem of separating FMN or FAD from their synthetic by-products has already been solved by use of FMN- or FAD-specific affinity column... 

A Cheddar-type cheese retains 48% of total solids of milk, 96% casein, 4% soluble proteins, 94% fat, 6% lactose, 6% H20, 62% calcium, 94% vitamin A, 15% thiamin, 26% riboflavin, and 6% vitamin C (National Dairy Council 1979). The lactose content varies in freshly prepared cheeses and decreases rapidly during ripening, completely disappearing in four to six weeks. The enzymes and ripening agents responsible for the rate and extent of fat and protein breakdown are fully discussed in Chapter 12, and vitamin variation is discussed in Chapter 7. 

Riboflavin, a yellow solid, has a low solubility of 100 mg /1 at 25°C. Three crystalline forms are known. One of these, the "readily soluble form," is ten times more soluble than the others and can be used to prepare metastable solutions of higher concentration. One crystalline form is platelike and occurs naturally in the tapetum (Box 13-C) of the nocturnal lemur. 

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