Isoflavones preparation

Four short-term (six months) human studies have been performed on the effects of different isoflavone preparations on bone mineral density (Alekel et ah, 2000 Potter et al., 1998, Clifton-Bligh et al., 2001 Hsu et al., 2001) and one on bone markers (Wagen et al., 2000). 

An extract of a red clover isoflavone preparation increased MCF-7 breast cancer cell proliferation rates (Bodinet and Freudenstein 2004). A standardized red clover isoflavone extract (9% isoflavones by dry weight) showed an affinity for both estrogen receptor (ER) a and P with a significantly stronger affinity for the ERP receptor in a yeast two-plasmid system (Dornstauder et al. 2001). 

The isoflavone 406 is prepared by the indirect a-phenylation of a ketone by reaction of phenylmercury(II) chloride with the enol acetate 405, prepared from 4-chromanone[371]. A simple synthesis of pterocarpin (409) has been achieved based on the oxypalladation of the oriho-mercurated phenol derivative 408 with the cyclic alkene 407[372,373]. 

Many stilbenelike thiophene compounds have been prepared for a study of estrogenic activity, especially by Buu-Hoi et al. Thiophene derivatives of nonhydroxylated stilbene types showed no significant activitywhereas weak estrogenic activity was found in 5-acetyl-, 5-propionyl-, and 5-benzoyl-2-(-stilbenyl)thiophene. 1-Bromo-l,2-diphenyl-2-(5-bromo-2-thienyl)ethylene (258) was found to inhibit body growth and to produce extensive testicular atropy in male rats. A thiophene analog of estrogenic isoflavones (259)... 

Doerge D.R., Churchwell M.I., and Delclos K.B., 2000. Online sample preparation using restricted-access media in the analysis of the soy isoflavones, genistein and daidzein, in rat serum using liquid chromatography electrospray mass spectrometry. Rapid Commun Mass Spectrom 14 673. 

Ma, X. et al.. Preparative isolation and purification of two isoflavones from Astragalus membra-naceus Bge. var. mongholicus (Bge.) Hsiao by high-speed countercurrent chromatography, J. Chromatogr. A, 992, 193, 2003. 

An important caveat is that the epidemiological evidence suggesting protection from hormone-dependent cancer by isoflavone phytoestrogens is based on foods rather than isoflavone extracts, which in the future could include isoflavone-containing therapeutics. Indeed, the preparation of isoflavone extracts from soy protein could well result in the loss... 

To test the validity of the bioassay itself we prepared a diet containing increasing amounts of rotenone, a compound derived from isoflavones and thus chemically not far removed from the soybean phytoalexins. Results in this case followed exactly the expected dose response curve (Table VII). Both survival and weight gain of larvae were drastically affected by increasing concentrations of rotenone. This experiment showed that the bioassay would be capable of detecting toxic effects of the phytoalexins on the soybean looper larvae, if such effects were acute. It showed also that the detoxification mechanisms in the soybean looper, a rather polyphagous insect, may permit it to adequately overcome the antibiotic effect of the isoflavonoid phytoalexins, but not that of the isoflavone rotenone. 

Enone formation-aromatization has been used for the synthesis of 7-hydro-xyalkavinone (716)[456], The isoflavone 717 was prepared by the elimina-tion[457]. The unsaturated j-keto allyl esters 718 and 719. obtained in two steps from myreene. were subjected to enone formation. The reaction can be carried out even at room temperature using dinitriles such as adiponitrile (720) or 1,6-dicyanohexanc as a solvent and a weak ligand to give the pseudo-ionone isomers 721 and 722 without giving an allylated product(458]. 

The ring closure of orf/io-substituted phenols is a route to production of benzo[6]furans on an industrial scale. The natural benzo[6 ]furan derivatives karanjin, pongapin, khellin and visnagin have been prepared via the respective o-hydroxyarylacetaldehydes, whilst an extension to the cyclization of o-hydroxybenzyl alkyl ketones has made it possible to synthesize polycyclic benzo[6]furans under the influence of demethylating and dehydrating media. Pterocarpans have been synthesized from suitable isoflavones by this route. 

Basic Protocol 2 Preparation of Individual Isoflavone Standards Basic Protocol 3 Gradient Separation and Identification of Isoflavones 11.6.5... 

Support Protocol 2 Sample Preparation for HPLC Analysis Support Protocol 3 Converting Glycosidic Isoflavones to Their Aglycones 11.6.10... 

In this protocol, commercially purchased isoflavone standards (Table II. 6.3) are dissolved in a suitable solvent to prepare solutions in a series of decreasing concentrations. The absorbances are then measured using a UV spectrophotometer set at the isoflavone s maximum wavelength ( max), and the concentrations of isoflavone standard solutions are calculated using published molar extinction coefficients. The spectrum is also scanned in order to evaluate the fine structure (see Background Information, Spectral fine structure). 

Acetic acid serves as a modifier to protonate glucosides of isoflavones in the mobile phase. While freshly prepared solvent A is preferred, solvent that is up to 2 days old is acceptable. 

The standard mixture can be prepared by taking a known amount of each standard solution into a sample vial and mixing well. The concentration of each isoflavone in the standard mixture can be calculated with the dilution factor. 

Use the area of each isoflavone peak and the calibration curves to calculate the isoflavone concentrations in the sample. Calculate the final concentration of isoflavones in soy food by considering the dilution factor during sample preparation. 

Quantification. For accurate quantification, a standard curve of peak area versus concentration should be constructed for each standard using the same chromatographic conditions (e.g., wavelength and solvent) as for the samples under analysis. The concentration range of standard curves should be determined according to both the isoflavone level of soy food samples and dilution factors during sample preparation such that the UV absorbance of the injected sample is within a range of 0 to 1. The appropriate standard curve can then be used to calculate the quantity of isoflavones represented by each HPLC peak in the sample. 

Traditionally HPLC methods were used for isoflavone analysis from foods [Wang et al., 1990], and gas chromatography/mass spectrometry (GC/MS) to determine isoflavones and their metabolites in human biological fluids including urine [Adlercreutz et al., 1991 Kelly et al., 1993], plasma [Adlercreutz et al., 1993], and feces [Adlercreutz et al., 1995 Kurzer et al., 1995]. HPLC with photodiode array (PDA) detection was introduced in 1994 to measure these analytes in human urine [Franke and Custer, 1994 Xu et al., 1994]. Compared to GC/MS, HPLC methods require fewer steps for sample preparation and analysis and demand less technician time and less expensive instrumentation. 

---