Chem 465 Organic Experiment 4

The Isolation and Chemistry of Hesperidin

The avonoid compounds can be regarded as C6-C3-C6 compounds, in which each C6 moiety is a benzene ring and the variation in the state of oxidation of the connecting C3 ring determines the properties and class of each compound. Flavonoids occur in all parts of plants, including the fruit, pollen, roots and heartwood. Numerous physiological activities have been attributed to them: thus, small quantities of avones may act as cardiac stimulants, others (eg hesperidin (I)), appear to strengthen weak capillary blood vessels, and highly hydroxylated avones act as antioxidants for fats. Flavonoid compounds and the related coumarins usually occur in plants as glycosides, in which one or more of the phenolic hydroxyl groups are combined with sugar residues. Acidic degradation of such a glycoside yields an aglycone, e.g. hesperetin (II), and the sugars. Hesperidin (I) was rst isolated in 1828 from the albedo (the spongy inner portion of the peel) of oranges, and has since been found in lemons and other citrus fruits. This experiment will explore the isolation of the natural product, and some of the differences in chemistry between the glycoside and the aglycone. Hesperetin will also be characterized by several spectroscopic methods.

2 Experimental 1. Hesperidin (I)

Chem 465 Organic

Hesperidin may be extracted from orange peel by the procedure below. It is important to have a good quantity of albedo and not just the outer orange coloured part of the peel. For this experiment, you are required to supply your own orange peels (try Orange Julius if necessary). Powdered dried3 orange peel (150 g) and petroleum ether (750 mL) are placed in a round bottomed ask and heated at reux for one hour. While hot, the mixture is ltered through a Buchner funnel. The powder is allowed to dry at room temperature and re-extracted with reuxing methanol (750 mL) for two hours. The hot solution is ltered, and concentration of the ltrate under reduced pressure provides a syrup. This syrup is crystallized by the procedure given below. Recrystallization of Hesperidin (I)1 The crude hesperidin is added to dimethylformamide (7 mL per g of syrup). The mixture is stirred vigorously at room temperature for 15 min, and then ltered to remove any insoluble material. To save some time and materials, dont try to recrystallize more than 7.5 g of the syrup. In a fumehood, the ltrate is added dropwise with stirring to a boiling solution of water (20 mL per g of hesperidin) and acetic acid (0.5 mL per g of hesperidin). Allow the mixture to cool. Where possible, longer term cooling of the mixture in the refrigerator may produce higher yields of product. The precipitated hesperidin is collected by suction ltration and washed with a little cold water. Obtain ir and uv spectra of hesperidin and carry out a ferric chloride test. Pure hesperidin has mp 252 - 254 oC. 2. Hesperetin (II)1 A mixture of hesperidin (0.7 g), methanol (25 mL) and concentrated sulfuric acid (0.4 mL) is stirred and heated at reux overnight. The resulting homogeneous solution is cooled, concentrated, and the residue is diluted with ethyl acetate (60 mL). The organic solution is washed with water (3 x 20 mL), and dried with magnesium sulfate. Keep the aqueous washings for an optional analysis of the sugar residues as described below. Concentrate the organic solution to obtain crude hesperetin as a yellow solid. Hesperetin is puried by the following procedure: Dissolve the crude product in minimum of acetone, and add the resulting solution to a vigorously stirred mixture of water (20 mL) and acetic acid (0.3 mL). Cool the mixture in an ice bath, collect the precipitated hesperetin and wash the crystals with water. Pure hesperetin has mp 220 - 221 oC. Analysis of the Products Carry out a ferric chloride test on both I and II. Explain the differences in terms of the structure of the two materials. Record the uv spectrum of both I and II in ethanol. It is not required to know the concentration. Compare and describe the behaviour of both I and II towards base by adding a small amount of solid sodium acetate to your uv sample, and recording the new spectrum. Consult the literature and predict the difference in the uv maxima before and after the addition of the base. Record the ir spectrum of hesperetin as a KBr disk. You may wish to record the ir spectrum of the glycoside as well.

Chem 465 Organic

Obtain a 1H nmr of hesperetin in acetone-d6 and pyridine-d5. Use approximately 20 mg of hesperetin in each sample. When ltering the sample into the nmr tube, lter through approximately 100 mg of anhydrous magnesium sulfate as well as the cotton. Explain the differences seen between the two solvents. Record a 13C nmr and a DEPT (Distortionless Enhancement by Polarization Transfer) spectrum of hesperetin in acetone-d6. This technique sorts the carbon signals based on the number of protons attached to the carbon giving the signal. In this spectrum, carbons with one or three protons attached (CH or CH3 groups) give positive signals, carbons with two protons attached (CH2) give negative signals, and carbons with no protons attached give no signal. References 1. 2. Seitz, C.T.; Wingard, R.E. J. Agric. Food Chem. 1978, 26, 278. A celite sandwich is prepared by pouring a slurry of celite (about 25 g in methanol) onto a piece of lter paper in a Buchner funnel and drawing the solvent through to the top of the celite pad. The sandwich is completed by topping with another piece of lter paper. An oven set at 50 oC is available for drying of the orange peel. Raadsveld, C.W.; Klomp, H. J. Chromatog. 1971, 57, 99. Personal communication, Garneau, F.X. Dept. Sc. Fondamenatales, Universit du Quebec Chicoutimi, July 1987. Horowitz, R.M.; Jurd, L. J. Org. Chem. 1961, 26, 2446.

3. 4. 5. 6.