Submitted to: Journal of the American Society for Horticultural Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 13, 1999
Publication Date: N/A
Interpretive Summary: Anthocyanins are naturally occurring compounds that impart most of the red, crimson and blue colors to various plant parts. In addition to their pigment properties, anthocyanins are known to be antioxidants. Antioxidants function within human and animal tissues to protect cells from the damaging effects of certain cancer-causing agents called free radicals. We conducted dan experiment to elucidate the anthocyanins in bean (Phaseolus vulgaris L. seed coats so we could further determine their potential as antioxidants. However, anthocyanins isolated and purified using standard methods are unstable and degrade at a predictable rate. Since these standard methods can take weeks for enough material to be obtained, degradation was occurring during the purification process, making it impossible to obtain pure compounds for use in an antioxidant assay. A method was developed that used two types of a procedure called chromatography for isolation and separation of anthocyanins. Initially, crude seed coat extracts containing anthocyanins were subjected to low pressure conditions and then further purified by a procedure called semi-preparative high pressure liquid chromatography. The coupling of these two chromatography systems allowed for rapid isolation of pure anthocyanin. With the new separation procedure scientists can now study the antioxidant potential of anthocyanins without worrying about degradation due to long isolation procedures. A knowledge of the antioxidant potential of beans provides a sound basis for the food advertising industry to promote the health benefits from eating beans. This puts bean growers in a good position to increase their profits through increased production necessary to meet a strong consumer demand.
Technical Abstract: A rapid high performance low pressure liquid chromatography (HPLPLC) method for the isolation of anthocyanins from black beans (Phaseolus vulgaris L.) was developed. A modified Waters 510 isocratic pump was coupled with a large C18 column and Waters 486 UV detector. The anthocyanins delphinidin 3-O-glucoside, petunidin 3-O-glycoside and malvidin 3-O-glucoside were recovered at a purity of 84-94% after separation on the HPLPLC C18 column. Amounts for the three semi-pure compounds obtained were 123.8, 26.5, and 18.4 mg respectively from 929.0 mg of crude acidic methanol seed coat extract. Further purification was achieved on a semi-preparative C18 column coupled to a Waters HPLC system. Structural determinations for these three compounds were based on UV-vis, HNMR spectra and retention times compared to published values. Using the purified anthocyanins as standards the concentrations of these compounds in Florida dry bean breeding line 5-593 were determined to be 477.53 +/- 30.98, 69.53 +/- 4.24, and 31.71 +/- 4.35 mg/100g fresh weight. The coupling of HPLPLC with HPLC allows for rapid purification of anthocyanins in quantities sufficient for NMR experiments and biological activity studies.