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The quantification of oligomeric proanthocyanidins in the inner and outer barks of selected B.C. conifers Seewald, Eric

Abstract

Oligomeric proanthocyanidins form the basis of neutraceutical anti-oxidant products sold under trade names such as Pycnognol™ and OPC's. These compounds fall in the class of compounds called condensed tannins and are essentially the hot water extracts of certain grape seed or pine species. The oligomeric proanthocyanidin fraction consists mostly of procyanidins, but includes a propelargonidins and prodelphinidins, as well. These compounds may form separate polymers, may be combined in a variety of conformations, and may be gallated or glycosylated. Although these compounds have been found to have many functions in plants, it is the applications in human medicine that has made them particularly interesting. Oligomeric proanthocyanidins have been shown to be antioxidant, free radical scavenging, antiinflammatory, and antihistamine. They have been studied for their prevention of cardiovascular diseases, tumors, and virus induced conditions, such as HIV. Traditionally, these compounds have been used by indigenous peoples almost exclusively for gastrointestinal disorders. Much of the research conducted into the physical properties and biological activity of this class of compounds has excluded reporting the quantity of the compounds extracted from the various tree and grape seed sources. This is largely because standardized quantification is not yet possible. Because proanthocyanidin biosynthetic pathway enzymology is not yet complete, the nomenclature for these compounds has not been definitively established. Compounds require identification before meaningful quantification can be made. However, as research in this field is still ongoing, quantification based on what is already known can be made on the medically active components of proanthocyanidins. The butanol-HCl (anthocyanidin), vanillic acid, and protein precipitation methods have been devised as methods for the quantification of tannins. However, all of these methods have deficiencies that make them unsuitable for the quantification of proanthocyanidin oligomers with confidence. The butanol-HCL method quantifies coloured proanthocyanidin hydrolysates relative to polymer sorghum or quebracho tannin. Different types of proanthocyanidins yield different colours and different colour intensities, and the colour intensity decreases in strength with the decreasing size of the oligomer. The vanillin acid method, which quantifies the coloured proanthocyanidin - vanillin adduct, actually measures the number of phloroglucinol-like A-rings associated with flavonoids. This reaction is quantified in catechin equivalents, and is sensitive to reaction time, temperature, light, age of the sample and even the water content in the reaction mixture, making this method very undesirable for testing various natural extracts. The protein precipitation method is unable to distinguish between condensed and hydrolyzable tannins. The method described herein is designed to quantify oligomeric proanthocyanidins by determining the occurrence of 10 signature procyanidins [OPC], identifying seven of the ten signature procyanins, and cumulatively measuring the concentration of the 10 signature peaks relative to a commercial oligomeric proanthocyanidin product, Indena brand LeucoSelect™. Because the quantification is made relative to an already commercialized product, the quantification results predict the amount of salable product in natural product extract. This method is reproducible, rapid, efficient, and can be used to screen for potential new sources of OPC-based products, or to compare the oligomeric proanthocyanidin content in various commercial products. When used to quantify the OPC content in bark extracts, this method was found to provide results similar to those in the literature. By using this method and screening various bark extracts, it was found that sample freshness played a role in the extractable amounts of proanthocyanidins. In future studies, fresh samples should be used as they most accurately represent the bark composition on live trees. The inner and outer bark quantities of proanthocyanidins were also seen to vary, with the inner bark quantity almost always being greater in concentration than that in the outer bark. Douglas-fir and interior Amabilis fir were found to be the best sources of oligomeric proanthocyanidins at 43.2 and 12.4 mg/g dry inner bark, respectively. The samples of lower mainland Amabilis fir, interior Lodgepole pine, and Caribbean pine from Venezuela, all showed signs of being potential sources of OPC products. Interior and lower mainland Sitka spruce and interior Western hemlock showed little potential as possible sources of OPC's. By comparing the commercially produced OPC containing products relative to the Indena brand LeucoSelect™ (made from grape seeds) the Capers brand grape seed extract was shown to be 77% similar to the Indena standard. Nu-Greens Prolong, which differed substantially, was shown to be only 7.2% similar to the Indena standard, raising questions about its quantification in units of 'Activity'. The SunForce Pycnogenol™ was found to be only 46.8 % similar, but is sourced from pine bark, which would seem to have a higher concentration of higher molecular weight OPC's, not included in the 10 signature peaks.

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