Proanthocyanidins: Extraction, Purification, and Determination of Subunit Composition by HPLC

Abstract

Proanthocyanidins are polymeric flavonoid compounds composed of flavan-3-ol subunits (UNIT I1.3), and are responsible for bitterness and astringency in some foods and beverages. This unit describes methods for extracting and purifying proanthocyanidins, and for determining their subunit composition by HPLC. Based upon HPLC results, the average degree of polymerization and the conversion yield for purified proanthocyanidins can be determined. This unit is composed of three separate procedures describing proanthocyanidin extraction from plant tissue (see Basic Protocol 1), purification (see Basic Protocol 2), and subsequent analysis by reversed-phase HPLC (see Basic Protocol 3). These protocols have been developed and used for analysis of grape skins, grape berries, and grape seeds. Without modification, wine, apples, and pears have also been analyzed using these procedures. BASIC PROTOCOL 1 EXTRACTION OF PROANTHOCYANIDINS This extraction method utilizes an aqueous acetone system to extract proanthocyanidins from whole plant tissue. Acetone is preferred over other extraction solvent systems (most notably methanol) because of its ability to solubilize proanthocyanidin-containing material that is insoluble in methanol. Materials Plant material containing proanthocyanidins 66% (v/v) aqueous acetone, HPLC grade Nitrogen gas Suitably sized Erlenmeyer flask with stopper or septum Aluminum foil Platform shaker Büchner funnel Whatman no. 1 filter paper Suitably sized round-bottom flask Rotary evaporator with vacuum pump or water aspirator, 40°C 1. Accurately weigh plant material (within ±0.1%) and place into an Erlenmeyer flask. Record the weight of the sample. As a general guideline, and based upon grape tissue, 5 to 10 g of plant tissue is placed into a 250-ml Erlenmeyer flask with 100 ml of extraction solvent. To ensure adequate mixing, Erlenmeyer flasks should not be filled to more than 80% of capacity (i.e., 200 ml in a 250-ml Erlenmeyer flask). An approximate yield for this step is 1 to 2 mg of proanthocyanidin extraction per gram of grape berry weight. 2. Add enough 66% (v/v) aqueous acetone to the Erlenmeyer flask to cover the plant tissue and record the volume to within ±0.5%. 3. Equip the Erlenmeyer flask with a stopper or septum and sparge the extraction system well with nitrogen. 4. Cover the flask with aluminum foil and shake 24 hr at room temperature on a platform shaker.

Relationship between the Sensory-Determined Astringency and the Flavanolic Composition of Red Wines

“…Acid-catalyzed depolymerization in the presence of Ph provided valuable information on PA subunit composition, aDP, average molecular weight (aMW), and total PA concentration of these wines ( Table 2). The aDP was calculated, as described by Kennedy; 26 briefly, it is determined by dividing the sum of all PAs subunits (flavan-3-olmonomers and flavanol-3-ol Ph adducts) to the sum of terminal units (flavan-3-ol monomers), in mole equivalents relative to catechin. The concentrations of flavan-3-ol monomers in wine before the phloroglucinolysis reaction were subtracted from the corresponding concentrations of flavan-3-ols after reaction to determine the terminal subunits.…”

Phenolic Fractions from Vaccinium vitis-idaea L. and Their Antioxidant and Anticancer Activities Assessment

“…The abundance of them in herbal raw materials is highly associated with health-protecting properties [ 42 ]. The last fractionation step with the 70% acetone as the most effective purification solvent—strong hydrogen bond breaker according to Kennedy—yielded bounded fractions of lingonberry leaves and fruits (L4 and F4, respectively) enriched with proanthocyanidins [ 43 ]. Proanthocyanidins from lingonberry have been reported to have very high antioxidant or anticancer activity in several studies [ 15 , 44 , 45 ].…”

Pilot Study of the Tart Cherry Juice for the Treatment of Insomnia and Investigation of Mechanisms

“…The method of Kennedy 25 was used to isolate and extract procyanidin-B2 from cherry juice. The method of Kim and Lee 26 was used to determine the levels of procyanidin B-2 in cherry juice.…”