## 2 Extraction of Peanut Skins
### Compound Identification
Although different flavonoids and their glycosides were found to be unique to the different colored peanut skins, they were found to have the same biosynthetic pathways for anthocyanins but with different modifications. Individual flavonoids were isolated and identified from the skins of black seed-coated peanuts after extraction with acidified water followed by a partition into ethyl acetate [29]. The extracts were fractionated using Amberlite XAD-7 HP resin to remove the most polar compounds followed by YMC(r) Gel ODS-AQHG resin to separate the hydrophilic ones and identified using High-Pressure Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (HPLC-ESI-MS/MS) and NMR spectroscopy. Three unique flavonoids (quercetin-methylpentoside, quercetin-feruloyl-hexoside, quercetin-3-dihexoside) and four anthocyanin (cyanidin-3-o-sphoroside, cyanidin-3-o-sambubiside, cyanidin-3-o-glucosyltrutinoside, cyanidin-3-o-xylovtioside) were identified. Using HPLC to determine the various compounds that compose peanut skins has advanced from initial reports of the oligomers [31]. Aqueous ethanol was the extractant for the analysis of peanut skins, and the method identified five phenolic acids (gallic acid, caffeic acid, p-coumaric acid, protocatechuic acid, ellagic acid), two stilbenes (piceid, resveratrol), and eight flavonoids (catechin, epicatechin, epigallocatechin, catechin gallate, epicatechin gallate, epigallocatechin gallate, proyanidin B2, quercetin). This report validated the methodology for the quantitation using the runner, Spanish, and Virginia market types. Concentrating specifically on peanut skins from the Virginia market type, a study compared the compounds extracted when either methanol, ethanol, acetone, or water at boiling temperature was used individually [32]. Using 100% acetone resulted in the highest amount of the smallest phenolic compounds. The study used HPLC-MS(r) to identify a range of compounds that were polymers of catechin and epicatechin with and without sugar moieties up to 9 catechins. These polymers were found mostly in the A form. An advanced study to determine the content and structural formation of the trimer and tetramer of the procyanidins used extraction with 30% methanol in water followed by 70% acetone in water [24]. The extracts were combined, and the solvent was evaporated to dryness. The dried material was dissolved in water and partitioned against ethyl acetate containing increasing amounts of aqueous ethanol (5%, 10%, 15%). The extracts were then loaded onto silica gel and rinsed with a series of solvents to purify and isolate the components. Identification was performed using NMR and electron circular dichroism spectroscopy (CD). The different polymers were tested for anti-inflammatory activity in a macrophage system and the tetramers were found to be the most effective with the dimers having little or no effect. This showed that biological systems could differentiate between the different forms of the procyanidins. As studies became more sophisticated to examine the composition of peanut skins in order to determine the source of bioactivity, multistep extraction schemes were used to selectively isolate certain compounds. A series of methanol and water mixtures was used to remove specifically the A-type procyanidins [33]. Drawing on previous research, the extraction scheme was created [18; 20; 21]. Peanut skins were extracted sequentially with 20% aqueous methanol, 70% aqueous methanol, and then finally 70% aqueous acetone. Each fraction was concentrated and then partitioned into ethyl acetate to remove soluble saccharides. The fractions were then analyzed by HPLC-MS, which revealed the 20% methanol fraction contained mostly oligomers of the A-type procyanidins and the 70% acetone fraction contained larger polymers. The use of peanut skins as a specific source of compounds for isolation has been described. Peanut skin has been a source of resveratrol and was extracted using 20% ethanol in water [34].