Oil and Fatty Acids in Seed of Eggplant (Solanum melongena L.) and Some Related and Unrelated Solanum Species

Department of Agriculture, Agricultural Research Service, Plant Genetic Resources Unit, 1109 Experiment Street, Griffin, Georgia 30223, USA Department of Chemistry, Gordon College, 255 Grapevine Road, Wenham, MA 01984 USA Department of Agriculture, Agricultural Research Service, Southeast Watershed Research Laboratory, P.O. Box 748, Tifton, Georgia 31793 USA Department of Agriculture, Agricultural Research Service, Bio-Oils Research Unit, 1815 N. University St., Peoria, IL 61604 USA


Introduction
Eggplant arose in Africa and was subsequently dispersed throughout the Middle East and thence to Asia (Weese and Bohs, 2010). The cultivated eggplant (S. melongena) and several of its related taxa form what is sometimes referred to as the S. melongena complex. This complex is composed of the Asian S. melongena eggplant (groups E, F, G and H), the African S. incanum eggplant (groups A, B, C and D) and (more recently) S. linnaeanum. Numerous studies have characterized the genetic, morphological and breeding characteristics of the members of this complex and space here does not permit a summarization of these; however, readers are referred to the publications of (Daunay et al., 1991;1999;Doganlar et al., 2002;Kahn, 1979;Lester and Hasan, 1991;Lester and Niakan, 1986) and the references cited therein. Two additional species of interest includeSolanum aethiopicum (scarlet eggplant) and Solanum macrocarpon (Gboma eggplant). These species are cultivated in Africa and elsewhere, but are not generally believed to be closely related to S. melongena (Whalen, 1984).
Eggplant is an important vegetable in Asia, Africa, the Middle East and, to a lesser extent, the Americas. Approximately 2×10 3 Ha of eggplant were planted in 2012 in the USA as compared to 8.0×10 5 Ha in China and 1.85×10 6 Ha globally (FAO, 2015). World production of eggplant in 2013 yielded an estimated 48.4×10 6 metric tons of fruit, most of which was produced in China (58%) and India (25%). Other leading producers included Iran, Egypt and Turkey, while the largest exporters of the crop in 2013 were Spain, Jordon and Mexico. On the basis of tonnage produced, eggplant production ranks sixth after tomatoes, watermelons, onions, cabbages and cucumbers (FAO, 2015).
Historically, eggplant and Solanum spp. in general, have not been cultivated for their seed or seed oil. Neither has breeding or selection been practiced with an aim towards increasing their seed yield or characterizing or altering their seed oil yield or composition. The genus Solanum contains a large number of species, many of which produce edible fruit or plant parts (D'Arcy, 1986). However, the literature contains little or no information on the seed oil characteristics of most of these. New uses for vegetable seed oil have prompted studies on the yield, fatty acid composition and physicochemical characteristics of oil derived from seed of tomato (Solanum lycopersicon) and pepper (Giuffre et al., 2015;Jarret et al., 2013). Yields of tomato seed oil averaged 20-23% (Giuffre et al., 2015) whereas seed oil yields among Capsicum spp. ranged from 11 to 36% (Jarret et al., 2013). Dhellot et al. (2006;Nzikou et al., 2007) proposed the use of seed oil of S. nigrum as a means of diversifying sources of lipids eaten by the peoples of Africa. Thus, this study was undertaken to examine the seed oil content in a genebank's holdings of eggplant (S. melongena), several of its related species and also in a variety of miscellaneous Solanum spp. for which seed were available. Also examined was the fatty acid composition of a subset of these materials.

Plant Material
All seed used in this study were obtained from the USDA/ARS Plant Germplasm Collection in Griffin, GA (Jarret et al., 1990). A total of 386 genebank accessions were examined. Prior to analysis, all seed (stored at -20°C in foil pouches) were brought to room temperature for a minimum of 24 h. Species examined are presented in Table 1.

Preparation of Oil Standards
Oil standards were prepared from S. melongena cv. Black Beauty (Eden Bros., Asheville, NC) essentially as described by Jarret et al. (2013). Table 1. Seed oil content (mean +/-standard deviation) in eggplant (Solanum melongena) and other Solanum species in the S-9 genebank Oil content

TD-NMR Analysis
Seed oil and moisture measurements were carried out on intact seed by TD-NMR as described by Krygsman and Barrett (2004) and Jarret et al. (2013) on a Bruker (Madison, WI) mq10 Minispec. NMR analyses were conducted on seed drawn from single inventories (01) without replacement. All samples were measured in triplicate and the results were averaged.

Isolation and Analysis of Fatty Acids
For isolation of fatty acids, replicate 100 mg seed samples were ground to a fine powder with a mortar and pestle in liquid nitrogen. Approximately 50 mg of ground powder was transferred into a 16×100 mm test tube and 5.0 mL of n-heptane (Fisher Scientific) was added to extract the oil. For conversion of Fatty Acids to Methyl Esters (FAME), 500 µL of 0.5 M sodium methoxide (NaOCH 3 ) in methanol solution was added to the test tube and mixed with the sample. The reaction was allowed to proceed for 2 h. Seven mL of distilled water was then added to separate the organic layer from the aqueous layer and residue (45 min). An aliquot of the organic layer (1.5 mL) containing the methyl esters was transferred to a 2.0 mL autosampler vial for GC analysis. FAME extracts were diluted 100-fold in heptane containing 25 µg mL −1 methyl nonadecanoate (C19:0) and analyzed with a Thermo Quest Finnigan DSQII GC-MS system (Thermo Fisher, San Jose, CA, USA) using C19:0 as an internal standard (ISTD). The mass spectrometer was operated in the electron impact mode and scanned at m/z = 50 to 400 during data acquisitions. Chromatographic separations were on a 30 m DB5 ® column, 0.25 mm i.d., 0.25 µ film (Agilent, San Jose, CA, USA). Helium carrier gas flow was held constant at 1.5 mL min −1 and injection port temperature was 220°C. Injection was in the splitless mode. Oven initial temperature of 60°C was held 1 min after injection and increased to 250°C at 8°C/min and held for 5 min. Peak assignments and quantitation were based on analysis of serial dilutions of a commercially available FAME mixture. The FAME mixture (GLC-10) and ISTD were purchased from Matreya LLC (Pleasant Gap, PA, USA).

Determination of Physicochemical Properties
Physicochemical properties including viscosity, viscosity index, oxidative stability and Gardner color, were determined as described previously (Jarret et al., 2013).

Statistical Analysis
A Pearson's co-efficient analysis and an analysis of variance (ANOVA) were performed on the data. Means were separated using Tukey's Studentized Range (HSD) Test. General statistical data were generated and analyzed using Sigma Plot 13 and SAS.
Though not cultivated, data from Solanum species other than S. melongena and S. aetheopicum are presented for comparative purposes. Seed oil content and fatty acid composition data are available at www.ars-grin.gov.

Results and Discussion
Seed Oil Content in S. melongena and Eggplant-Related Spp.
Seed oil content in 305 genebank accessions of S. melongena ranged from 17.7 to 28% (mean = 23.7%, standard deviation = 2.1). Oil contents in seed of its related species S. incanum and S. linnaeanum were 22.3, 25.6 and 23.4%, respectively. Oil content in seed of the S. aethiopicum (scarlet eggplant), S. anguivi and S. macrocarpon were 25.3, 23.2 and 23.4%, respectively. We were unable to locate published values for seed oil content in these species and so a direct comparison cannot be made of values obtained in the present study, with previous studies. However, the average values for these species are quite close to those reported for tomato (20-23%) and pepper (ave. = 23.5%) (Giuffre et al., 2015;Jarret et al., 2013). In addition to being consumed as a vegetable (with the exception of S. linnaeanum), these species are reported to possess phytochemical and/or medicinal properties (Oyeyemi et al., 2015).

Seed Oil Content in Miscellaneous Solanum Spp.
It has been suggested that the genus Solanum contains approximately 1500 to 2000 species (Vander Burgt and van Medenbach de Rooy, 1996)-though the actual number is unknown. Limited research has been conducted on many of the non-cultivated species; yet, some data are available. Nolasco et al. (2001) reported a yield of 20.6% from seed of S. sisymbriifolium. The four genebank accessions of S. sisymbriifolium included in the present study averaged 29.3%. Dhellot et al. (2006;Nzikou et al., 2007) reported oil yields of between 34.5 to 37.5% and 37.1 to 38.8%, respectively, from seed of S. nigrum. (synonym-S. americanum Mill.). The six samples of S. amercanum available for analysis in the current study averaged 34% oil. Other reports in the literature include S. elaeagnifolium Cav (2.95% - Feki et al., 2013), S. ferox L.
(2.7%- Garg and Gupta, 1966), S. platanifolium Hook. (6.5% - Puri and Bhatnagar, 1975) and S. argentinum Bitter and Lillo (21. 1% -Lucini et al., 1994). Unfortunately, seed of these later species were not available for analysis. Zygadlo (1994) analyzed sterols in seed of 13 Solanum species-but oil yields were not determined. Our analysis of the oil content of the 25 remaining misc. Solanum spp. revealed maximum and minimum values of 27.5 and 42.8%, respectively and with a mean of 26.5% ±7.2. The highest oil content was found in seeds of S. aviculare (42.8%) and S. laciniatum (35.8%). Solanum aviculare is a soft-wooded Australian species sometimes referred to as poropora. It is a coastal lowland shrub bearing (toxic) green immature fruit and yellow to orange mature fruit. Solanum laciniatum (Tasmanian kangaroo apple) is a closely related species, also native to Australia and New Zealand. The lowest average oil content was found in S. capsicoides, a weed species native to eastern Brazil.
Fatty Acids in Seed of S. melongena and its Related Spp.
Data on the seed fatty acid composition of eggplantrelated species is lacking in the scientific literature. As noted previously, Grosso et al. (1991) examined the fatty acid composition of various Solanum species including the common cultivated eggplant. However, no eggplantrelated species were included in that study. They did however, note the uncommon occurrence of high (> 1.0%) levels of C14:0 in S. diflorum Vell. and S. elaeagnifolium Cav.Data on fatty acid composition for more distantly related species such as S. elaeagnifolium, S. argentinum, S. platanifolium, S. lycopersicon and others have been published (Puri and Bhatnagar, 1975;Grosso et al., 1991;Lucini et al., 1994;Lazos et al., 1998;Ceron et al., 2012;Feki et al., 2013). As Table 2 indicates, the fatty acid composition of the five eggplantrelated species examined does not differ significantly from that of S. melongena. In all instances, C18:2n-6 was the predominant fatty acid followed by C18:1n-9, C16:0 and C18:0. The high concentration of linoleic acid makes these oils susceptible to oxidation (Feki et al., 2013). Inadequate seed supplies prevented the analysis of the full range of species listed in Table 1.
We examined several physicochemical parameters of the seed oil of eggplant cv. Black Beauty-a widely planted and commercially significant cultivar. 'Black Beauty' seed oil had a pour point of -12°C, viscosities of 28.8 (40°C) and 7.3 (100°C), a viscosity index of 240, an oxidation onset temperature of 160°C and a Gardner Color of 6+. These may be compared to oil extracted from seed of pepper cv. California Wonder which had a pour point of -9°C, viscosities of 25.1 and 6.6 at 40 and 100°C, respectively, a viscosity index of 239 and a Gardner color of 6+ (Jarret et al., 2013).

Conclusion
Seed oil of eggplant varied from 17.2 to 28.0% among 305 genebank accessions, with a mean of 23.7%. Seed oil content in other Solanum species varied from 11.8% in S. capsicoides to 44.9% in S. aviculare. The predominant fatty acid in seed of eggplant and several of its related species was in the order of: Linoleic acid >oleic acid > palmitic acid. Levels of linoleic acid ranged from 57% in S. aethiopicum to 74.5% in S. anguivi. Oleic and palmitic acid levels ranged from 11.3% in S. anguivi to 25.2% in S. aethiopicum and from 8.4% in 11.2 in S. melongena to 11.2%, respectively. Oil extracted from seed of S. melongena cv. Black Beauty had a pour point of -12°C, viscosities of 28.8 (40°C) and 7.3 (100°C), a viscosity index of 240, an oxidation onset temperature of 160°C and a Gardner Color of 6+, similar to oil from tomato and pepper.