Idenfication of Nutraceutical Phenolic Compounds in Bambara Groundnuts (Vigna subterranea L. Verdc) by HPLC-PDA-ESI-MS

Aim: The aim of the study was to explore the nutraceutical potential of bambara groundnuts (Vigna subterranea L. Verdc) commonly grown in Zambia based on the phenolic phytochemical profiles. Methodology: Two market classes of bambara groundnuts (red and brown) commonly grown in Zambia were screened in raw dry form. The study employed the High Performance Liquid Chromatography-Photo Diode Array-Electrospray Ionization-Mass Spectrometry (HPLC-PDA-ESIMS) to screen for phenolic phytochemical profiles of the 70% methanol extracts from bambara groundnuts Results: HPLC-PDA-ESI-MSbased identification revealed the presence of various phenolic compounds, mainly phenolic acids and flavonoids. In both the red and brown bambara groundnuts, the following phenolic compounds were tentatively identified: Quinic acid, (E) GC-hexoside, catechin glucoside, catechin, epicatechin, medioresinol, p-coumaric acid, salicylic acid, caffeic acid Original Research Article Nyau et al.; BJAST, 6(1): 77-85, 2015; Article no.BJAST.2015.068 78 derivative and catechin dimer. The red bambara groundnuts revealed the following phenolic compounds that were absent in the brown: myricetin hexoside, quercetin-3-O-rutinoside and quercetin-3-O-glucoside. Conclusion: The nuts contain various polyphenolic compounds, mainly from the class of flavonoids. It is suggested that consumption of bambara groundnuts could possibly offer some health benefits since they contain phytochemical constituents that have been reported to possess protective functions. These data indicate that bambara groundnuts studied have the potential for use as nutraceuticals.

Phenolic acids includes hydroxybenzoic acids (gallic and ellagic acids being major ones) and hydroxycinnamic acids (most common being coumaric, caffeic and ferulic acid). Added to this is quinic acid, a conjugate of caffeic acid and chlorogenic acid [6]. Tannins are polyphenolic compounds that constitute hydrolysable and nonhydrolysable tannins. They are complex polyphenols that can be degraded to sugars and phenolic acids by both enzymatic and nonenzymatic hydrolytic processes [7].
Plant-derived phenolic compounds are important as nutraceutical constituents in our diet. They have antioxidant properties and may protect against major clinical conditions such as heart disease and cancer in which reactive oxygen species (i.e., superoxide anion, hydroxyl radicals and peroxy radicals) are involved [8,9]. Plant phenolics represent one of the major groups of compounds acting as primary antioxidants or free radical terminators [10,11].
This study was undertaken to explore the nutraceutical phenolic profiles of the bambara groundnut (Vignea subterranean L. verdc) market classes grown in Zambia. Whilst the nutritional value of bambara groundnuts is well known, their potential as nutraceuticals has not been exploited. Bambara groundnuts remain uncharacterised in many aspects and have not been the subject of sustained research [12]. Since under-utilised crops are grown for subsistence and contribute to the food security of many of the world's poorest people, attempts to improve them rarely attract interest from international agencies or commercial sponsors [13]. Studies on the nutraceutical properties of bambara groundnuts are essential to establish their potential for use in the functional foods and nutraceutical industry. Additionally, this information is necessary for strategies aimed at promoting consumption of bambara groundnuts, which are slowly drifting into the category of "neglected species" or "forgotten crops" of Africa.

Sample Collection
Popularly cultivated market classes of bambara groundnuts (red and brown) were procured directly from the farmers in the Eastern region of Zambia immediately after harvest. The two market classes were botanically identified as Vigna subterranea L. Verdc by the National Plant Genetic Resources Centre Gene bank at Mount Makulu, Lusaka, Zambia. In order to make the samples representative, an attempt was made to collect the seeds of each market class from 15 farmers in the area with not less than 0.5 kg per farmer. Both market classes are landraces that have become well adapted to the local climate and soils, and indigenous knowledge about the germplasm is well preserved in the communities.

Preparation of the Extracts
Raw dry bambara groundnuts and common beans were ground into powder of the same consistency using a coffee grinder (Braun, Mexico). The 70% methanol extracts were obtained using Ultrasound-Assisted Extraction (UAE) from the seed flour [14]. Approximately 15 g of seed powder in 150 ml of 70% methanol was sonicated for 30 minutes at 25°C using the Eumax UD500SH 40 kHz ultrasonic bath. After extraction, the mixture was centrifuged at a speed of 10,000 rpm for 15 minute in Beckman Coulter JE centrifuge. The resulting supernatant first concentrated to 30 ml by evaporation under reduced pressure in a rotary evaporator (Buchi R-210 model, Switzerland) to remove methanol. The extract was then frozen at -80°C and freeze dried to obtain a powdered methanolic extract using the Telstar LyoQuest -85 freeze dryer. The freeze dried extracts were stored at -4°C until further analysis.

HPLC -DAD-ESI-MS Instrumentation and Chromatographic Conditions
The freeze dried 70% methanolic extracts of bambara groundnuts were analysed using a Waters ZMD 4000 system that was equipped with a Waters 2690 HPLC, Waters 996 photodiode array, ZMD mass spectrophotometer, 717 Plus autosampler, and a quaternary pump (Waters Corp, Milford, MA, USA). Separations were carried out on a 300x3.9 mm, 4 µm reversed phase Nova-Pak C18 (Waters) column that was maintained at 40°C. The photodiode array detector (PDA) was linked directly to a sprayer needle where ions were generated by electrospray ionisation (ESI) in a negative mode. The mobile phase A consisted of 5% (v/v) acetonitrile/water, containing 0.1% (v/v) formic acid and mobile phase B consisted of 100% acetonitrile containing 0.1% (v/v) formic acid. The sample was injected at a volume of 25 µl. The elution profile consisted of a stepwise linear gradient from 0% to 28% solvent B for 22 minutes with a flowrate of 0.3 ml/min. The PDA dectector was set to a scanning range of 200 to 700 nm and the UV-Vis absorption spectra were recorded online during the HPLC analysis. Phenolic acids and flavonols were detected at 280 and 360 nm respectively. Continuous mass spectra data were recorded on a full scan negative ionisation mode for a mass range of m/z 85 to 1000. The capillary voltage was set at 2.5 kV, the cone at 20 V and the extractor at 5 V. Nitrogen gas was used for nebulising and drying at different fragmentation voltages. Data acquisition was controlled using MassLynx 4.1 (Micromass, Waters Corp., Beverly, MA, USA).

Preparation of the Samples for HPLC -DAD-ESI-MS Analysis
Preparation of the test solution for HPLC-DAD-ESI-MS was done according to the procedure by Gülçin et al. [15] with slight modifications. One hundred mg of the freeze dried 70% methanolic extract was dissolved in 5 ml of ethanol-water (50:50 v/v). 100 µl of the prepared extract was transferred into a 5 ml volumetric flask and diluted to the volume with ethanol-water (50:50). From the final solution, an aliquot of 1.5 ml was transferred into a capped autosampler vial and 25 µl of the sample was injected into the HPLC-DAD-ESI-MS system. Identification of phenolic compounds was accomplished using UV spectra and ESI-MS spectral data and by comparison with published data reported in the literature. Authentic standards were also used where available by comparing their chromatograms with those of the samples. The available standards were t-ferrulic acid, gallic acid, salicylic acid, pcoumaric acid, epicatechin and catechin.

Phenolic Compounds Identified by HPLC-PDA-ESI-MS in Brown Bambara Groundnuts
Ten phenolic compound were identified based on co-chromatography with available authentic standards and mass spectra obtained in the negative mode by using their fragmentation pattern and data from published literature [16,17]. The gradient solvent system used for the analysis of phenolic compounds is summarised in Table 1. The chromatogram of the 70% methanol extract of brown bambara groundnuts is presented in Fig. 1 and the identified compounds are summarised in Table 2 [19]. The presence of flavanol compounds in free and conjugated forms has been reported as one of the principal phenolics in common legumes [20].
The red bambara groundnuts contained the following phenolic compounds that were not detected in the brown market class:  [23]. A compound with similar fragmentation pattern has been reported previously in strawberries [24]. The majority of the phenolic compounds identified in bambara groundnuts were flavonoids. Various health benefits have been described previously for flavonoids. An inverse correlation between flavonoid intake and total plasma cholesterol concentrations has been shown [25]. Flavonoid intake has also been reported to have a protective effect against coronary heart disease [26,27]. Cocoa beans have historically been used as a treatment for diarrhoea [28]. The nature of the active ingredient, nor the exact mechanism of action was not known but the recent research attributes the antidiarrhoea effect to the flavonoids present in cocoa [28]. Flavonoids like quercetin have been shown to have anti-inflammatory properties and do so by inhibiting the cyclooxygenase pathway [29]. Quercetin has also been shown to inhibit the growth of helicobacter pylori bacteria in in-vitro studies [27]. Methyl-3-(+)-catechin interferes with the formation of histamine in gastric mucosa and hence produces the protective effect [30]. Most flavonoids have antiviral effects against Herpes simplex virus, respiratory syncytial virus, parainfluenza virus, and adenovirus [29]. Flavonoids have also been shown to have free radical scavenging properties [31]. In a summary, one may speculate that bambara groundnuts may be useful as nutraceuticals due to various phenolic compounds from the flavonoids class that they contain.

CONCLUSION
The study has shown that bambara groundnuts contain various polyphenolic compounds, mainly from the class of flavonoids. Based on this, it is suggested that consumption of bambara groundnuts could possibly offer some health benefits since they contain phytochemical constituents that have been reported to possess protective functions. These findings have demonstrated that bambara groundnuts have the potential for use in the nutraceutical industry. Consumption of bambara groundnuts can be as good as other commonly consumed legumes and this is an opportunity for dietary and crop diversification on the part of the consumers and farmers respectively.