Antioxidant Properties of Pecan Shell Bioactive Components of Different Cultivars and Extraction Methods

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Nearly 300 million pounds of pecans valued at over 500 million dollars are produced annually, in the U.S. (NASS, 2018). The edible seed, or kernel of the pecan is highly desired for its nutritive and sensory properties. High consumption has been associated with reduced risk for cardiovascular disease, Alzheimer's, Parkinson's, and lower oxidative stress in cells.
The shell protective layer constitutes nearly 50% of the mass of a pecan nut.
Currently, uses for nut shell are limited, but include particle board fill, lost circulation material following oil drilling, and mulch for gardening (Worley, 1994). In many cases, the shell is a waste problem. Potential novel applications for pecan shells has emerged with rising demands for natural, or non-synthetic food products. Previous works have shown that pecan shell is a rich source of phenolic compounds. These compounds have antimicrobial and antioxidant properties that can be exploited for use in natural food products.  Consumer consciousness to potential health risks associated with synthetic chemical usage in the production or manufacturing of food products is a major market driving force in the United States. In a survey conducted by Nielsen (2015) 29% of U.S. respondents ranked "all natural" and "no artificial colors" as very important in food purchasing decisions.
Other popular consumer food trends include organic, minimally processed, and fresh. Value added products that provide health promoting benefits are also gaining popularity. In interferences, concentrate analyte to one phase, and provide analytical reproducibility independent of sample matrix variation (Smith, 2003). The selection of an extraction procedure is dependent on several factors such as the nature of the sample, characteristics of the target compounds, feasibility, and the overall purpose for extraction. Azmir et al.
(2013) discusses different methods used to extract bioactive compounds from plants.
Traditional methods are based on the extraction power of water or organic solvents, along with agitation, and heat to penetrate samples and bind to the analyte. The typical procedure to extract plant bioactive components involves: 1. Sample processing to dry and reduce particle size with grinding to increase surface area. 2. Treatment with various solvents which bind the analyte of choice 3. Centrifugation or filtration to remove insoluble materials. 4.
Concentrating the extracts by evaporating or lyophilizing the solvent. This step is required for isolated components to be later use in downstream applications.
The efficacy of traditional solvent extraction methods is dependent on several variables including the presence of interfering components, sample characteristics (i.e. particle size, stability, and chemical make-up), and the extraction parameters (i.e. time, temperature, pressure, agitation, and solvent choice)(Azwanida, 2015). The most critical factor to consider is the solvent polarity, which is generally described by the polarity index or dielectric constant (Snyder, 1974

Challenges with application of preservatives in food products
There are significant technical challenges involved with the application of any new food preservative. The compounds first must be isolated or concentrated and characterized. A delivery system must be developed. Then, the treatment dosage must be optimized. In any case, the food matrix under evaluation may react with the preservative and produce undesirable changes to appearance or flavor characteristics. The nature of a food, such as its pH, storage conditions or hydrophobicity may decrease the effectiveness of the preservative.
Potential adverse reactions are a major concern for any new concentrated bioactive component. Therefore, they must be cautiously evaluated for toxicity, prior to application on any product intended for use in humans. Lucera (Worley, 1994). Around November, full maturation has occurred and the fleshy hull splits. The developed nut is dropped from the tree and awaits harvesting. Following harvesting the pecan nuts are typically treated with a conditioning step with either chlorinated or boiling water. Post-harvest treatment steps loosen the shell layer from edible seed or kernel (NMSU, 2005).
Pecans are categorized as either native or improved varieties (cultivars). Native or "wild" pecans typically have thick shells and a low kernel percentage or shell out weight of around 30%. (Worley, 1994). These properties make them less desirable for commercial cultivation. Rosa, Alvarez-Parrilla, and Shahidi 2011 evaluated the effect of growing region.
Acetonic extracts (80% v/v) from nut shells and kernels were obtained from pecans cultivated in North, Central, and Southern regions of Chihuahua, Mexico and analyzed by colorimetric assays. Pecan shells from the southern region were significantly higher in percent yield, total phenolics, flavonoids, and condensed tannins. This geographical affect was not observed in analyses of kernels.

Knowledge gap
Shifts in social perceptions about the safety of synthetic products, has catapulted a new wave of plant bioactive research. Improved separatory and spectrometric techniques have aided in the discovery, and characterization of thousands of plant bioactive components.
Many of which have potential to be used as natural antimicrobials and antioxidants and nutraceuticals, among other things. There is an abundance of pecan characterization studies.
However, the kernel has overshadowed shell research. This is due to the kernel's edible nature, and status as a healthy food.   (Worley, 1994). As it stands, they provide very little to no revenue for pecan shellers and can be a significant disposal issue.
The natural foods sector has undergone significant growth over the past decade (Statista, 2019). This is partly due to consumers consciousness about potential health risks associated with synthetic ingredients. In response, demands are shifting away from food products preserved by conventional chemical or physical methods, in favor of "natural" or  3.75 ml aqueous methanol (80% V/V) in a 15 ml tube for 24h at room temperature. The resulting slurry was gently mixed and then slowly transferred using a graduated pipette into a 10 mm x 100 mm Omnifit EZ glass column until the resin bed was packed to the 10 cm line.
When the 10 cm line was reached, mobile phase (80% aqueous methanol) was added to cover the top of the resin bed and the column was sealed until further use. Crude Caddo ethanolic extract was weighed on analytical balance and placed in a micro-centrifuge tube.
The extract was diluted with 1 ml mobile phase (50 mg/ml) and vortexed to mix. After mixing, diluted extract was filtered through a 0.45 µm filter. The column bed was washed by passing 10 ml mobile phase at flow rate of 0.5 mL min -1 using a Bio-Rad Econo Gradient Pump prior to extract elucidation. Aliquots of 300 µL of diluted extract were gently loaded on top of the LH-20 column bed with a micro-pipette and eluted with 10 ml of 80% aqueous ethanol at 0.5 mL min -1 and five 2 ml fractions were collected in 15 ml centrifuge tubes using a Waters fraction collector (WFC 43030). The column was washed with 10 ml mobile phase after the 5 fractions were collected and before the next sample was injected. This process was repeated 2 times for a total of 3 injections. Similar extract fractions were mixed together and analyzed with a VWR UV-3100PC UV/VIS scanning spectrophotometer. Absorbance spectrums were collected from 800nm to 240nm at a scan rate of 5 nm/s. An absorbance spectrum signal from the mobile phase was collected as a reference blank and was subtracted from the signals collected for the 5 fractions. An absorbance spectrum of Gallic acid was collected and used as a free phenolic reference.

FIA-ESI-MS
Flow injection analysis mass spectrometry using an Advion expression L CMS mass spectrometer was performed on acid hydrolyzed Nacono ethanolic extracts to confirm potential compounds identified using RP-HPLC-DAD. A 5 µL volume of extract was manually injected and ionized with either electrospray ionization (ESI) with a typical fragmentation setting with acetonitrile (75% v/v) as a mobile phase. Positive and negative ions from 50-1200amu were recorded in the mass spectrums. Background noise was collected and subtracted from the total ion count chromatograms.

Statistical model
The effect of extraction method was evaluated under the assumptions that total phenolic content (TPC) or free-radical scavenging activity of aqueous and ethanolic pecan shell extracts from corresponding cultivars were equal (H0: µaqueous = µethanolic). The claim that either TP or DPPH of ethanolic and aqueous extracts from corresponding pecan cultivars were different was tested using a two-sided paired t-test (P≤0.05) on replication means (Ha: µaqueous ≠ µethanolic). This t-test is appropriate for our data set because it allows you to determine if a difference exists between two values that correspond to a common group. In      are breed to more resistant to environmental stresses and produce nuts with thin shell walls and kernels that are high and lipid and resist oxidation over long storage times (Worley, 1994 It is concluded that the phenolic and antioxidant properties of pecan shell components are dependent on numerous factors in combination. Pecans cultivated in Louisiana were found to be rich in antioxidant components. Ethanol was found to be better than distilled water as a solvent to extract phenolics from pecan shell. The antioxidant activity of extracts obtained through distilled water or ethanol extraction was highly dependent on cultivar.   The treatment of lignocellulose with dilute acid in a polar solvent cleaves ester and ether linkages to produce free monomeric phenols (Hagerman, 2002). Furthermore, cleaved ester and ether bonds can reassociation into more complex polymeric structures. These modifications limit the reproducibility of pecan shell characterization studies (Gosselink, 2011).
In the present study, Nacono and Caddo crude extracts were extracted with acidified methanol (1% HCl v/v) to free, polymeric or bound form phenolics. The soluble components were analyzed by RP-HPPLC-DAD with the same method used to analyze their crude constituents. Acid hydrolysis removed interfering components in the chromatograms, resulting in the detection of two prominent and fully resolved peaks for all extracts ( Figure   3.2). 2.6E+08 HNA = Acid hydrolyzed aqueous extracts from the Nacono cultivar HNE = Acid hydrolyzed ethanol extracts from the Nacono cultivar HCA = Acid hydrolyzed aqueous extracts from the Caddo cultivar HCE = Acid hydrolyzed ethanol extracts from the Caddo cultivar CNA = Crude aqueous extract from the Nacono cultivar CNE = Crude ethanol extract from the Nacono cultivar A peak at 4.9 min with a maximum absorption wavelength of 280nm was common in all extracts but was most abundant in aqueous extracts. This peak closely resembled gallic acid with Rt 5.0 min and max absorption at 272nm. The second major component eluted at Rt 6.3 with maximum absorption at 280 nm, which was not consistent with phenolic standards. It is hypothesized that this peak is a phenolic product derived from acidified methanol extraction. The quantification of these peaks was not attempted, however retention times, and relative abundance is reported in Table 3 In the present study, protonated and deprotonated ions produced using electrospray ionization with a typical fragmentation setting of acid hydrolyzed Nacono pecan shell extracts were monitored simultaneously with ion mode switching every second. Spectral data was digitally processed with Advion data express software. Background signal was subtracted from the peak ion chromatogram signal to improve the spectral resolution of the mass spectrums.  1.0E+06 0.5 5.1E+05 *n.d.=not determined was detected in low abundance (peak area 0.5 %) in the positive ion mode at m/z 593. Mass spectrums of deprotonated ions between 300-1200 m/z showed evidence of highly polymerized components. There was a low abundance of components greater than 500 u detected in the positive ion mode. Various phenylpropanoid derivatives were the main components in ethanolic pecan shell extracts.

Conclusions
Among 20 tested cultivars, shell extracts from Caddo provided highest levels of phenolics and antioxidant activity (Folin-Ciocalteu and DPPH). Extracts obtained by solidliquid extraction with ethanol were significantly higher in phenolics, compared to those obtained using distilled water; however, no significant difference was observed in Characterization of crude aqueous and ethanolic extracts of the Caddo and Nacono pecan cultivars was not achieved by reverse phase high performance liquid chromatography (RP-HPLC). Chromatograms of crude extracts resulted in a single broad-shouldered peak.
This was mostly attributed to interfering materials attributed to lignocellulosic and other glycoside bound components. Crude extracts were extracted with acidified methanol (1% HCL), which resulted in the removal of the interfering material and allowed for the elution of two components in either extract. The first and most abundant peak was attributed to gallic acid, while the other peak did not resemble phenolic standards.
Acid hydrolyzed Nacono ethanolic extracts were further analyzed by flow injection electrospray ionization mass spectrometry with detection in the positive and negative ion modes. The major components identified between 100-1200 m/z were lignin degradation products with varying degrees of polymerization. Monolignols corresponding to fragmentation of g-structure dilignols were numerous. The antioxidant activity of pecan shell extracts is attributed to a wide variety of bioactive compounds from the class of phenylpropanoids. The significance of these finding is the potential to create new revenue streams for shell by-product, thereby increasing the economic value of the Louisiana pecan crop. Figure S1. Absorbance spectrum (240 -350 nm) of Caddo ethanolic extract fractions 1-3 separated by lipophilic LH-20 Sephadex resin. Figure S2. Total ion chromatogram of positive and negative ions produced using electrospray ionization of Nacono ethanolic extract using a normal fragmentation setting.