Effect of Different Solvents on the Extraction of Phytochemicals in Colored Potatoes

Colored potatoes are an interesting 
alternative to the traditional, white-creamed potatoes, 
due to their high phytochemical content, which have been proved to have 
antioxidative properties. The extraction of polyphenols is highly dependent on 
using the appropriate solvent systems. Therefore, polyphenol extraction in 
colored potatoes [Vermillion Fingerling (VF); Jester Potato (JP); Magic Molly 
(MM); Blue Belle (BB); All Blue (AB)] using different solvents [Solvent A (24% 
water, 67% ethanol 9% acetic acid); Solvent B (5% acetic acid); and Solvent C 
(95% water, 5% acetic acid, 0.5 g sodium bisulfate)] may have significant 
effects on extraction efficiency and phytochemical content. Total phenolic 
content (TPC), Total flavonoid content (TFC), Total anthocyanin content (TAC) 
and antioxidant activities [Trolox Equivalent Antioxidant Capacity (TEAC) and 
Ferric reducing-antioxidant power (FRAP)] were determined. Solvent A extracted significantly (p mg GAE/100g) compared to 
the other solvents. VF and JP displayed higher TFC (14.53 and 19.46 mg CE/100g, 
respectively) when Solvent C was utilized. VF and JP extracts with Solvent C 
displayed the highest FRAP value compared to Solvent B. MM displayed the 
highest TAC using Solvent A and C. Potato variety resulted in high 
variability in polyphenols content and antioxidant activity. The utilization of colored potatoes or extracts in the food industry could provide innovative 
products and a functional alternative to the traditional.


Introduction
Solanum tuberosum, potato, is categorized as a tuberous starchy vegetable from the nightshade, Solanaceae family [1]. Potato has become one of the predominant staple foods in the world [2]. Potatoes contain several polyphenols such as phenolics (chlorogenic acid derivatives), flavonoids, (anthocyanins) and carotenoids [3]. Cinnamic acid and its derivatives, more specifically chlorogenic acid, are the major phenolic acids found in potatoes [4]. Chlorogenic acid can range from 50% to 95% of the total phenolic acid content of potatoes [4]. Studies have shown that chlorogenic acid inhibits nitrosamine formation in A549 human lung cancer cells [5] [6].
Colored potatoes are now the rave among consumers. These potato varieties have gained notoriety because they are different, and an interesting choice compared to the traditional white-cream/fleshed potatoes. The unusual colors of these potatoes lend to their high polyphenol content. These vibrant (red, purple, and blue) colored potatoes are noted for their high anthocyanins contents [4].
Research has shown that anthocyanins can protect against free radicals, lower LDL cholesterol thus reducing the risks of heart disease. Anthocyanins also have anti-inflammation and cancer properties [7] [8] [9]. The colors of these potatoes are attributed to the acylated glucosides of pelargonidin, malvidin, petunidin, peonidin, and delphinidin [10]. Predominantly, red-fleshed potato contains high amounts of pelargonidin and those with blue and purple flesh contain acylated glucosides, petunidin and peonidin with small amounts of delphinidin and malvidin [11]. According to Lachman [7], petunidin has potent antioxidant potential due to the number of free hydroxyl groups when compared to pelargonidin, peonidin, or malvidin. Other types of flavonoids include catechin, epicatechin, erodictyol, kaempeferol, quercetin, naringenin, and rutin [4]. According to Moser et al. [12], colored potatoes can provide up to 20 mg of phenolics per serving, thus making them one of the prominent sources of polyphenols in the diet.
Polyphenols from colored potatoes are of major interest due to their antioxidant properties and potential applications in food technology to enhance functionality [13], i.e., for preventing oxidation in certain foods [14] [15]. A major step in retrieving these compounds is through extraction. Several solvents have been used to extract phytochemicals from plants including methanol, water, acetone, ethanol, hexane, and chloroform [16]. To ensure convenient separation of phytochemicals from food and plant matrices, the uses of single solvents (ethanol, methanol, acetone, diethyl ether, etc.) and binary systems or hydro-organic solvents (such as methanol: water or ethanol: water) are usually required [15] [17] [18]. Concerning their structure, it is widely indicated that phenolic compounds create polymerized derivatives of various dissolubility, hence impacting their solubility in solvents of different polarity [14] [15] [17] [18]. Thus, it is crucial to select the best solvent and extraction systems that allow optimum extraction or yield of antioxidant components and other poly- Blue (AB). The study will screen the optimum solvent system for extracting phytochemicals from the colored potato cultivars.

Preparation of Samples for SPE (Solid-Phase Extraction)
Sep Pak Vac 6cc C18 cartridge was conditioned with ethanol followed by water, both at pH 7 before phenolics acids fractionation. Following conditioning step, 2 mL of previously prepared SLE extracts were passed through the column and washed with 5 mL of water (pH 7). Afterwards, 20 mL 20% (v/v) ethanol/water was used to elute polyphenols. The extracts obtained was stored at −80˚C and later used for the analysis.

Determination of Polyphenols Content and Antioxidants
Total Phenolics Content (TPC) of Colored Potatoes: The TPC of potato extracts was determined by a modification of the Folin-Ciocalteu (FC) colorimetric method [21]. The sample was incubated for 90 min then absorbance was taken at 750 nm and compared to a gallic acid standard curve. The results were expressed as mean (mg gallic acid equivalents/100g potato).
Total Flavonoids Content (TFC): The TFC of colored potatoes were determined using a modified aluminum colorimetric method [21]. The sample was mixed with distilled water in a 96 well plate followed by 5% sodium nitrate solution. After 5-minute incubation, 10% aluminum chloride solution was added and incubated for another 5 minutes before 1 M NaOH was added. The mixture was adjusted with distilled water and the absorbance was measured at 510 nm. Results were expressed as mg CAE/ 100g of potato.
Total Anthocyanins Contents (TAC): The TAC was determined using a pH differential method [22]. Potassium chloride buffer (0.025 M, pH = 1.0) and sodium acetate buffer (0.04 M; pH 4.5) were the two buffer systems utilized. In a 96 well microplate, 10 μL of the extract was added to 290 μL each of buffer 1 (pH 1.0) and 2 (pH 4.5). The testing solution was equilibrated for 30 min. The absorbance was measured at 520 nm and 700 nm. Total anthocyanin content was calculated according to the following formula: Molecular Weight (MW) = 449.2 g/mol cyanidin-3-glucoside; Dilution Factor (DF); molar extinction coefficient (ε) = 26,900 L·mol -1 ·cm -1 . Monomeric anthocyanin results are expressed as cyanidin-3-glucoside equivalents in mg/g. Ferric Reducing Antioxidant Power (FRAP): The FRAP assay was determined following the modified methods by Gajula et al. [21]. Potato extracts were added to FRAP reagent (300 mM acetate buffer pH 3.6, 10 mM 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) in 40 mM HCL and 20 mM ferric chloride (FeCl 3 ·6H 2 O). Samples were incubated for 10 minutes under restricted light at 37˚C, then absorbance was read at 590 nm. The absorbance was compared to ferrous sulfate standard (0.1 mM1 -0 mM). Trolox Equivalent Antioxidant Capacity (TEAC): The TEAC was determined according to the protocol by Rice-Evans, Miller (1994). The ABTS solution was diluted with deionized water. Potato extracts were added to ABTS solution and the absorbance reading was carried out for 6 minutes are 1-minute intervals. Absorbance was read at 734 nm.
Statistical Analysis: Results from this study were expressed as means (±SEM) for three replications. Data was evaluated using the SAS statistical software and comparison was performed using a one-way analysis of variance (ANOVA) followed by Tukey's test. A p-value of ≤0.05 was considered statistically significant.

Effect of Solvent Systems on Polyphenolic Content of Color Potatoes
The total phenolics content (TPC) and total flavonoids content (TFC) of colored potatoes (VF, JP, MM, BB, and AB) are shown in Table 1 The results of TAC using the pH differential method are shown in Figure 2. MM displayed the highest (p < 0.05) TAC (70.22 -152.95 mg C3G/g) with all three solvents followed by AB > VF > JP. > BB. Based on the results, the extraction efficiency of TAC was influenced by extraction solvent properties. The best solvent systems were found to be solvents A and C, while Solvent B had the lowest (p < 0.05) extraction yield in MM (70.22 mg C3G/g), VF (24.08 mg C3G/g) and JP (7.26 mg C3G/g). There was no significant difference in the extraction efficiency between Solvent A and C (6.94 mg C3G/g) in AB and MM. Solvent C had the highest TAC in JP (22.20 mg C3G/g) and VF (55.96 mg C3G/g) compared to other solvents. TAC in MM was increased (p < 0.05) when compared to VF, JP, and AB by 59% -91%; 49% -89% and 51% -86% with solvents A, B and   C, respectively. As expected, TAC was barely detected (Solvent A) or not detected (Solvents B and C) in BB due to the color of this variety.

Correlation between Functional Components and Antioxidant Activity
A positive correlation (p ≤ 0.05) was noted between functional components and antioxidant activity of colored potatoes Table 3

Discussion
The aim of the current study was to evaluate the efficiency of acidified solvents on the extraction of polyphenolic compounds in colored potatoes. The solubility of polyphenols is reliant on the polar properties of the extracting solvents [23].
Research suggests that the best solvent system for the extraction of phytochemicals is hydroalcoholic mixtures such as water and ethanol [24]. Although the solvent polarities are a critical aspect, it is also crucial to evaluate the extraction efficiency of low toxic solvents, such as ethanol and acetic acid, for possible industrial applications, especially as they relate to human consumption acceptability [25]. It has also been suggested that the addition of acids, like acetic acid to hydroalcoholic solution creates an acidified environment, which leads to better extraction efficiency of antioxidant compounds such as polyphenols [25]. For these reasons, this study evaluated the proficiency of three extracting solvents: Solvent A (24% water, 67% ethanol 9% acetic acid); Solvent B (5% acetic acid in 95% water); and Solvent C (95% water, 5% acetic acid, 0.5 g sodium bisulfate).
Based on present observation, the solvent efficiency varied based on the potato variety. For example, Solvent C was more efficient when extracting anthocyanins and phenolics from all the potato varieties, albeit MM for anthocyanins and, BB and AB for phenolics, respectively. According to Hosseini [26], water, due to its dipole momentum of 1.95, is more efficient when extracting polyphenols than ethanol. Furthermore, studies have suggested that organic acids, such as acetic acid, disrupt the cell membranes allowing for faster polyphenol extraction, especially as it relates to anthocyanin extraction [18] [26] [27]. Partial hydrolysis of polyphenols from insoluble complexation with polysaccharides is said to be one mechanism by which acidified aqueous alcohols improve extraction efficiency [18] [26]. Research conducted by Sipahli et al. [28] suggests that pigment retention was more stable in an acetic acid solvent system than citric and formic acid. Aliphatic or aromatic acids such as acetic acid can be used in the chemical acylation of anthocyanins, thus leading to an increased in stability when compared to unacetylated anthocyanins [29]. This could be a major benefit to the food industry by not only addressing extraction efficiency but also the stability of polyphenol compounds. Issues regarding the stability of natural colorant such anthocyanins have been a major problem for the food industry, considering that anthocyanins are highly valuable compounds. A study by Złotek et al. [23] indicated addition of acetic acid to aqueous acetone mixture intensified the extraction of phenolic compounds from basil leaves. Furthermore, results from Imen et al. [30] suggested that ethanolic solutions yielded the highest TPC in horseradish. Due to increased consumption, it can be supposed that most of the daily polyphenol consumption comes from potatoes than from fruits and vegetables [31]. Colored potatoes have gained popularity because of their color and rich phytochemical contents when compared to traditional white-cream colored potatoes [32] [33]. The levels of polyphenols in purple-blue potatoes are two to three times higher than in yellow-fleshed potatoes [33] [34]. The results of this study suggested that darker colored potatoes (MM and AB potatoes) displayed the highest polyphenolic content as well as antioxidant activity when compared to the mixed (JP) and the yellow (BB) colored potatoes. Contrary to the results, Madiwale [31] observed that the mixed colored variety had the highest polyphenolic content and antioxidant activity when compared to purple, red and yellow-colored potatoes. Furthermore, Kalita [35], showed that mixed colored potatoes had the higher phenolic content than purple tubers and red tubers. The results of this study indicated that yellow potatoes have lower polyphenol content than colored potatoes. Similarly, Lee et al. [36] reported that yellow potatoes (Seohong and Jaseo) had significantly higher polyphenol content when compared to white potatoes (Superior). Brown [37] reported that yellow and orange flesh potatoes contain 5 -16-fold (800 -2000 µg/100 g FW) more phytochemicals than that observed in white potatoes (50 -350 µg/100g FW) [37]. The current study showed that MM potatoes, with its deep purple flesh and skin had more polyphenols compared to the other varieties. Similar results were reported in studies conducted by Madiwale [31], where Purple Majesty and purple-fleshed clones displayed significantly (p < 0.05) higher total phenolic and anthocyanin content than Atlantic (white), Yukon Gold (yellow), and two yellow clones.
Both FRAP and TEAC assays are used to determine the antioxidant capacity in several food products [38]. The antioxidant activities in potatoes utilized in this study were varied, as observed with the polyphenolic content. Purple (MM), blue (AB), and red-colored (VF) potatoes displayed higher antioxidant activity than yellow (BB) and mixed colored (JP) potatoes. Similar results were reported by Bellumori [39], who observed that blue-violet potato showed a higher antioxidant capacity than the yellow-colored varieties. Based on the results of the current study, colored potatoes are a good source of phytonutrients that can participate as an antioxidant via a single electron transfer mechanism (SET) [40]. Polyphenols including certain flavonoids and anthocyanins protect against blood clotting; act as chemical messengers, and play a significant role in protecting against vascular dementia and Alzheimer's disease [41]. It is hypothesized that polyphenols and anthocyanin compounds in colored potatoes could decrease inflammation and oxidative stress via mechanisms such as antioxidant, antimutagenic, and antimicrobial activities thus, potentially leading to reduced risks of heart disease, hypertension, diabetes, and cancer [39].

Conclusion
The results indicated that solvent type and potato variety resulted in high variability in polyphenols content and antioxidant activities. The present study showed that Solvent C was the best extracting solvent for TPC TFC and TAC in selected potatoes. Solvent C was composed of water, acetic acid and sodium bisulfate all of which are utilized in the food industry for polyphenols extractions. Sodium bisulfate is a chemical additive used as a leaven agent and pH lowering agent used in several food applications without adding the sour taste. Meanwhile acetic acid is a weak organic acid used as a natural preservative, and lower pH agent with adding a sour taste. Anthocyanins stability depends heavily on pH and several other factors; thus, the combination of these compounds used in Solvent C has great potential to extract nutraceuticals that could be incorporated into foods as additives. This is important information as it relates to maximizing the food industry application of colored potatoes as a source of nutraceuticals. As the trend for natural sources for food color continues to rise, this study proved that the selected colored potatoes such as MM and AB are good sources of anthocyanins, a natural coloring agent. Furthermore, colored potatoes are a great source of polyphenol compounds with antioxidant activities and, could have the potential to significantly reduce oxidative.