Assessment of Bioactive Compounds , Physicochemical Composition , and In Vitro Antioxidant Activity of Eggplant Flour

Mailing Address: Gláucia Maria Moraes de Oliveira Hospital Universitário Clementino Fraga Filho – UFRJ Rua Prof. Rodolpho P. Rocco, 255 8o andar, sala 6,Cidade Universitária. Postal Code: 21941-913, Rio de Janeiro, RJ – Brazil E-mail: glauciam@cardiol.br Assessment of Bioactive Compounds, Physicochemical Composition, and In Vitro Antioxidant Activity of Eggplant Flour Mauara Scorsatto,1 Aline de Castro Pimentel,1 Antonio Jorge Ribeiro da Silva,2 Kebba Sabally,4 Glorimar Rosa,3 Gláucia Maria Moraes de Oliveira2 Programa de Pós-gradução em Medicina/Cardiologia Universidade Federal do Rio de Janeiro;1 Instituto de Pesquisa de Produtos Naturais Universidade Federal do Rio de Janeiro;2 Instituto de Nutrição Josué de Castro Universidade Federal do Rio de Janeiro,3 Rio de Janeiro, RJ – Brazil; School of Dietetics and Human Nutrition McGill,4 Montreal – Canada

In addition to being rich in fiber and having low lipid content, it contains a variety of phytochemicals, such as polyphenols, which provide important health benefits. 3One way to avoid losses and capitalize on the nutritional characteristics of eggplant is processing it into flour.Eggplant flour (EF) is a highly desirable food ingredient to enrich the diet; however, there are few data on its chemical composition. 4udies have shown that phenolic compounds of eggplant have the potential to reduce intestinal glucose Bioactive compounds and antioxidant activity of eggplant flour Int J Cardiovasc Sci.2017;30(3):235-242 Original Article absorption and provide cellular antioxidant protection, preventing oxidation and diabetes complications, 5 especially those phenolic compounds present in eggplant peel. 6In addition, the eggplant peel is rich in anthocyanins and has therapeutic potential for the treatment of hyperlipidemia and prevention of atherogenic cardiovascular diseases by inhibiting lipid peroxidation. 7onçalves et al. 8 conducted a literature review comprising 25 articles on the species S. melongena and concluded that this species has a mitigation effect on dyslipidemia, particularly hypercholesterolemia, when used in the form of juice made from the fruit with peel.
Cooking methods tend to reduce the content of polyphenols in fruits and vegetables, for example by simply removing their peel, since the higher concentrations of these substances are often present in the external parts. 9Even though several studies have addressed the eggplant in its different forms (in natura, juices, teas, extracts), studies that present a broad analysis of EF produced from the whole fruit dehydrated in an oven are scarce.The authors consider that this process is more accessible to the general public than the freeze-drying process.
The goal of the present study was to assess the physicochemical composition of EF prepared from the whole fruit dehydrated in an oven, characterizing qualitatively and quantitatively phenolic compounds, niacin, and saponins, in addition to assessing in vitro antioxidant activity using 2.2-diphenyl-1-picrylhydrazyl radical (DPPH) and Ferric Reducing/Antioxidant Power (FRAP) assays.

Standards and reagents
High-Performance Liquid Chromatography (HPLC) reagents and standard chlorogenic acid 3878, caffeic acid 0625, ferulic acid 12.870, and rutin 5143 were purchased from Sigma Chemical Co.(St.Louis, MO, USA).The samples were analyzed using a Varian HPLC system, with a tertiary gradient pump, variablewavelength UV/VIS absorbance detector, and an automatic sampler with a refrigerated compartment (Varian Canada Inc., Mississauga, ON, Canada).The Folin-Denis reagent (Fluka) was purchased from Sigma-Aldrich Brazil LTDA.

Preparation of samples
The samples of EF used for analysis were purchased in stores of Rio de Janeiro, Brazil.According to the manufacturer, the eggplants produced in the State of Rio de Janeiro had originally been sanitized, sliced, and dehydrated in an oven at a temperature between 62°C and 70°C for approximately ten hours.Subsequently, they were placed in containers, sealed until complete cooling and the next day they were ground and packaged immediately.The final result was a dark beige thick powder.Samples from different lots were homogenized and the analyses were performed in triplicate.

Physicochemical Analysis
Moisture content was determined by weight loss in an oven set at 105°C until constant weight. 10he determination of fixed mineral residue (FMR) was based on the burning of organic matter in a muffle furnace at 550ºC until the ashes were white or slightly grey.The ashes allowed the analysis of specific minerals since they were composed of macro-and micronutrients-and trace elements. 10oteins were determined through Kjeldahl digestion process, by which organic matter was decomposed by sulfuric acid and nitrogen was transformed into ammonia. 10pids were determined through continuous diethyl ether extraction in a Soxhlet apparatus, followed by the removal of the ether through evaporation. 10e analysis of crude fiber content was conducted in an industrial digester (Marconi MA-444/CI) subjecting the samples to acid digestion with 1.25% sulfuric acid solution, followed by alkaline digestion with 1.25% sodium hydroxide. 10tal carbohydrates were estimated by difference, subtracting the values obtained for moisture, proteins, lipids, ashes, and fibers from 100.
Total titratable acidity (TTA) was determined by titration with 0.1 N NaOH, using alcoholic solution of phenolphthalein to determine color change. 10tal dietary fiber was determined using the method proposed by Prosky et al., 11 in addition to soluble and insoluble dietary fiber. 12,13e determination of minerals (K, Ca, Na, Mg, Cu, Fe, Mn, and Zn) was performed through atomic absorption spectrophotometry, using an Analytik Jena spectrophotometer (Model ContrAA® 700).

Original Article
Niacin concentrations were determined using the method proposed by Horwitz et al. 14 Total phenols were determined using Folin-Ciocalteu reagent. 15ponins content was determined using the doublesolvent extraction gravimetric method described by Harborne. 16,17 vitro antioxidant activity Sample extraction 18 The EF was weighted (500 mg) and placed in 15-mL plastic tubes to which 10 mL of 80% methanol were added.The mixture was vigorously shaken using a vortex mixer for two minutes.The samples were then centrifuged at 5000 rpm for 15 minutes and the supernatant was filtered using Whatman filter paper (0.45 µm).The waste was re-extracted two more times with additional 10 mL of 80% methanol.The three extracts were combined to carry out the DPPH and FRAP assays and the analysis of phenolic acids through HPLC assay.

Scavenging method of DPPH free radical 19
Ferric reducing/antioxidant power 20

Analysis of phenolic acids and flavonoids through HPLC
The samples were analyzed using a Gemini-NX reversephase (RP)-HPLC column (100 x 4.5 mm) (Phenomenex, CA, USA), using 1 mL/min solvent flow rate, and detection was at 215 nm.Gradient elution was carried out with a mixture of the following solvents: Solvent A: 0.05% trifluoroacetic acid (TFA) in 10% aqueous acetonitrile (ACN); and solvent B: 0.05% TFA in 60% aqueous ACN (v/v), starting with 100% solvent A and reaching 40% solvent A and 60% solvent B in 30 minutes.
Phenolic acids and flavonoids were identified through comparisons with the retention time (RT) and UV spectra of authentic standards analyzed under identical analytical conditions.Ascorbic acid, tyrosine, chlorogenic acid, caffeic acid, ferulic acid, and rutin were analyzed.The same standards were used for calibration curves to quantify these substances in the EF.The results are presented as mean and standard deviation.

Statistical analysis
Data are expressed as mean ± standard deviation for continuous variables.The linear regression analysis was performed to estimate ascorbic acid, tyrosine and phenolic acids in EF, after analysis of assumptions for the use of the regression analysis.The statistical analysis was performed using the Statistical Package for Social Sciences, version 21 (SPSS Inc., Chicago, IL, USA).

Results
The results of the centesimal composition are shown in Table 1 and the results of minerals, niacin, soluble phenolic compounds, and saponins are shown in Table 2.

Activity of DPPH free radical elimination
The increase in DPPH radical scavenging in a dose-dependent manner due to the elimination of the methanolic extract of EF is 455.6 ± 3.27 ascorbic acid equivalent (mg).The results of the determination of antioxidant activity performed using the FRAP 486.8 ± 86.8 ascorbic acid equivalent (mg).

Discussion
The Brazilian Government Health Authority has shown a growing interest in the development and promotion of strategies for the prevention of cardiovascular diseases, mainly for a better control of the known risk factors, such as smoking, obesity, sedentary lifestyle, hyperglycemia, hypertension, and hypercholesterolemia. 21 Thus, the search for healthy and low-cost alternatives that can contribute to the health of the population becomes imperative.
A study conducted by Perez and Germani 4 showed that EF had high fiber content (44%).A similar result was obtained by Possetti and Dutra (2011), 22 who found 45% fibers.In addition to low content of lipids (1.99%),   there were 6.2% ashes, 25.54% carbohydrates, and 8% proteins.Even though fibers do not supply nutrients to the body, they are essential in the diet.They promote a number of health benefits in view of which the use of EF is considered a potential food ingredient.
In the present study we found 39.2% total fibers, 1.85% lipids, 4.7% ashes, 23.1% carbohydrates, and 13.34% proteins, in addition to a good mineral content, highlighting manganese, zinc, and copper.It is observed that, although there are small variations between the different studies, the EF features around 40% fibers and low lipid content.When compared with wheat flour, it was observed that EF featured higher levels of protein, ashes, total dietary fiber, and total sugars, whereas carbohydrate content was lower.In this way, the high dietary fiber content, the high water absorption capacity,   and the ease of grinding make EF a good alternative to be mixed with wheat flour.This mixed flour can be used in the preparation of bakery products (cookies, breads, cakes, and pasta), expanding the number of products to supplement the daily fiber intake. 4 analysis performed with the eggplant pulp showed a concentration of 62.5 mg gallic acid equivalent/100 g in the fresh pulp. 23This value is lower than that found in the present study with EF prepared from the whole fruit, which was 1,540 mg gallic acid equivalent/100 g in the flour.This is an expected result, since eggplant skin is rich in polyphenols and 100 g EF represent 1,000 g of the fruit.Another study showed greater presence of total phenols in extracts prepared from the eggplant peel (2,200 µg/g) than in extracts made from the pulp (390 µg/g). 24e results of a study assessing the centesimal composition of 20% eggplant extract showed that nutrient concentrations found in the present study were much larger than those found by those authors: 0.118% proteins; 0.052% ashes; 0.0366% lipids; 0.038% insoluble fibers; 0.025% soluble fibers; and 256.66 mg/dL total polyphenols.Even though the amount of polyphenols was significant, they were not able to protect mice against the oxidation of LDL-cholesterol, probably due to the presence of biogenic amines, especially histamine.These results do not support the popular use of S. melongena extract as hypocholesterolemic agent.On the other hand, Guimarães et al. 25 assessed S. melongena powder and found 15.09% protein, 1.42% lipids, 13.89% fibers, 0.22% calcium, and 0.31% phosphorus, and qualitative tests assessing composition showed positive results for the presence of polyphenols and saponins.These results are similar to those found in the present study regarding proteins and lipids (13.34 and 1.85%, respectively); however, far below with respect to fiber (39.19%,i.e., fiber concentration in EF was 2.8 times greater) and calcium concentrations.
A study on extracts prepared from the pulp and the peel of eggplant showed a greater presence of soluble phenolic compounds in the peel than in the pulp, and the ability to inhibit DPPH radical was greater in all the extracts prepared from the pulp of eggplant than in those prepared from the peel.These results suggest that greater phenolic content allows moderate antioxidant activity linked to free radical scavenging potential. 5he present study showed similarity between the results of DPPH and FRAP, thus revealing the antioxidant capacity of EF, possibly due to the important presence of phenolic compounds.
A comparison performed between raw, grilled for 4 to 5 minutes on both sides, and boiled in water for 10 minutes over moderate heat 100 ºC and freeze dried after the process, showed the following concentrations of total polyphenols/100 g dry matter: raw = 910 mg; grilled = 1,803 mg; and cooked = 1,991 mg.The chlorogenic acid concentrations were as follows: 154 mg; 549 mg; and 439 mg, respectively.The concentrations of caffeic acid were: 12.8 mg; 23.3 mg; and 33.9 mg, i.e., the grilling and boiling processes increased polyphenols concentrations.The authors also observed higher free radical scavenger capacity in boiled and grilled samples. 26The amount of total polyphenols was much greater in the present study, i.e., 1,540 mg/100 g EF, probably due to the longest period during which the eggplants were exposed to heat at a constant temperature of approximately 70 ºC.
It is important to highlight that the polyphenols commonly present in the human diet are not necessarily the most active within the body.This fact is due to low bioavailability, since they are little absorbed by the intestine, highly metabolized, or eliminated quickly.Furthermore, the metabolites found in blood and specific organs, which result from a liver or digestive activity, may differ from ingested substances in terms of biological activity. 9Variations in phenolic content concentrations between several studies occur due to different growing conditions. 27study assessed the hypolipidemic effect of three capsules containing 360 mg dry extract of S. melongena L. (eggplant) or 360 mg placebo/day in 28 women with dyslipidemias under nutritional control during 90 days.The authors concluded that the dry extract of S. melongena L. had a modest effect on lipid profile, with no hepatotoxic effect or adverse reactions; however, it should be noted that the clinical response found in the study did not reach the values established by the III Brazilian Guidelines on Dyslipidemias and Atherosclerosis. 28eeze-dried eggplants exhibited the following phenolic acids determined through HPLC assay: caffeic; p-coumaric; ferulic; gallic; protocatechuic; and p-hydroxybenzoic.The amount of hydroxycinnamates (caffeic, ferulic, p-coumaric, and chlorogenic acids) was from 9 g/g to 12 ug/g fresh matter. 29In the present study, the EF had the following hydroxycinnamates: caffeic, ferulic, and chlorogenic acids, totaling 1,96 mg/g flour.
The major limitation of the present study is a lack of comparison between different forms of EF preparation, to analyse how it would change the composition of EF.

Original Article
However, it is worth mentioning that the EF was produced by hand, thus making the procedure easier, without the need of a freeze dryer, or difficult-to-access equipment.An electric oven is sufficient, allowing low-cost access to nutrients and phenolic compounds present in this flour.
The present study demonstrates that EF is a good addition to the diet of the general population, since it is a source of fiber, minerals, phenolic compounds, saponins, ascorbic acid, tyrosine, and phenolic acids, in addition to exhibiting an important antioxidant activity observed by means of in vitro assays.However, it is observed that although several studies have assessed the eggplant in different ways, the studies on EF are limited to inserting it mixed with other flours to prepare cakes and cookies.No studies were found that had actually assessed the effect of EF on humans in order to know if it would have some hypolipidemic effect or could reduce oxidative stress.Clinical trials are needed to answer these questions.

Conclusion
The EF analyzed exhibited high fiber content and low lipid content.Also, it had good mineral content, highlighting manganese, zinc, and copper, in addition to phenolic compounds and saponins with important in vitro antioxidant capacity.In view of the results, the EF is a good addition to the diet of the population, since it enriches the diet and brings health benefits.

Figure 1 -
Figure 1 -Phenolic compounds and antioxidants extracted from the eggplant flour: a ascorbic acid; b tyrosine; c chlorogenic acid; d caffeic acid; and e ferulic acid.

Table 2 -Minerals, niacin, phenolic compounds, and saponins present in eggplant flour
1Results are expressed in terms of gallic acid equivalent (mg).