Influence of Soil Composition on the Profile and Content Of

agronomy

Article Inﬂuence of Soil Composition on the Proﬁle and Content of Polyphenols in Habanero Peppers (Capsicum chinense Jacq.)

Julio Oney-Montalvo 1, Alberto Uc-Varguez 1, Emmanuel Ramírez-Rivera 1,2 , Manuel Ramírez-Sucre 1 and Ingrid Rodríguez-Buenﬁl 1,* 1 Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C. Sede Sureste, Tablaje Catastral 31264 Km. 5.5 Carretera Sierra Papacal-Chuburna Puerto, Parque Cientíﬁco Tecnológico de Yucatán, Mérida 97302, Mexico; [email protected] (J.O.-M.); [email protected] (A.U.-V.); [email protected] (E.R.-R.); [email protected] (M.R.-S.) 2 Tecnológico Nacional de Mexico/Tecnológico Superior de Zongolica, Departamento de Innovación Agrícola Sustentable Km. 4 Carretera S/N, Tepetlitlanapa, Zongolica 95005, Mexico * Correspondence: [email protected]; Tel.: +52-3333455200 (ext. 4011)

Received: 10 July 2020; Accepted: 18 August 2020; Published: 21 August 2020

Abstract: Capsicum chinense Jacq. obtained the designation of origin in 2010 due to the unique organoleptic properties given by the characteristics of soils in the Peninsula of Yucatán. So, the aim of this work was to investigate the effect of soil composition on the profile and concentration of polyphenols, antioxidant activity, and its relationship with the degree of maturity in habanero pepper (Capsicum chinense Jacq.). Pepper plants were grown in three soils named according to the Maya classification as: K’ankab lu’um (red soil); Box lu’um (black soil); and Chich lu’um (brown soil). The crops were cultivated in four different dates. The peppers were analyzed for antioxidant activity, profile and content of polyphenols. The results indicated that peppers grown in black soil had the highest concentration of total polyphenols (122.78 12.60 mg of gallic ± acid 100 g 1), catechin (61.64 7.55 mg 100 g 1) and antioxidant activity by DPPH (86.51 0.82%). − ± − ± Physicochemical characterization indicated that black soil has the highest concentration of organic matter (10.93 0.23%), nitrogen (52.01 7.05 mg kg 1), manganese (5.24 0.45 mg kg 1) and electric ± ± − ± − conductivity (2.32 0.16 d Sm 1) compared to the other soils evaluated. These results demonstrate ± − that the physicochemical composition of soils could be related to the biosynthesis of polyphenols in the habanero pepper.

Keywords: polyphenols; soil; maturity; antioxidant activity; habanero pepper

1. Introduction Habanero pepper (Capsicum chinense Jacq.) is considered the main fruit species in the Yucatan Peninsula in Mexico, this pepper is internationally recognized for having a superior quality than those grown in other parts of the world due to its longer shelf life and high pungency [1]. These characteristics allowed to obtain the designation of origin in 2010 (Chile habanero de la península de Yucatán) by the “Mexican Institute of Industrial Property” (IMPI) and positioned it as a socio-economic reference in the region [2]. The soil and the climatic characteristics of the Yucatan Peninsula are considered factors that give properties to the habanero pepper grown in this region as flavor and color, which differentiates it from other peppers. Traditionally, pepper plants grown in the Yucatan Peninsula are cultivated in three main soils, named according to the Mayan classification as: (1) K’ankab lu’um (red soil); (2) Box lu’um (black soil); or (3) Chich lu’um (brown soil) [3]. These soils are characterized for having a different

Agronomy 2020, 10, 1234; doi:10.3390/agronomy10091234 www.mdpi.com/journal/agronomy Agronomy 2020, 10, 1234 2 of 14 chemical and physical composition. The red color in the soil seems to be related to the formation of hematite produced by the oxidation of iron, whereas the black color could be associated with the high content of organic material. The red soil is characterized for being a rocky soil with a lower organic matter content compared to other soils in the area (black and brown soils), and with the ability to retain less moisture than the Box lu’um (black soil), which is characterized by being in the upper zones of the micro relief, making it higher in moisture retention, in addition to have high contents of organic matter and assimilable phosphorus. The Chich lu’um (brown soil) is characterized by being a gravel soil of reddish brown to black colors, in addition to have the ability to retain more moisture [3]. Phenolic compounds are considered one of the most important groups of secondary metabolites distributed all over the plants; these compounds have been widely studied in recent years due to different pharmacological effects, highlighting their antioxidant and anti-inflammatory activity [4]. Despite habanero pepper (Capsicum chinense Jacq.) having been mainly studied for its high content of capsaicinoids, which classifies it as one of the hottest peppers in the world [5], in literature are also found works studying the profile and content of polyphenols of habanero peppers [6]. In other work, Campos et al. [7] have shown habanero pepper as an important source of polyphenols, recommending its consumption and proposing the use of habanero pepper extracts to increase nutritional value. In peppers, profile and content of polyphenols are mainly related to plant genotype [8], but the degree of maturity and soil composition may play an important role [9,10]. Soils are a complicated physical, chemical and biological system that strongly influences the growth, development and quality of plants, affecting the synthesis and accumulation of secondary metabolites [11]. A clear example is the work done by Meckelmann et al. [12], who demonstrated that Peruvian peppers cultivars grown in different geographical location (subjected to different soils, temperatures, irrigations and 1 altitudes) showed different content of total polyphenols (27.7 mg of gallic acid g− in Chiclayo and 1 13.4 mg of gallic acid g− in Piura). On the other hand, a study has shown a significant effect of the degree of maturity in the concentration of metabolites in habanero pepper, reporting changes in the 1 1 concentration of polyphenols (782 5.8 mg 100 g− in immature peppers and 759 2.3 mg 100 g− in ± 1 ± mature peppers) and the antioxidant activity (97.1 0.97 µg mL− in immature peppers and 287 1 ± ± 2.57 µg mL− in mature peppers) [13]. Another example of this effect is shown in the work carried out by Howard et al. [14], who determined a higher concentration of total polyphenols, total flavonoids, luteolin and quercetin in mature habanero peppers. Accordingly, this work aimed to estimate the effect of soil composition on the profile and concentration of polyphenols, antioxidant activity, and its relationship with the degree of maturity in the habanero pepper.

2. Materials and Methods

2.1. Plant Growth Conditions The crops of habanero peppers (Capsicum chinense Jacq. ‘Jaguar’) were cultivated in four diﬀerent dates of the year, 3 March 2017 (Crop 1), 2 May 2017 (Crop 2), 18 September 2017 (Crop 3) and 14 March 2018 (Crop 4), habanero pepper plants had an average growing cycle of 8 months, the date of harvest was selected based on the availability of peppers (>100) grown in the three soils and with the two degrees of maturity. The crops were harvested at 142 post-transplant day (PTD). The plants were developed in a greenhouse in Sierra Papacal, Yucatán in Mexico (CIATEJ, Sede Sureste). The greenhouse had a north-south orientation, a ridge height of 7.0 m, with a triple-layer plastic cover (25% shade), and lateral walls of high-density plastic anti-trips screens. The crops were composed by 300 polyethylene bags, ﬁlled with 12 kg of soil each (100 polyethylene bags with red soil, 100 with brown soil and 100 with black soil). Habanero plants were planted after 48 days from germination. Data Loggers were placed along the greenhouse to monitored temperature and relative humidity in the crops. Agronomy 2020, 10, 1234 3 of 14

Water from a local well was used for irrigation. Electric conductivity of the water oscillated from 2.8 to 3.4 mS. For fertilization, the methodology of Martínez-Estévez et al. [15] was used, which is recommended for habanero pepper cultivated in the soils of Yucatan. The fertilizer used after 10 post-transplant days (PTD) was the Triple 18 Ultrasol® (SQM, Santiago de Chile, Chile), that is a commercial fertilizer with a formula of 18N-18P-18K% (nitrogen-phosphorus-potassium); it was applied with the irrigation twice a week (12 g dissolved in 20 L of water). The micronutrients were applied spraying the commercial product Bayfolan® Forte (Bayer CropScience, Edo. de Mexico, Mexico) (24 mL diluted in 16 L of water) superficially on the leaves once a week. After 20 post-transplant days and before floral initiation, a growth regulator containing gibberellin, cytokinin, and auxin (Biozyme® TF, Arysta LifeScience, Guatemala, Guatemala) was applied (16 mL diluted in 16 L of water) once a week. Irrigation was applied sporadically (about twice a week) during the first 15 days after the transplant; subsequently, the irrigation frequency was maintained at 2 L per polyethylene bag, every third day.

2.2. Physicochemical Characterization of the Soil The physicochemical characterization of the soils was performed using the methods described below, analyzing three samples from each of the soils studied for each determination.

2.2.1. PH Measurement For pH measured, 10 g of soil were weighed, then mixed with 10 mL of distilled water for 5 s and left to suspend for 30 min [16]. Finally, the pH of the soils was performed by a pH-meter Thermo Scientiﬁc Orion Star A211.

2.2.2. Determination of the Percentage of Organic Matter The percentage of organic matter was determined by the Walkley and Black method [17]. For this, to 5 g of soil 10 mL of K2Cr2O7 (0.17 M) was added followed by 20 mL of H2SO4 (85%). The mix was shaken and leaved to stand for 30 min. Subsequently it was added with 300 mL of distilled water, 10 mL of H3PO4 and 1 mL of diphenylamine solution (0.5 g in 20 mL of distilled water and 100 mL of 1 H2SO4). Finally, it was titrated with FeSO4 (1 mol L− ) until the mixture turns green.

2.2.3. Determination of the Ion Exchange Capacity

1 Initially, 2.5 g of soil were mixed with 50 mL of ammonium acetate (1 mol L− ). The excess of sodium cations was then washed with ethanol. Subsequently, an ammonium acetate solution was added, replacing adsorbed sodium cations with ammonium cations. The solution was stirred and then centrifuged at 3500 rpm for 30 min. Finally, the concentration of displaced sodium was measured by ﬂame photometer at 596 nm [18].

2.2.4. Nitrogen Quantiﬁcation The content of nitrogen in the soils was quantiﬁed with the Kjeldhal method [19]. For this, 1 g of soil was digested with 500 mg K2SO4 and 2.0 mL of CuSO4/H2SO4 Kjeldahl digestion solution at 200 ◦C until the fuming starts. After, the temperature is raised at 400 ◦C. The digestion is continued at this temperature during 30 min. The total digestion time was 3 h. Later the sample was alkalinized with NaOH and steam distilled. The ammonia liberated was absorbed in a solution of boric acid (2%) and nitrogen contents was determined by titration with HCl (10 mN).

2.2.5. Phosphorus Quantification The content of phosphorus in the soils was quantified by the Olsen method [20]. For this, 1 g of soil was weighed and added with 20 mL of NaHCO3 0.5 M (pH 8.5) and shaken for 30 min at Agronomy 2020, 10, 1234 4 of 14 room temperature. The extracts were filtered through Whatman No. 42 filter paper and analyzed for phosphorus by colorimetry.

2.2.6. Mineral Determination The concentrations of sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), copper (Cu), iron (Fe), zinc (Zn) and manganese (Mn), were quantiﬁed using a microwave induced Plasm Atomic Emission Spectroscopy (MP-AES, 4200MP-AES, Agilent Technologies, New Castle, DE, USA) connected to a nitrogen generator (Peak Genius 3055, Agilent Technologies, New Castle, DE, USA). 1 All multielement solutions were diluted in a range of concentrations of 0.1–5 mg L− using Cu, Fe, 1 1 Mn and Zn (50 mg L− ) and Ca, K, Mg, and Na (500 mg L− ) multielement standard solutions (Agilent Technologies, New Castle, DE, USA) [21].

2.3. Sample Preparation The peppers were collected at 142 PTD and classify by soil (red, brown and black) and the degree of maturity (green and orange); in the same harvest it was possible to have immature (green) and mature (orange) peppers. As received, the peppers were dried in an oven (Felisa) by gravity convection at 65 ◦C for 72 h according to the methodology established by Gomez et al. [22]. Then, the peppers were milled with a mortar and pestle and, ﬁnally, sieved at # 35 mesh (500 µm). The resulting powder was stored in a sealed plastic bag at room temperature and protected from light. Three samples of peppers were analyzed by each combination of soil, maturity stage and crop.

2.4. Extraction of Polyphenols About 500 mg of habanero pepper powder were mixed with 2.5 mL of solvent (80% MeOH: 20% H2O) in a falcon tube. The mix was sonicated for 30 min at room temperature and, then, centrifuged at 4700 rpm for 30 min. The supernatant was collected and ﬁltered through a 0.2 µm PTFE ﬁlters and analyzed by UPLC.

2.5. Determination of Antioxidant Activity by DPPH The antioxidant activity was determined by 2,2,1-diphenyl-1-picrylhydrazyl (DPPH) radical inhibition [23]. The extracts used were those obtained from the extraction of polyphenols (procedure previously described). The DPPH solution was prepared in MeOH and diluted to a concentration with an absorbance of 0.7 0.002 at 515 nm. To 3.9 mL of DPPH, solution with the adjusted absorbance ± was added (100 µL of the pepper extract), the mixture was stirred and allowed to stand for 30 min for a subsequent reading in the spectrophotometer at 515 nm. The percentage of DPPH was calculated using the following formula: ! Acontrol ASample %DPPH = − 100 (1) Acontrol × where AControl is the absorbance of the control (0.7 Abs) and ASample is the absorbance of the sample.

2.6. Determination of Antioxidant Activity by ABTS For the determination of antioxidant activity the ABTS method was used [24]. The ABTS substrate working solution was prepared adding 25 µL of 3% hydrogen peroxide solution to 10 mL of ABTS substrate solution. For test samples, to 30 µL of the pepper extract (previously diluted 1:50 with methanol) 60 µL of myoglobin working solution and 450 µL of ABTS substrate working solution were added, leaving to stand for 5 min at room temperature. Subsequently, 300 µL of stop solution were added and the mixture was lead to stand at room temperature for one hour; then, the absorbance of the sample was read at 405 nm in the spectrophotometer. Previously, a calibration curve was prepared Agronomy 2020, 10, 1234 5 of 14 with Trolox standard at diﬀerent concentrations (0.015, 0.045, 0.105, 0.21 and 0.42 mM) to determine antioxidant activity in Trolox units.

2.7. Determination of Total Polyphenols The method used to quantify total polyphenols was the Folin Ciocalteu method [25]. For the determination of total polyphenols, 25 µL of extract was diluted in 25 µL of water, then 3 mL of deionized water and 250 µL of Folin de Ciocalteu were added, allowing to stand for 5 min. After, 750 µL of 20% Na2CO3 and 950 µL of deionized water were added, stirred, and allowed to stand for 30 min to measure the absorbance at 765 nm in a spectrophotometer.

2.8. Analysis by UPLC-DAD For analysis of polyphenols, UPLC Acquity H Class (Waters, Milford, MA, USA) with diode array detector (DAD) was used. The column was an Acquity UPLC HSS C18 (100 Å, 1.8 mm, 2.1 50 mm) × (Waters, Milford, MA, USA). The quantification of polyphenols was done with the following method: 1 flow speed of 0.5 mL min− with a column temperature set at 45 ◦C and injection volume of 2 µL. The DAD performed the measurement at 280 nm. The polyphenols were separated using acetic acid (0.2%) as solvent A and acetonitrile with acetic acid (0.1%) as solvent B. The elution conditions were: 0–10 min from 1% B to 30% B; 10–12 min 30% B; 12–15 min from 30% B to 1% B. The polyphenols were quantified by external calibration prepared with 16 standards of polyphenols (gallic acid, protocatechic acid, catechine, chlorogenic acid, coumaric acid, cinnamic acid, rutin, luteolin, quercetin, kaempferol, vanillin, myrcetin, diosmin, hesperidin, neohesperidin, naringenin, apigenin and diosmetin), all standards were purchased from Sigma-Aldrich® (St. Louis, MO, USA). First a stock 1 solution at a concentration of 1 mg mL− was prepared from all standards, and then the calibration 1 curve was prepared in the range of 1 to 75 µg mL− . The polyphenols were identified in the samples with a comparison of the retention time of the standards.

2.9. Statistical Analysis Analysis of variance (ANOVA) was used with α = 0.5% to analyze all data. The interaction graphs were made with all the factors: soil (red, brown and black) and degree of maturity (green and orange), to evaluate the effect of the interaction of these factors in the concentration of total polyphenols, catechin, and antioxidant activity (DPPH and ABTS). The results presented in Table1, Table2, and Table A1 were evaluated by descriptive and dispersion statistics, being the values presented the means standard ± deviation. The statistical test used for separation of means was the last significant difference test with 95% confidence level. All statistical analyses were performed with the software Statgraphics Centurion XVII.II-X64 (Statgraphics Technologies Inc., the Plains, VA, USA).

Table 1. Results of the physicochemical analysis evaluated to the three soils used for the cultivation of habanero pepper (Capsicum chinense Jacq.).

Red Soil Brown Soil Black Soil pH 7.67 0.02 a 7.54 0.02 c 7.62 0.03 b ± ± ± Electric conductivity (d Sm 1) 2.00 0.05 b 2.03 0.05 b 2.32 0.16 a − ± ± ± Organic matter (%) 5.15 0.05 c 8.30 0.14 b 10.93 0.23 a ± ± ± Sodium (mg kg 1) 11.04 0.28 a 10.19 0.21 a 11.04 0.85 a − ± ± ± Potassium (mg kg 1) 353.35 20.23 a 380.07 10.12 a 387.07 4.34 a − ± ± ± Calcium (mg kg 1) 2075.28 29.70 a 1688.12 9.45 c 1823.9 54.22 b − ± ± ± Magnesium (mg kg 1) 779.23 12.71 a 672.77 33.12 c 738.13 16.69 b − ± ± ± Nitrogen (mg kg 1) 33.25 2.47 b 26.67 1.89 c 52.01 7.05 a − ± ± ± Phosphorous (mg kg 1) 11.00 2.38 a 7.87 1.11 c 8.98 0.79 b − ± ± ± Copper (mg kg 1) 2.09 0.05 a 2.13 0.04 a 2.09 0.21 a − ± ± ± Agronomy 2020, 10, 1234 6 of 14

Table 1. Cont.

Red Soil Brown Soil Black Soil Iron (mg kg 1) 6.27 0.21 a 3.61 0.52 b 4.07 0.16 b − ± ± ± Zinc (mg kg 1) 1.17 0.49 a 1.02 0.23 a 1.40 0.27 a − ± ± ± Manganese (mg kg 1) 2.62 0.19 c 4.35 0.18 b 5.24 0.45 a − ± ± ± Note: Values presented are the means standard deviation; a, b, c means groups of responses, values followed by different letters in the same row indicate± statistically significant differences using least significant difference (LSD) test at p 0.05. ≤

Table 2. Results of the quantiﬁcation of polyphenols in mature (orange) and immature (green) habanero pepper (Capsicum chinense Jacq.) cultivated in diﬀerent soils.

Red Soil Brown Soil Black Soil Polyphenol (mg 100 g 1) − Green Orange Green Orange Green Orange Gallic acid 0.58 0.37 b 1.09 0.19 a 0.91 0.43 ab 0.94 0.21 ab 0.57 0.30 b 0.89 0.20 ab ± ± ± ± ± ± Protocatechuic acid 8.60 0.42 b 18.13 8.05 a 8.59 1.03 b 24.36 5.15 a 7.67 0.70 b 16.16 5.61 a ± ± ± ± ± ± Catechin 38.99 11.55 b 50.43 4.11 b 39.31 10.35 b 42.71 10.38 b 35.31 10.10 b 61.64 7.55 a ± ± ± ± ± ± Chlorogenic acid 16.41 8.40 b 32.55 8.69 a 17.24 9.72 b 37.88 9.69 a 15.68 9.55 b 34.99 7.69 a ± ± ± ± ± ± Coumaric acid 3.22 0.72 b 5.58 1.26 a 3.85 0.72 b 4.72 1.03 ab 3.21 0.53 b 3.69 0.69 b ± ± ± ± ± ± Cinnamic acid 3.69 0.76 ab 4.09 1.11 ab 3.05 0.49 b 5.16 0.95 a 2.89 0.86 b 3.68 0.20 b ± ± ± ± ± ± Rutin 18.83 4.41 b 27.31 5.05 a 22.89 3.40 ab 23.60 5.88 ab 17.75 4.35 b 29.14 6.33 a ± ± ± ± ± ± Quercetin + Luteolin 5.14 2.24 a 1.98 0.67 b 4.26 1.93 a 4.02 3.01 a 3.67 2.29 ab 2.04 0.58 b ± ± ± ± ± ± Kaempferol 1.86 0.62 de 2.45 1.07 cd 3.68 0.60 c 6.37 0.87 b 1.19 0.17 e 9.55 0.95 a ± ± ± ± ± ± Vanillin 2.14 0.54 a 2.49 0.76 a 2.08 0.29 a 2.09 0.81 a 2.02 0.34 a 2.34 0.91 a ± ± ± ± ± ± Myricetin 5.65 1.89 a 3.92 1.41 b 7.81 2.33 a 5.47 2.10 a 6.76 2.06 a 4.46 1.30 b ± ± ± ± ± ± Diosmin + hesperidin 6.16 1.60 a 2.68 0.63 b 6.89 1.66 a 2.79 0.56 b 5.69 2.46 a 2.55 0.62 b ± ± ± ± ± ± Neohesperidin 3.32 0.75 a 2.54 0.66 ab 4.43 1.55 a 2.59 0.49 ab 4.14 1.32 a 1.65 0.42 b ± ± ± ± ± ± Naringenin 10.61 3.85 a 6.55 2.66 ab 4.02 1.25 b 2.65 1.64 b 1.57 0.41 b 1.01 0.51 b ± ± ± ± ± ± Apigenin 1.03 0.59 c 3.17 1.35 b 7.82 4.35 ab 12.04 5.59 a 0.83 0.28 c 3.92 1.64 b ± ± ± ± ± ± Diosmetin 1.05 0.14 c 1.41 0.31 bc 1.49 0.49 bc 7.87 6.30 b 1.09 0.23 c 19.01 7.13 a ± ± ± ± ± ± Gliseofulvin 7.89 0.61 b 21.49 7.18 a 7.61 0.76 b 23.16 3.93 a 5.77 0.43 c 20.41 3.26 a ± ± ± ± ± ± Sum of polyphenols 135.17 14.24 d 187.86 18.93 b 148.93 14.89 d 208.42 31.01 b 115.81 23.62 c 217.13 28.04 a ± ± ± ± ± ± Note: Values presented are the means of polyphenols over the four crops standard deviation; a, b, c, d means groups of responses, values followed by different letters in the same row indicate± statistically significant differences using least significant difference (LSD) test at p 0.05. ≤

3. Results

3.1. Analysis of the Physicochemical Characteristics of Soils Results of physicochemical characterization of soils used for the cultivation of habanero pepper are shown in Table1. The red soil was characterized for having the highest concentration of phosphorus (11.00 2.38 mg kg 1), calcium (2075.28 29.70 mg kg 1), magnesium (779.23 12.71 mg kg 1), ± − ± − ± − and iron (6.27 0.21 mg kg 1), but the lowest concentration of organic matter (5.16 0.04%). ± − ± On the other hand, the black soil had the highest concentration of organic matter (10.93 0.23%), ± nitrogen (52.01 7.05 mg kg 1), manganese (5.24 0.45 mg kg 1) and the best electric conductivity ± − ± − (2.32 0.16 d Sm 1). The brown soil had the lowest concentration of nitrogen (26.67 1.89 mg kg 1), ± − ± − and an intermedium quantity of organic matter (8.30 0.14%). However, parameters such as sodium, ± potassium, copper, and zinc did not show significant differences between the three soils evaluated, being statistically equivalent. Measurements realized by data loggers placed in the greenhouse indicated that the crop 1 had temperatures and relative humidity of 23–54 ◦C and 16–91% respectively, while crop 2 reported temperatures of 21–47 ◦C and relative humidity of 18–90%; on the other hand, crop 3 had temperatures and relative humidity of 15–44 ◦C and 21–92% respectively, and finally the temperatures and relative humidity of the crop 4 was of 22–47 ◦C and 27–92% respectively.

3.2. Quantification of Polyphenols by UPLC-DAD Results obtained from the quantification of the polyphenols in habanero pepper are presented in Table2. It is observed that the profile of polyphenols changes in each one of the evaluated soils Agronomy 2020, 10, 1234 7 of 14 as well as by the degree of maturity of the fruit. The polyphenol that was found in the highest concentration under the evaluated conditions was catechin (61.64 7.55 mg 100 g 1), followed by ± − chlorogenic acid (37.88 96.9 mg 100 g 1), rutin (29.14 6.33 mg 100 g 1) and protocatechuic acid ± − ± − (24.36 5.15 mg 100 g 1), all of them in mature pepper. ± − Black soil was characterized by reporting the highest concentration of catechin (61.64 7.55 mg 100 g 1), rutin (29.14 6.33 mg 100 g 1), kaempferol (9.55 0.95 mg 100 g 1) ± − ± − ± − and diosmetin (19.01 7.13 mg 100 g 1); while brown soil had the highest concentration of ± − chlorogenic acid (37.88 96.9 mg 100 g 1), protocatechuic acid (24.36 5.15 mg 100 g 1) and ± − ± − apigenin (12.04 5.59 mg 100 g 1). On the other hand, red soil was characterized for having the ± − highest concentration of naringenin (10.61 3.85 mg 100 g 1). Polyphenols like protocatechuic acid ± − (16.16 5.61–24.36 5.15 mg 100 g 1), chlorogenic acid (32.55 8.69–37.88 9.69 mg 100 g 1) and ± ± − ± ± − gliseofulvin (20.41 3.26–23.16 3.93 mg 100 g 1) were not statistically different by the soil factor, ± ± − in contrast to the degree of maturity, since they were found in a higher concentration in mature pepper (orange) in comparison with the immature pepper (green). Also, diosmin + hesperidin did not had differences by the soil but the immature pepper (green) showed the highest concentration of this polyphenol (5.69 2.46–6.89 1.66 mg 100 g 1). The polyphenols quercetin and luteolin were ± ± − determined together because separation of the peaks by UPLC was not evident, the same happens with diosmin and hesperidin. In the AppendixA (Table A1), the average values of the individual polyphenols in each of the crops are shown, observing changes in the concentration of polyphenols. Crop 3 presented the highest concentration of catechin (68.55 14.56 mg 100 g 1) and rutin (34.67 6.89 mg 100 g 1), while crop 1 ± − ± − reported the highest concentration of chlorogenic acid (48.55 5.84 mg 100 g 1) and crop 4 the highest ± − concentration of protocatechuic acid (21.75 1.45 mg 100 g 1). ± − 3.3. Antioxidant Activity and Total Polyphenols The Figure1 present the results of antioxidant activity by DPPH (Figure1A) and by ABTS (Figure1B) of habanero pepper ( Capsicum chinense Jacq.) cultivated in the three different soils with two degrees of maturity, and also the results of the quantification of total polyphenols (Figure1C). The mature habanero peppers grown in black soil had the highest antioxidant activity using the DPPH method (86.51 0.82%) and the highest concentration of total polyphenols (137.65 13.99 mg of gallic 1 ± ± acid 100 g− ); whereas, the antioxidant activity by ABTS shows significant differences only for green peppers. In the AppendixA (Table A1) are presented the average values of the determination of total polyphenols, and the antioxidant activity by DPPH and ABTS in each crop. Crop 3 developed the highest concentration of total polyphenols (132.33 4.45 mg of gallic acid 100 g 1) and antioxidant ± − activity by DPPH (86.36 0.10%), followed by the crop 1, 2 and 4 respectively. ± 3.4. Statistical Analysis The p values obtained after carrying out the multifactorial analysis of variance showed that for all the variables studied, the number of crop had a significant effect (Table3). The crop was the only factor that had significant effect (p < 0.05) in all the response variables evaluated. While the soil showed significant effect just on the antioxidant activity, total polyphenols, and the concentration of catechin, coumaric acid, vanillin, naringenin and apigenin. On the other hand, the degree of maturity showed significant effect on all variables except the content of gallic acid, naringenin, quercetina + luteolina, vanillin and apigenin. All the response variables evaluated were significantly affected by at least one of the factor interactions, except for cinnamic acid and diosmetin. Figure2 represents the graphs of interaction of the factors of degree of maturity and soil in di fferent response variables: catechin concentration, total polyphenols, and antioxidant activity (by DPPH and ABTS methods). Interaction of degree of maturity and soil had an effect in the concentration of catechin (Figure2A), total polyphenols (Figure2C), and the antioxidant activity by DPPH method (Figure2B); the highest concentration and antioxidant activity was observed in mature habanero peppers grown in Agronomy 2020, 10, x FOR PEER REVIEW 7 of 15

100 g−1). Polyphenols like protocatechuic acid (16.16 ± 5.61–24.36 ± 5.15 mg 100 g−1), chlorogenic acid (32.55 ± 8.69–37.88 ± 9.69 mg 100 g−1) and gliseofulvin (20.41 ± 3.26–23.16± 3.93 mg 100 g−1) were not statistically different by the soil factor, in contrast to the degree of maturity, since they were found in a higher concentration in mature pepper (orange) in comparison with the immature pepper (green). Also, diosmin + hesperidin did not had differences by the soil but the immature pepper (green) showed the highest concentration of this polyphenol (5.69 ± 2.46–6.89 ± 1.66 mg 100 g−1). The polyphenols quercetin and luteolin were determined together because separation of the peaks by UPLC was not evident, the same happens with diosmin and hesperidin. In the Appendix A (Table A1), the average values of the individual polyphenols in each of the crops are shown, observing changes in the concentration of polyphenols. Crop 3 presented the highest concentration of catechin (68.55 ± 14.56 mg 100 g−1) and rutin (34.67 ± 6.89 mg 100 g−1), while crop 1 reported the highest concentration of chlorogenic acid (48.55 ± 5.84 mg 100 g−1) and crop 4 the highest concentration of protocatechuic acid (21.75 ± 1.45 mg 100 g−1).

3.3. Antioxidant Activity and Total Polyphenols The Figure 1 present the results of antioxidant activity by DPPH (Figure 1A) and by ABTS (Figure 1B) of habanero pepper (Capsicum chinense Jacq.) cultivated in the three different soils with two degrees of maturity, and also the results of the quantification of total polyphenols (Figure 1C). The mature habanero peppers grown in black soil had the highest antioxidant activity using the DPPH method (86.51 ± 0.82%) and the highest concentration of total polyphenols (137.65 ± 13.99 mg Agronomy 2020 10 of gallic acid, 100, 1234 g−1); whereas, the antioxidant activity by ABTS shows significant differences8 only of 14 for green peppers. In the Appendix A (Table A1) are presented the average values of the blackdetermination soil. On theof total other po hand,lyphenols, the antioxidant and the activityantioxidant calculated activity by by the DPPH ABTS and method ABTS (Figure in each2D) crop. had anCrop eﬀ ect3 developed only in green the highest peppers. concentration The highest antioxidantof total polyphenols activity was (132.33 developed ± 4.45 mg in peppers of gallic grown acid 100 in blackg−1) and and antioxidant brown soils. activity by DPPH (86.36 ± 0.10%), followed by the crop 1, 2 and 4 respectively.

Figure 1. Results of the determination of antioxidant activity by DPPH (A), ABTS (B) and quantification Figure 1. Results of the determination of antioxidant activity by DPPH (A), ABTS (B) and of total polyphenols (C) in habanero pepper (Capsicum chinense Jacq.) cultivated in three soils and quantification of total polyphenols (C) in habanero pepper (Capsicum chinense Jacq.) cultivated in three with two degrees of maturity. Different letters indicate statistically significant differences using least soils and with two degrees of maturity. Different letters indicate statistically significant differences significant difference (LSD) test at p 0.05. The error bars on the figure represent standard deviation. using least significant difference (LSD)≤ test at p ≤ 0.05. The error bars on the figure represent standard deviation. Table 3. p values of the different factors evaluated and their respective interactions for each of the

quantiﬁed polyphenols, total polyphenols, and antioxidant activity (DPPH and ABTS).

Factors Polyphenol A: Crop B: Soil C: Maturity AB AC BC ABC Gallic acid 0.0082 * 0.1886 0.1780 0.0087 * 0.0004 * 0.0098 * 0.0186 * Protocatechuic acid <0.0001 * 0.0554 <0.0001 * 0.0273 * <0.0001 * 0.0408 * 0.0446 * Catechin <0.0001 * 0.0030 * <0.0001 * 0.0101 * <0.0001 * 0.0001 * 0.0557 Chlorogenic acid <0.0001 * 0.4529 <0.0001 * 0.4435 0.0001 * 0.6287 0.5767 Coumaric acid <0.0001 * 0.0367 * 0.0020 * 0.0130 * 0.3795 0.0559 0.0454 * Cinnamic acid 0.0002 * 0.2930 0.0074 * 0.0773 0.8104 0.1530 0.0751 Rutin <0.0001 * 0.8034 0.0009 * 0.1927 0.0004 * 0.0821 0.7323 Quercetin + Luteolin <0.0001 * 0.3981 0.0547 0.0057 * 0.1779 0.1947 0.0364 * Kaempferol <0.0001 * 0.0923 0.0049 * 0.0372 * 0.0009 * 0.0477 * 0.0173 * Vanillin <0.0001 * 0.0176 * 0.0601 0.0012 * <0.0001 * 0.1425 0.0002 * Myricetin <0.0001 * 0.0120 * <0.0001 * 0.0934 <0.0001 * 0.6941 0.0983 Diosmin + hesperidin 0.0004 * 0.7484 <0.0001 * 0.0017 * 0.0004 * 0.7132 0.3122 Neohesperidin <0.0001 * 0.1930 <0.0001 * 0.0006 * 0.0162 * 0.0223 * 0.0060 * Naringenin <0.0001 * 0.0116 * 0.1455 0.0011 * 0.0791 0.4938 0.6582 Apigenin 0.0001 * 0.0220 * 0.2184 0.0036 * 0.2671 0.9389 0.9991 Diosmetin 0.0129 * 0.1751 0.0438 * 0.1515 0.0171 0.1780 0.1402 Gliseofulvin <0.0001 * 0.1719 0.0019 * 0.0415 * <0.0001 * 0.0386 * 0.0239 * Total polyphenol <0.0001 * <0.0001 * <0.0001 * <0.0001 * <0.0001 * <0.0001 * <0.0001 * Antioxidant activity (DPPH) <0.0001 * <0.0001 * <0.0001 * <0.0001 * <0.0001 * <0.0001 * <0.0001 * Antioxidant activity (ABTS) <0.0001 * <0.0001 * <0.0001 * <0.0001 * <0.0001 * <0.0001 * <0.0001 * Note: (*) = Signiﬁcant eﬀect. Agronomy 2020, 10, 1234 9 of 14 Agronomy 2020, 10, x FOR PEER REVIEW 9 of 15

Figure 2. Interaction graphs of the effect of the factors degree of maturity and soil on different response Figure 2. Interaction graphs of the effect of the factors degree of maturity and soil on different variables: (A) concentration of catechin, (B) antioxidant activity by DPPH, (C) concentration of total response variables: (A) concentration of catechin, (B) antioxidant activity by DPPH, (C) concentration polyphenols and (D) antioxidant activity by ABTS. The error bars on the figure represent the standard of total polyphenols and (D) antioxidant activity by ABTS. The error bars on the figure represent the error (p 0.05) of the analysis of interaction of the factors degree of maturity and soil. standard≤ error (p ≤ 0.05) of the analysis of interaction of the factors degree of maturity and soil. 4. Discussion 4. Discussion The black soil is characterized by having the highest concentration of organic matter (10.71 0.40%), ± nitrogenThe (52.50black soil9.90 is characterized mg kg 1), magnesium by having (779.23the highest12.71 concentration mg kg 1) of and organic electric matter conductivity (10.71 ± ± − ± − (2.260.40%),0.15 nitrogen d Sm (52.501) compared ± 9.90 mg to thekg−1 other), magnesium two soils (779.23 evaluated ± 12.71 (Table mg1 ).kg Previous−1) and electric works conductivity such as the ± − carried(2.26 ± 0.15 out byd Sm Nú−1ñez-Ram) comparedírez to et the al. [other26] showed two soils that evaluated antioxidant (Table activity 1). Previous and concentration works such of as total the polyphenolscarried out by are Núñez-Ramírez affected by nitrogen et al. [26] content showed in soils that usedantioxidant for cultivation. activity and While concentration Sinkoviˇcet of al. total [27] associatepolyphenols an increaseare affected in the by amount nitrogen of content polyphenols in soils content used for with cultivation. a higher contentWhile Sinkovi of organicč et matteral. [27] andassociate nitrogen, an increase and with in athe lower amount amount of polyphenol of minerals.s content The characteristic with a higher chemical content composition of organic matter of the blackand nitrogen, soil mentioned and with (especially a lower theamount high nitrogenof minerals. content The andcharacteristic organic matter) chemical is a factorcomposition that has of been the shownblack soil an increasementioned of the (especially activity ofthe the high enzyme nitrogen phenylalanine content and ammonia-lyase organic matter) PAL is [ 28a ].factor This that enzyme has hasbeen an shown important an increase role in theof the biosynthesis activity of of the polyphenols enzyme phenylalanine in the species ammo of thenia-lyase genus Capsicum PAL [28]., and This is responsibleenzyme has of an the important phenylalanine role in beingthe biosynthesis transformed of topolyphenols 4-coumaril-CoA, in the species a compound of the thatgenus is laterCapsicum used, asand a precursoris responsible in the of synthesis the phenylalanine of other polyphenolic being transformed compounds to 4-coumaril-CoA, present in peppers a compound like naringenin, that is quercetin,later used kaempferol,as a precursor apigenin in the andsynthesis luteolin of [ot29her,30 ].polyphenolic compounds present in peppers like naringenin,The amount quercetin, of total kaempferol, polyphenols apigenin content and (Figure luteolin2C) was [29,30]. also higher in mature habanero peppers grownThe in amount the black of soil, total which polyphenols coincides content with the (Figure results 2C) obtained was also from higher the analysis in mature of antioxidant habanero activity.peppers Thesegrown results in the can black be associated soil, which to thecoincides process with of maturation the results of theobtained fruit, the from effect the of analysis the degree of ofantioxidant maturity onactivity. the concentration These results ofcan polyphenols be associated has to beenthe process previously of maturation studied by ofHoward the fruit,et the al .[effect14], reportingof the degree an increase of maturity in the concentrationon the concentration of these of compounds polyphenols during has thebeen maturation previously process studied of theby habaneroHoward pepper.et al. [14], The reporting same effect an was increase observed in in the the antioxidantconcentration activity of these measured compounds by DPPH during and ABTS the assay,maturation phenomenon process previouslyof the habanero reported pepper. by Ghasemnezhad The same effect et was al. [observed31] whofound in the thatantioxidant the antioxidant activity measured by DPPH and ABTS assay, phenomenon previously reported by Ghasemnezhad et al. [31] who found that the antioxidant activity changes positively with the maturation of bell pepper

Agronomy 2020, 10, 1234 10 of 14 activity changes positively with the maturation of bell pepper (Capsicum annum). This could be due to changes in the biosynthesis of polyphenols caused by the process of maturation [13]. The change in the concentration of polyphenols due to maturation is occasioned by the accumulation of NO3 and PO4, two structural nutrients that form organic compounds in the plant (such as polyphenols). Concentration of NO3 and PO4 in peppers increase through a maturation process contributing to the biosynthesis of polyphenols and increasing their concentration [32]. The reason why the soil has a significant effect on the antioxidant activity and on the content of total polyphenols in habanero peppers also may be related to the different concentrations of minerals (Table1), because these minerals are available for the plant and have been linked to the production of phenolic acids and polyphenols [33]. The effect of minerals as Ca, Mg, Mn and Cu presented in the soils has been shown to have a negative correlation with the content of polyphenols [34], since low Cu, Mn, Ca, Fe, Zn and Mn contents are associated with high concentrations of NO3 and PO4, which as previously mentioned, are precursors for polyphenol biosynthesis. [32]. On the other hand, it has also been observed that the PAL enzyme responsible for the synthesis of polyphenols in the habanero pepper increases its activity in high concentrations of potassium (K) and phosphorus (P). It was found that black and red soils presented the highest amount of these minerals in comparison to the brown soil [35]. The K and P play an important role in the activation of numerous enzymes, protein synthesis and osmoregulation, for this reason are included in fertilization to increase crop production [36]. The quantification of polyphenols by UPLC showed that catechin is the compound with the major concentration (61.64 7.55 mg 100 g 1). It coincide with the results reported by ± − Troconis-Torres et al.[37], who analyzed the concentration of different polyphenols developed in mature 1 habanero pepper, finding catechin as the major flavonoid with a concentration of 52.25 mg 100 g− . 1 Moreover, other compounds with a high concentration were chlorogenic acid (37.88 9.69 mg 100 g− ) 1 ± and rutin (29.14 6.33 mg 100 g− ); these two polyphenols were reported in similar concentrations ± 1 in habanero pepper by Sherova et al. [6], quantifying 45.72 mg 100 g− of chlorogenic acid and 1 29.54 mg 100 g− of rutin. Other works have reported similar concentrations of the other polyphenols found in lower concentrations in this study. For example, Troconis-Torres et al. [37] quantified gallic 1 1 1 acid (16.67 mg 100 g− ), protocatechuic acid (0.06 mg 100 g− ), and vanillin (9.59 mg 100 g− ); Sherova 1 1 et al. [6] quantified kaempferol (1.47 mg 100 g− ), coumaric acid (1.45 mg 100 g− ), cinnamic acid 1 1 1 (1.25 mg 100 g− ) and myricetin (55.24 mg 100 g− ); and luteolin (0.87 mg 100 g− ) and quercetin 1 (4.63 mg 100 g− ) were studied by Howard et al. [14]. Polyphenols mentioned above are determined to be characteristic of the species of peppers, and the variations in the concentration and profile of individual polyphenols can be explained by environmental conditions, such as the physicochemical characteristics of the soil that could affect the expression of the genes responsible for the production of enzymes that have a role in the biosynthesis of polyphenols [38]. For example, the higher concentration of the major polyphenol (catechin) could be associated to the concentration of organic matter, nitrogen, potassium and phosphorus in the regulation of the enzymes: phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR) and leucoanthocyanidin reductase (LCR). These are considered enzymes that regulate the biosynthesis of catechin and other individual polyphenols [39]. The organic matter and nitrogen play an important role because they are nutrients necessary for the synthesis of aminoacids that are later used to form these enzymes, on the other hand potassium and phosphorus can be used as cofactors by the enzymes mentioned. The crop showed significant differences in all the response variables evaluated (Table3). In the AppendixA (Table A1), the concentration of the di fferent polyphenols evaluated, and the antioxidant activity are presented. Crop 3 presented the highest concentration of total polyphenols (132.33 4.45 mg of gallic acid 100 g 1), catechin (68.55 14.56 mg 100 g 1), ± − ± − rutin (34.67 6.89 mg 100 g 1) and antioxidant activity by DPPH (86.36 0.10%) and ABTS ± − ± (20.86 0.76 mg of trolox g 1), followed by crops 1, 2 and 4 respectively. These could be due to ± − different environmental conditions in each crop. Temperature is considered one of the main factors that Agronomy 2020, 10, 1234 11 of 14 affect the production of polyphenols and compounds with antioxidant activity because they influence structural genes involved in the biosynthesis of polyphenolic compounds [40]. The higher concentration of polyphenols in crop 3 may be due to the lowest temperatures reported since previous works have reported a negative relationship between temperature and levels of phenolic compounds [41]. This is because exposure to high temperatures inhibits photosynthesis in the plant, decreasing the availability of carbohydrates and slowing down the biosynthesis of polyphenols in the fruit [42]. Although the soil (nutrients and fertility) had less influence on the amount and profile of polyphenols present in the peppers compared to number of crop and degree of maturity (Table3), that factor showed an e ffect on polyphenols like catechin, coumaric acid, vanillin, myricetin, naringenin, and apigenin. This behavior demonstrate that soil composition has also an important role in the biosynthesis of polyphenols. The results obtained from the quantification of polyphenols in habanero pepper (Table2) and interactions of maturity and soil on the antioxidant activity determined by DPPH (Figure2B) suggest that catechin is the main polyphenol that influences the antioxidant activity in the habanero pepper, along with other compounds that have been shown to contribute to the antioxidant activity of habanero peppers such as vitamin C and capsaicinoids [43]. The mature habanero peppers grown in black soil had the highest antioxidant capacity determined by the DPPH (86.51 0.82%) method, as can be seen ± in Figure1A. On the other hand, the results of the determination of the antioxidant activity by ABTS method (Figure1B) showed significant di fferences only for green peppers. The work previously carried out by Morozova et al. [44], showed that the type of soil had a significant effect on the content of capsaicinoids, reporting that habanero peppers grown in red soil had a higher concentration of capsaicinoids. This information and the results obtained in the present work demonstrate that the physicochemical composition of the soil affects the production of one type of metabolites over others. This knowledge could be used in the agroindustry to provide habanero pepper with characteristics that give added value to the product.

5. Conclusions The results indicated that the profile and content of polyphenols change by effect of the soil and the degree of maturity. The polyphenols with the highest concentration in mature habanero pepper regardless the kind of soil were catechin, chlorogenic acid and rutin. The mature peppers grown in black soil had the highest concentration of total polyphenols (122.78 12.60 mg of gallic acid 100 g 1), ± − catechin (61.64 7.55 mg 100 g 1), kaempferol (9.55 0.95 mg 100 g 1), diosmetin (19.01 7.13 mg ± − ± − ± 100 g 1) and the highest antioxidant activity (86.51 0.82%) measured by DPPH method. This effect − ± may be due the highest concentration of organic matter (10.93 0.23%), nitrogen (52.01 7.05 mg kg 1) ± ± − and electric conductivity (2.32 0.16 d Sm 1) of the black soil. This characteristic chemical composition ± − of the black soil probably increase the activity of the phenylalanine ammonia-lyase (PAL), an enzyme that has an important role in the biosynthesis of polyphenols in the species of the genus Capsicum, transforming phenylalanine to 4-coumaril-CoA, a compound that is a precursor in the synthesis of polyphenolic compounds. This knowledge could be used by the agroindustry to select black soil to produce habanero pepper with a high content of polyphenols with a commercial interest for the functional food sector.

Author Contributions: This study is a result of the full collaboration of all the authors. However, Conceptualization, J.O.-M. and I.R.-B.; field methodology, A.U.-V.; analytical methodology, J.O.-M. and I.R.-B.; software, E.R.-R.; validation, I.R.-B. and M.R.-S.; formal analysis, J.O.-M. and E.R.-R.; investigation, I.R.-B. and M.R.-S.; resources, I.R.-B.; data curation, J.O.-M.; writing—original draft preparation, J.O.-M. and I.R.-B.; writing—review and editing, I.R.-B.; visualization, M.R.-S.; supervision, I.R.-B.; project administration, I.R.-B.; funding acquisition, I.R.-B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Council of Science and Technology of Mexico (CONACYT) that financed the project No. 257588 and the scholarship 715424 for Julio Enrique Oney Montalvo. Conflicts of Interest: The authors declare that they have no conflict of interest. Agronomy 2020, 10, 1234 12 of 14

Appendix A

Table A1. Average values of the determination of individual polyphenols, total polyphenols, and the antioxidant activity by DPPH and ABTS in each of the crops.

1 Polyphenols (mg 100 g− ) Crop 1 Crop 2 Crop 3 Crop 4 Gallic acid 1.22 0.68 a 0.84 0.56 ab 0.93 0.57 ab 0.34 0.40 b ± ± ± ± Protocatechuic acid 10.39 0.84 c 9.56 0.94 c 13.98 0.83 b 21.75 1.45 a ± ± ± ± Catechin 18.98 16.17 b 53.38 3.66 a 68.55 14.56 a 38.02 10.93 b ± ± ± ± Chlorogenic acid 48.55 5.84 a 26.67 21.35 ab 16.81 8.69 b 11.15 7.37 b ± ± ± ± Coumaric acid 4.67 2.33 a 3.92 0.52 ab 5.32 1.20 a 2.26 0.87 b ± ± ± ± Cinnamic acid 5.01 0.84 a 4.41 0.42 a 3.13 0.47 b 2.50 0.14 c ± ± ± ± Rutin 22.85 6.49 ab 17.21 6.24 b 34.67 6.89 a 18.28 9.99 b ± ± ± ± Quercetin + Luteolin 4.50 1.35 a 6.79 2.14 a 1.88 0.77 b 0.89 0.14 c ± ± ± ± Kaempferol 12.75 1.24 a 1.77 0.66 b 1.12 0.14 b 1.09 0.10 b ± ± ± ± Vanillin 2.28 0.45 ab 1.27 0.68 b 3.69 1.07 a 1.53 0.43 b ± ± ± ± Myricetin 8.57 1.37 a 7.17 3.13 a 5.40 3.76 ab 1.58 0.86 b ± ± ± ± Diosmin + hesperidin 3.68 1.16 ab 4.65 1.10 a 6.94 2.64 a 2.58 1.01 b ± ± ± ± Neohesperidin 3.77 1.77 a 4.24 2.35 a 2.91 1.91 ab 1.52 0.64 b ± ± ± ± Naringenin 17.04 1.52 a 1.11 0.30 b 0.68 0.10 b 0.78 0.22 b ± ± ± ± Apigenin 17.19 1.71 a 0.48 0.20 b 0.60 0.22 b 0.93 0.49 b ± ± ± ± Diosmetin 17.22 27.93 a 1.01 0.37 b 1.32 0.15 b 1.74 0.11 b ± ± ± ± Gliseofulvin 2.23 1.81 c 4.83 2.31 c 31.76 3.46 a 23.09 6.43 b ± ± ± ± Total polyphenol (mg of gallic acid 100 g 1) 115.11 2.20 b 107.62 2.34 c 132.33 4.45a 78.27 3.38 d − ± ± ± ± Antioxidant activity by DPPH (%) 85.99 0.20 b 85.31 0.09 c 86.36 0.10 a 84.60 0.21 d ± ± ± ± Antioxidant activity by ABTS (mg of trolox g 1) 20.09 0.75 a 18.61 0.57 b 20.86 0.76 a 19.32 0.47 b − ± ± ± ± Note: a, b, c, d means groups of responses. Different letters in the same row indicate statistically significant differences using least significant difference (LSD) test at p 0.05. ≤

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