Compostos da fruta e da geleia de cambuci

Phenolic compounds of cambuci fruit (Campomanesia phaea O.Berg.) and determination of antioxidant activity of cambuci jam

Nutrição Ensino Médio Concurso Tecnologia de Alimentos Química dos Alimentos
Compostos da fruta e da geleia de cambuci
Milena B.
em 13 de Maio de 2019

Phenolic compounds of cambuci fruit (Campomanesia phaea O.Berg.) and determination of antioxidant activity of cambuci jam

 M. BAGETTI a, M.A. PRADO a*

a a : Laboratory Food Analysis, Department Food Science, Faculty Food Engineering – University of Campinas - UNICAMP, 80 , Monteiro Lobato St, Brazil - Artigo parte de minha tese e doutorado 
 

Abstract

The consumption of fruits and vegetables has several beneficial effects on our health with respect to the prevention of chronic degenerative diseases such as arteriosclerosis and cancer. However, little is known about the bioactive or phytochemical compounds present in some Brazilian native fruits—for example, the cambuci fruit (Campomanesia phaea O. Berg.)—and little data exist with regard to their antioxidant activities. The aim of this work was to determine the phenolic compound content of the cambuci fruit by means of two methods of extraction: ultrasonication and homogenization using the Ultra-Turrax mixer by employing aqueous ethanol (80%) and acetone (80%) solvents. Furthermore, phenolic compound content of cambuci jam was determined by using response surface methodology, and the antioxidant activity of the cambuci jam was measured. The extraction employing the Ultra-Turrax mixer with aqueous acetone (80%) resulted in a larger amount of phenolic compounds (232.12 ± 10.4 mg gallic acid equivalents (GAE)·100 g−1 in dry basis). The highest content of phenolic compounds extracted from cambuci jam was 322.5 mg GAE·100 g−1 in dry basis. The cambuci jam exhibited the best antioxidant activity as determined by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assay method and the phenolic content of the cambuci jam correlated positively with antioxidant activity for DDPH and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) assays. The best result for the antioxidant activity obtained using response surface methodology varied widely among the different methodologies. The cambuci jam, and hence cambuci fruit, proved to be a rich source of phenolic compounds, which showed positive correlations with antioxidant capacity. The confirmation of antioxidant activity in cambuci fruit jam and the phnolic compounds of fruit demonstrate the importance of the preservation and utilization of cambuci, a fruit currently in danger of extinction. 

Key-words: phenolic compound; campomanesia phaea Berg; jam.

RESUMO

O consumo de frutas e vegetais tem apresentado vários efeitos benéficos para nossa saúde, relacionados com a prevenção de doenças crônicas degenerativas, entre elas, arteriosclerose e câncer. Contudo, algumas frutas nativas brasileiras apresentam poucos estudos na literatura sobre os compostos bioativos ou fitoquímicos bem como sua atividade antioxidante, como por exemplo, o cambuci (Campomanesia phaea O.Berg.). O objetivo deste trabalho foi determinar o conteúdo de compostos fenólicos da fruta cambuci utilizando dois sistemas de extração: ultrassom e Ultra-Turrax,  com o emprego de etanol 80% e acetona 80%. Além disso, determinar o teor de compostos fenólicos de geleia de Cambuci, com o uso de metodologia de superfície de resposta medir a atividade antioxidante de geleia de cambuci, avaliando a influência das variáveis tempo, temperatura e concentração de etanol. A extração que utilizou Ultra-turrax com acetona 80% resultou em maior teor de compostos fenólicos (232, 12 ± 10,4 mg de equivalentes de ácido gálico (GAE).100g-1   em base seca). O maior teor de compostos fenólicos encontrado em geleia de Cambuci foi de 322.5 mg GAE.100g-1  em base seca. A geleia apresentou melhor atividade antioxidante com o método 1,1-diphenyl-2-picrylhydrazyl (DPPH). Os melhores resultados encontrados utilizando a metodologia de superfície de resposta variaram amplamente entre as diferentes metodologias antioxidantes utilizadas (ABTS e DPPH). Geleia de cambuci provou ser uma boa fonte de compostos fenólicos, que apresenta correlação positiva com a capacidade antioxidante. A confirmação da atividade antioxidante em extratos da geleia e os compotos fenólicos presentes na fruta cambuci demonstram a importância da preservação e utilização do Cambuci, uma fruta em risco de extinção.

Palavras-chave: compostos fenólicos, campomanesia phaea O. Berg, geleia

 

1. Introduction

                  Food and beverages rich in flavonoids and other phenolic compounds have been associated with reducing the risk of disease in several epidemiological studies (Huxley & Neil, 2003; Hertog et al., 1993; Lin et al., 2007). Investigations demonstrate that the antioxidant activity of plant tissue is related to the defense against oxidative stress and in other human disorders, including arteriosclerosis and aging processes (Manostroi, 1995; Stajner, 1998). There have also been reports of a correlation between the intake of phenolic compounds, such as flavonoids, and the possibility of reduction of certain types of cancer (Lopez-Otin & Diamandis, 1998; Middleton et al., 2000). Furthermore, the concentration of these compounds has been positively correlated with antioxidant activity (Meyers et al., 2003; Rekika et al, 2005).

          The cambuci fruit (Campomanesia phaea O. Berg.) is a member of the Myrtaceae family, which is one of the most important plant families (Donadio, 1997). However, little information exists on the phenolic and flavonoid composition of the fruit and its jam in the literature. The main ingredients used in jam preparation are fresh fruits (juice or pulp), sugar, pectin, and citric acid. Although fruit processing may reduce the total amount of anthocyanin and other antioxidant phenolic compounds, studies have shown that a significant amount of these compounds were found in blackberry jam after three months of storage at room temperature in the presence of light (Mota, 2006).

            The optimization of extraction and specific analysis methodologies is fundamental, because small details, such as time and temperature of extraction can affect the reliability of the results. Despite the abundant literature about the phenolic content in food, there is little work describing modifications in extraction procedures employing specific matrices and/or critical sample preparation conditions for quantification (Souza et al., 2009).          

          Ultrasonically assisted extraction (ultrasonication) is one of the non-conventional extraction techniques that has increased in popularity, especially during the last 25 years, because of the development of ultrasound devices (Vinatoru, 2001). Some authors have proposed that the efficacy of ultrasound extraction is attributed to the fact that ultrasound waves promote better contact of the solvent with the analyte (Virot et al., 2009; Wang et al., 2008; Wang & Weller, 2006). However, the effects of ultrasound on the yield and kinetics of extraction can differ depending on the nature of the plant material to be extracted (Wang & Weller, 2006). Ultrasound also has a mechanical effect, allowing better penetration of the solvent in the sample, which increases the contact surface area and improves solute diffusivity to the environment (Wang et al., 2008; Virot et al., 2009; Rostagno, Palm, & Barroso, 2003).

         Soluble phenolic compounds are found compartmentalized in the plant cells within vacuoles (Beckamn, 2000) in free or conjugated form, while insoluble phenolics are bound to cell wall structures, or esterified with arabinose or galactose residues of pectin, or hemi-cellulosic components (Faulds & Willianson, 1999). However, with jam processing, the liberation of compounds bound to these cell wall structures tends to occur, because the process involves loss of water.

The Folin–Ciocalteau (FC) method has the advantage of being simple, fast, and robust; it is easily automated (Huang & Prior, 2005) for the estimation of the phenolic compound concentrations in food, despite its disadvantages, namely, interference from other substances that react with the FC reagent, such as sugars, aromatic amines, ascorbic acid, organic acids, and various inorganic substances. Amakuraet al. (2000) previously used the FC method to determine the influence of jam processing on the radical scavenging activity of the phenolic content in berries.

           The aim of this work was to determine the phenolic compound content of the cambuci fruit undergoing two methods of extraction: ultrasound and Ultra-Turrax mixing, using aqueous ethanol (80%) and acetone (80%) solvents. Furthermore, the phenolic compound content in cambuci jam was determined by using response surface methodology; in addition, measurements of the antioxidant activity of cambuci jam were performed and correlated with phenolic compound concentrations.

2 Material and Methods

 

 

           2.1 Material

The cambuci fruit (Figure 1) harvested in Rio Grande da Serra, São Paulo, (SP, Brazil) consisted of a mix of completely ripe fruits from plants belonging to a cooperative of cambuci and derivate producers (Cooper Cambucy, Rio Grande da Serra, SP, Brazil). The sanitized samples were placed in cold storage and transported to the State University of Campinas. Twenty kilograms of fresh cambuci fruit and twelve jars of cambuci jam (40 g each) (Figure 2) were acquired from the same harvest (2011) as prepared by Cooper Cambucy. The batches of fruits and jam were frozen at −38 ºC. The peeled fruits and pulp were sanitized in a similar manner as in fruit jam formulation processing. 

 

2.2. Reagentsand standards

            The reagents and standards used were Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), soda ash, FC reagent, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid diammonium salt (ABTS), ethanol and acetone. All reagents were purchased from Sigma-Aldrich, Synth, or Dinamica.

 

2.3. Extraction from the pulp cambuci

 

The fruit samples (10 g) were extracted using two different solvents: ethanol (80% in water) and acetone (80% in water), using two different systems: Ultra-Turrax (Turatec TE-102-TECNAL, 4000 rpm) and ultrasound according to the method of Shin et al. (2008) (Figure 2). A quantity of sample (10 g) was mixed with solvent and extracted at room temperature for 5 min; this process was repeated twice. After extraction, the extracts were filtered through a Whatman No. 1 paper filter and the volume was adjusted to 100 mL. The total phenolic compounds were measured using the FC reagent (Singleton et al., 1999) and the calibration curve expressed in mg of gallic acid equivalents (GAE).

 

  2.4. Determination of phenolic compounds in jam

Response surface methodology was applied to determine the optimal conditions for cambuci jam extraction via experimental planning of a rotatable central composite design, totaling 17 extractions (Table 1). Jam samples (10 g) were placed in an Erlenmeyer flask with 100 mL of extraction solvent and subjected to ultrasound (model USC-1880, 40 KHz, Unique, Ind. e Co. Ltd., Brazil) under different conditions (Figure 3). Temperature, ethanol concentrations, and time were varied in accordance with the studies of Morelli (2012).

The total phenolic compound was determined using the FC reagent (Singleton et al., 1999). The constructed calibration curve was expressed in mg of GAE per 100 g of the dry basis sample, using the extracting solution of the central point of a rotatable central composite design (50% de ethanol)

  2.5. Antioxidant activity of the cambuci jam

 

 2.5.1 Determination of antioxidant activity through the DPPH• radical

The determination of antioxidant capacity of the sample by the reduction of the DPPH free radical was conducted according to Brand-Willians & Cuvelier (1995). The analyses were performed by adding 3.9 mL of a DPPH solution methanolic and 100 μL of the sample extract in each test tube, or the same volume of solution used in extract samples to obtain a control solution. Absorbance readings were acquired at room temperature after a reaction time of 90 min in a dark environment. The analyses were repeated three times and expressed in (µmol Trolox) (100 g −1 f.w.).

 

2.5.2 Determination of antioxidant activity through ABTS+(TEAC) assay

 

            The determination of antioxidant capacity of the sample by the reduction of the ABTS+radical cation (Trolox equivalent antioxidant capacity (TEAC) assay) was conducted according to the procedure described by Re et al. (1999). An aqueous stock solution of potassium persulfate (K2S2O8; 2.45 mM) and a stock solution of ABTS (7 mM) were prepared and used to dilute extracts such the assay results would be between 20 and 80% of the control absorbance. For 3 mL of the diluted ABTS solution, 30 uL of sample extract and standard solutions were added to build the Trolox calibration curve at concentrations of 0.4 to 1.5 µM.

 

2.6 Statistical analysis

          Results were analyzed by one-way analysis of variance (ANOVA) for cambuci fruit and experimental planning of the central rotatable composite design was realized for cambuci jam.

 

3. Results and discussion

              In this study, the system employing the Ultra-Turrax mixer with aqueous acetone (80%) resulted in a larger amount of extracted phenolic compounds (232.12 ± 10.4 mg GAE·100 g−1) (Figure 5). This amount was significantly different (more than double) from that obtained for the Ultra-Turrax/ultrasound systems using aqueous ethanol (80%); however, there were no significant differences within 5% errorwith the system using acetone and ultrasound. During extraction under agitation, as with Ultra-Turrax, bubble formation and cavitation occurred, resulting in an increase in yield. According to Gan and Latiff (2011), acetone promotes a larger yield in phenolic compounds than ethanol, methanol, or hexane/ethyl acetate systems. However, this solvent is also more toxic and can promote the extraction of non-phenolic compounds or tannins, possibly more than ethanol.

Figure  5. Content of phenolic compounds of the fruit under different solvents and  extraction  systems. 1:  80% Ethanol  in ultra- turrax, 2: 80% Ethanol in ultrasound, 3: 80% Acetone in ultra- turrax, 4: 80% Acetone in ultrasound  -  *mg GAE.100g dry basis.

 

 

       The phenolic content determined by the FC method was slightly lower than that previously found in cambuci by Genovese et al. (2008) (246 mg GAE·100 g−1 f.w.), and by Lima et al. (2002) in other fruits of the Myrtaceae family such as the purple pitanga (Eugenia uniflora L.; 325 mg catechin·100 g−1 f.w.) and red pitanga (257 mg catechin·100 g−1 f.w.). It must be considered that the fruits exhibit quantitative and qualitative variations in the content of these components due to intrinsic factors (cultivation, variety, phase of maturity) as well as non-intrinsic factors (climatic and edaphic conditions) (Melo et al., 2008), in addition to the extraction system and extracting solution employed. According to Gonçalves et al. (2010), the frozen pulp of cambuci presents a high content of quercetin glycosidic derivatives and is considered a rich source of ellagic acid.

              The highest phenolic compound content obtained from cambuci jam was 322.5 mg GAE·100 g−1 in dry basis, under the following extraction parameters in ultrasound method: ethanol 20.2%, temperature 37 ºC, and extraction time 24 min  (Table 2).            

           The highest antioxidant activity as determined by the DPPH assay (44.50 ± 1.00  µmol·g−1 in dry basis) was obtained with the following extraction conditions: ethanol 50%, temperature 65 ºC, and extraction time 15 min (Table 2). Best antioxidant activity results for the ABTS assay (0.034 µmol.g−1 in dry basis) were obtained under the following conditions: ethanol 20.2%, temperature 37 ºC, and extraction time 6 min (Table 1). In this study, the antioxidant activities of cambuci jam using the DPPH method were higher than that of ABTS. The best result for the DPPH method was higher that found in the study by Falcão et al. (2007) using a jam wine model system(Vitis labrusca) and Refosco (Vitis vinifera L.), while activities were less when the ABTS methodology was used. The correlation coefficient found for phenolic compound content of cambuci jam and antioxidant activities using DPPH and ABTS methods were positive (0.82 and 0.80, respectively). Antioxidants methods (DPPH and ABTS) also showed a positive correlation between them (0. 80).

 

 

Table 2. Phenolic compounds and antioxidant activity of jam cambuci prepared extracted different conditions of concentration of ethanol, time and temperature.

Assays

Phenolics

mgGAE*.100g-1

dry basis

 

DPPH

µmol trolox.g -1

dry basis

 

TEAC**

µmol trolox.g-1

dry basis

 

Ethanol %

Temperature ºC

Time min.

 

 

 

20.2

37

6

303.83± 3.75

34.85±1.10

0.034±0.18

20.2

37

6

280.41±13.80

33.13±1.10

0.030±0.01

79.8

58

6

322.03±2.16

34.74±0.60

0.033±0.04

79.8

58

6

276.74±1.34

32.45±2.40

0.030±0.03

20.2

37

24

322.56±0.28

36.62±0.90

0.029±0.04

79.8

37

24

292.88±3.43

31.65±2.10

0.028±0.01

20.2

58

24

286.07±0.57

31.92±2.60

0.038±0.04

79.8

58

24

312.59±1.91

21.15±1.40

0.028±0.04

50

47

15

295.24±2.68

31.10±2.40

0.026±0.06

50

47

15

314.79±1.23

33.15± 0.90

0.029±0.10

50

47

15

263.79±1.22

35.28±1.60

0.028±0.01

0

47

15

239.07±1.50

27.21±1.90

0.026±0.00

100

47

15

150.85±0,28

12.47± 1.10

0.019±0.04

50

30

15

316.99±2,34

41.78±4.20

0.030±0.06

50

65

15

319.96±1.65

44.50± 1.00

0.032±0.02

 

50

 

47

 

0

280.70±3.51

     32.80±2.10  

0.029±0.01

 

50

 

47

 

30

316.93±3.16

     33.97±0.10

0.031±0.04

0

47

15

239.07±1.50

27.21±1.90

0.026±0.00

100

47

15

150.85±0,28

12.47± 1.10

0.019±0.04

50

30

15

316.99±2,34

41.78±4.20

0.030±0.06

50

65

15

319.96±1.65

44.50± 1.00

0.032±0.02

 

50

 

47

 

0

280.70±3.51

     32.80±2.10  

0.029±0.01

 

50

 

47

 

30

316.93±3.16

     33.97±0.10

0.031±0.04

 

Table  3.   Best results for jam cambuci extraction phenolic compounds and antioxidant activity

                       

                           ethanol (%)                    temperature ( ºC)                              time (min)

 

Phenolic*                    20,2                                    37                                               24

 

DPPH**                      50                                      65                                               15

 

ABTS**                       20,2                                     37                                               6

*mgGAE.100g-1dry basis 

** µmol trolox.g -1dry basis                       

                    

The optimum parameters of ethanol concentration, time, and temperature for extracts of cambuci jam are shown in Table 3.Contrary to the results of Morelli (2012), the temperature variable made a more significant impact on the antioxidant capacity.

For both ABTS and DPPH methods, the interaction of ethanol concentration and time parameter with temperature had a positive influence in antioxidant activity jam cambuci. In DPPH method, when extraction time and temperature was reduced to less than 6 min and less than 37 ºC, respectively, the DPPH assay showed higher antioxidant concentration at high ethanol concentration; however, it passed through the central point (50%). Nevertheless, the cambuci jam can generally be considered as a rich source of compounds with antioxidant activity; since jam has more shelf life than the fruit. Futhermore, the ultrasound methodology is a efficient alternative for the extraction of phenolic compounds, because it reduces time of analysis.

 

4. Conclusion

 

           The results demonstrate the cambuci fruit as a source of phenolic compounds that needs to be better utilized. The cambuci jam presented as a source de phenolic compounds with best antioxidant activity by DPPH method. The highest result found for content phenolic compounds of cambuci jam were with ethanol 20.2%, temperature 37ºC and 24 minutes. Phenolic content of cambuci jam correlated positively with antioxidant activity for DDPH and ABTS.  The best result found using response surface methodology for antioxidant activity ranged between antioxidant methodologies,  being for DPPPH : ethanol 50%, 65ºC and 15 minutes  and for ABTS with ethanol 20.2%,  37ºC and 6 minutes and  varied widely antioxidant activity found in the different methodologies.  The antioxidant activity verified in fruit jam and that the extraction by ultrasound and ultra-turrax of fruit cambuci can be good alternatives. 

 

 

 

 

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