Hydroalcoholic extraction of antioxidant compounds in Japanese grape pseudofruits

Gisiéli Carla Morandin1, Sabrina Vicentini Schaefer2, Adrieli Maiandra Piccinin do Amaral3, Elisandra Rigo4,

Georgia Ane Raquel Sehn5, Darlene Cavalheiro6*

DOI: https://doi.org/10.35699/2447-6218.2023.45007

Abstract

The pseudofruit of Japanese grape is rich in sugar and phenolic compounds but little explored for extracting antioxi-

dant compounds. The objective of the study was to extract and evaluate the antioxidant capacity of these pseudofruits

harvested at two maturation stages: the development phase (DP) and the mature phase (MP). The pseudofruits were

evaluated regarding the centesimal composition and physicochemical characteristics and submitted to extraction with

100% water, 50% ethanol, and 100% ethanol. For the extracts, we determined the phenolic compound content and

the antioxidant activity using radical capture methods ABTS and DPPH. Pseudofruits may be considered a good source

of dietary fiber, regardless of the maturation stage, proving to be a promising raw material for use in foods. Moreover,

pseudofruits in the MP presented an increase in the contents of soluble solids, reducing and non-reducing sugars,

and titratable acidity, attributed to the formation of the galacturonic acid during the maturation process. Among the

solvent used, extraction with 50% ethanol resulted in a larger phenolic compound content and better antioxidant

activity, especially for the pseudofruits in the DP, characterizing them as a vegetable matrix of excellent antioxidant

capacity and with potential for application in foods and drugs.

keywords: ABTS. Phenolic compounds. DPPH. Hovenia dulcis. Maturation.

Extração hidroalcóolica de compostos antioxidantes em pseudofrutos da uva Japão

Resumo

O pseudofruto da uva Japão é rico em açúcares e compostos fenólicos, mas ainda pouco explorado para extração de

compostos antioxidantes. O objetivo do estudo foi extrair e avaliar a capacidade antioxidante desses pseudofrutos

colhidos em dois estádios de maturação: a fase de desenvolvimento (FD) e a fase madura (FM). Os pseudofrutos foram

avaliados quanto à composição centesimal e características físico-químicas e submetidos à extração com água 100%,

etanol 50% e etanol 100%. Para os extratos, determinamos o teor de compostos fenólicos e a atividade antioxidante

pelos métodos de captura de radicais ABTS e DPPH. Os pseudofrutos podem ser considerados uma boa fonte de fibra

alimentar, independentemente do estádio de maturação, mostrando-se uma matéria-prima promissora para utilização

em alimentos. Além disso, os pseudofrutos na FM apresentaram aumento nos teores de sólidos solúveis, açúcares

redutores e não redutores e acidez titulável, atribuídos à formação do ácido galacturônico durante o processo de

maturação. Dentre os solventes utilizados, a extração com etanol 50% resultou em maior teor de compostos fenólicos

1Universidade do Estado de Santa Catarina, Departamento de Engenharia de Alimentos e Engenharia Química.Pinhalzinho, SC. Brasil.

https://orcid.org/0000-0002-4003-9706

2Universidade do Estado de Santa Catarina, Departamento de Engenharia de Alimentos e Engenharia Química. Pinhalzinho, SC. Brasil.

https://orcid.org/0000-0002-9515-2298

Universidade do Estado de Santa Catarina, Departamento de Engenharia de Alimentos e Engenharia Química. Pinhalzinho, SC. Brasil.

https://orcid.org/0000-0002-0759-0432

4Universidade do Estado de Santa Catarina, Departamento de Engenharia de Alimentos e Engenharia Química. Pinhalzinho, SC. Brasil.

https://orcid.org/0000-0002-5405-5168

5Universidade do Estado de Santa Catarina, Departamento de Engenharia de Alimentos e Engenharia Química. Pinhalzinho, SC. Brasil.

https://orcid.org/0000-0002-3780-3670

6Universidade do Estado de Santa Catarina, Departamento de Engenharia de Alimentos e Engenharia Química. Pinhalzinho, SC. Brasil.

https://orcid.org/0000-0003-2556-0277

*Autor para correspondência: darlene.cavalheiro@udesc.br

Recebido para publicação em 03 de março 2023. Aceito para publicação em 26 de abril de 2023

e-ISSN: 2447-6218

Caderno de Ciências Agrárias está licenciado

com uma Licença Creative Commons

Atribuição - Não Comercial 4.0 Internacional

CADERNO DE CIÊNCIAS AGRÁRIAS

Agrarian Sciences Journal

Morandin, G. C. et al.

Cad. Ciênc. Agrá., v. 15, p. 01–06, DOI: https://doi.org/10.35699/2447-6218.2023.45007

e melhor atividade antioxidante, principalmente para os pseudofrutos da FD, caracterizando-os como uma matriz

vegetal de excelente capacidade antioxidante e com potencial para aplicação em alimentos e fármacos.

Palavras-Chave: ABTS. Compostos fenólicos. DPPH. Hovenia dulcis. Maturação.

Introduction

Hovenia dulcis T. (Rhamnaceae), commonly

known as the Japanese grape, produced pseudofruits,

and these edible parts have antialcohol, liver protec-

tion, anti-fatigue, anti-steatosis, and anti-inflammatory

functions, moreover polysaccharides with hypoglycemic

activity (Yang et al., 2022). Thus, pseudofruits can be

explored, as they present biocompounds with potential

for application in functional foods and pharmaceutical

products.

Extraction is the first step to separate biologically

active compounds from natural products, and solvent

extraction is the most widely used method (Tileuberdi et

al., 2022). The main solvents used are of organic origins,

such as methanol and ethanol, or also an aqueous solvent,

and these may be used separately or combined (Sarfarazi

et al., 2019; He et al., 2023). Among the variety of solvents

that may be used, ethanol presents some advantages. It

is produced by biotechnology, does not generate toxic

residues, its flammability degree is intermediary, and it

is considered safe for human health, so it may be used

for extracting vegetable compounds for application in

the most diverse industries such as food, pharmaceutics,

and cosmetics, among others (Nikolić et al., 2023).

Therefore, with the objective of obtaining more

knowledge about the functional potential of the pseu-

dofruits of H. dulcis as an alternative source of bioactive

compounds for the industry, this study performed the

hydroalcoholic extraction with pseudofruits harvested

at two maturation stages and determined the phenolic

compound content and the antioxidant capacity of the

extracts. Moreover, the pseudofruits were evaluated for

their centesimal composition and physicochemical cha-

racteristics.

Material and Methods

Sample collection

The pseudofruits were harvested from the same

tree in the city of Chapecó, Santa Catarina, Brazil (la-

titude 27°05’08.7” S and longitude 52°36’58.9” W), in

two periods: February, the development phase (DP), and

April, the mature phase (MP). After the harvesting, the

pseudofruits were selected, sanitized in running water

then immersed in a 200 ppm hypochlorite solution for

15 minutes. Next, they were separated into two batches:

the first was used for characterizing the pseudofruits

in natura, while the second was frozen at -86 °C in an

Ultrafreezer (ULT 335/710 D Vertical, Indrel) involved

in a dark-colored plastic packaging until the antioxidant

capacity analyses were performed.

Characterization of the pseudofruits in natura

The centesimal composition of the pseudofruits

was determined through moisture, ash, lipid, protein, and

total dietary fiber analyses, as per methodologies 934.06,

940.26, 920.39, 960.52, and 985.29 by AOAC (2016),

respectively. The carbohydrate content was calculated

by difference.

For the pH, titratable acidity, soluble solids, redu-

cing and non-reducing sugars, the samples in natura were

crushed in a mixer (250W, Walita) for 30 s. To determine

the pH, 10 g of the crushed samples were quantified on

an analytical balance and diluted in 100 mL of water and

the reading was carried out with a previously calibrated

pH meter (MPA210, Tecnopon). The titratable acidity

was determined as per methodology no. 962.19 (AOAC,

2016). For determining the total soluble solids, 3 to 4

drops of the samples homogenized in 10 mL of water

were transferred to the refractometer prism (RFM 732,

Bellingham Stanley). The analyses of reducing sugars in

glucose and non-reducing sugars in sucrose were carried

out employing the Lane-Eynon method, using Fehling’s

solution (AOAC, 2016).

Extraction of antioxidant compounds

For extracting the antioxidant compounds, the

methodology proposed by Larrauri et al. (1997) was

followed with modifications. The extractions were car-

ried out in batch duplicate with extractor solvents 100%

water, 100% ethanol, and ethanol-water (50:50, v/v)

in the pseudofruit-solvent proportion of 1.5:10 (w/v).

We used a shaker incubator (Luca-223, Lucadema) at

30±1 °C with an agitation speed of 40 rpm for 60 min.

The mixtures were filtered with quantitative filter paper

‘Whatman’ no. 40, and the filtrate was stored in an amber

bottle involved with aluminum foil at 4.0+1 °C for 24 h

in a refrigerator (CRD37EB, Consul) until the analyses

were performed.

Determination of the total phenolic compounds and

antioxidant activity through the capture of free radi-

cals DPPH and ABTS+

The quantification of the phenolic compounds

was carried out in triplicate using the Folin-Ciocalteu

colorimetric method (Singleton and Rossi, 1965). The

Hydroalcoholic extraction of antioxidant compounds in Japanese grape pseudofruits

Cad. Ciênc. Agrá., v. 15, p. 01–06, DOI: https://doi.org/10.35699/2447-6218.2023.45007

quantitative evaluation of the antioxidant activity was

determined by the free radical capture of DPPH metho-

dology (Rufino et al., 2007a). The results were expressed

in EC50. The total antioxidant activity was determined

by the free radical capture of ABTS according to the

method described by Rufino et al. (2007b). The results

were expressed in µM Trolox.g-1 of dry sample.

Statistical analysis

The results of the quantifications that presented

a null hypothesis occurrence probability lower than 5%

(p<0.05) applying an ANOVA were considered statisti-

cally different, followed by multiple comparisons by the

Tukey test. All analyses were conducted using the trial

version of software trial STATISTICA 14 (Statsoft).

Results and Discussions

Characterization of the pseudofruits in natura

Significant differences (p<0.05) were observed

between the two maturation stages (Figure 1) for all

pseudofruit characterization analyses performed (Table

1), except for the protein content. For the total dietary

fiber content, a reduction was observed from the DP to

the MP. This change may be attributed to the increase

in the solubility of the pectic polysaccharides, with this

being an alteration observed in the maturation of fleshy

fruits (Liu et al., 2023).

Table 1 – Characterization (dry base) of Japanese grape pseudofruits at different maturation stages

Maturation stage

Analyses

Values expressed as averages

standard deviation. †inserted in the carbohydrate fraction; Averages on the same line followed by equal letters do

not present significant differences according to the Tukey test (p<0.05).

Carbohydrates constitute the majority compo-

nent of the pseudofruits of the Japanese grape. During

the maturation, an increase occurred in contents of total

soluble solids and reducing and non-reducing sugars due

to the hydrolysis of the starch, which is present in greater

amounts in immature fruits (Ali et al., 2022). Over the

maturation stage, an increase in pH and titratable acidity

was observed. In fruit maturation, there is usually an

increase in the ratio of soluble solids to titratable acidity

resulting from the increase in the soluble solid content

and decrease in titratable acidity; however, in this study,

this reduction was not observed.

Evaluation of the hydroalcoholic extracts of pseu-

dofruits

The study of the extraction conditions, among

them the type of solvent and concentration thereof, is

extremely important to quantify the antioxidant poten-

tial of vegetable extracts (Table 2). It was observed that

the content of total phenolic compounds (TPC) differed

significantly (p<0.05) among the three solvent systems

evaluated, being higher for solvent 50% ethanol (v/v)

in both maturation stages (DP and MP).

The physicochemical characteristics of the diluted

ethanolic solvent system possibly resemble, to a higher

degree, the characteristics of most phenolic compounds

present in the samples evaluated. The type of solvent and

the polarity may affect the transference of electrons and

hydrogen atoms, which is a key aspect in evaluating the

phenolic compounds and antioxidant capacity (Gonzá-

lez-Cardoso et al., 2023).

The values for TPC obtained in the extractions

with different systems were influenced by the maturation

Development phase

Mature phase

Moisture (%)

64.45 ± 0.20

70.18 ± 0.93

Ash (%)

1.96

0.08

1.52 ± 0.09

Protein (%)

7.19

0.62

7.97

0.48

Lipids (%)

1.39

0.07

1.17 ± 0.06

Total dietary fiber (%)†

61.74 ± 3.66

41.66 ± 3.68

Carbohydrates (by difference) (%)

89.46

89.34

Total soluble solids (°Brix)

4.50 ± 0.10

10.10 ± 0.20

Reducing sugars (%)

5.33 ± 0.18

7.97

0.18

Non-reducing sugars (%)

2.84 ± 0.67

6.54

0.16

Titratable acidity (%)

4.17 ± 0.11

5.00

0.21

6.49 ± 0.01

6.68

0.04

Morandin, G. C. et al.

Cad. Ciênc. Agrá., v. 15, p. 01–06, DOI: https://doi.org/10.35699/2447-6218.2023.45007

of the pseudofruits. Regardless of the extractor solvent

used, the DP presented a higher content (p<0.05) of

phenolic compounds than the MP. This difference bet-

ween the stages was more significant when the solvent

used was 100% ethanol, with the TPC content in the DP

being 3.5 times that of the MP. It is worth stressing that

the hydroalcoholic extract (50% ethanol) of the Japanese

grape pseudofruit proves to be an alternative for extracting

a high amount of phenolic compounds.

Figure 1 – Maturation stages of the Hovenia dulcis pseudofruits

Table 2 – Total phenolic compounds and antioxidant activity of the extracts of Japanese grape pseudofruits in different

maturation stages, obtained with different solvents.

Analysis Maturation Solvents

TPC

EC50

ABTS

Values expressed as averages

standard deviation. DP: development phase; MP: mature phase; Different lower-case letters on the same line and

different upper-case letters on the same columns, for the same analysis, indicate significant differences from the others (p<0.05); TPC: total phenolic

compounds expressed in mg of gallic acid equivalent per 100 g of dry sample; ABTS expressed in µM of Trolox per g of dry sample; EC50: extract

concentration in µg/mL capable of reacting with 50% of the radical present in the DPPH solution.

For the ABTS and EC50 analyses (extract con-

centration in µg/mL capable of reacting with 50% of the

radical present in the DPPH solution), in which lower

EC50 values result in higher antioxidant activity, the

extraction with 50% ethanol (v/v) rendered more con-

siderable antioxidant activity in both maturation stages

(Table 2). The lowest efficiency was found for the 100%

ethanol solvent, while water presented intermediary ef-

ficiency. When a higher number of OH groups is added,

stemming from ethanol, an increment in the hydrogen

bonds between the solute and water occurs, and, with

this, there is an increase in the solubility of some bioac-

tive compounds of Hovenia dulcis (Martins et al., 2013).

However, possibly, when a larger number of OH groups is

added (100% ethanol), the solubility of such compounds

decreases considerably.

The antioxidant activity was significantly diffe-

rent (p<0.05) between the two maturation stages for all

solvents used, being higher for the DP. These results can

be attributed to the phenolic compound content; however,

as the fruits mature, their phenols undergo oxidation

processes by polyphenol oxidase and participate in the

biosynthesis of anthocyanins, which accumulate during

fruit maturation, increasing the antioxidant activity or

keeping it high (Schwartz et al., 2009). Non-phenolic

stage

100% Water

50% Ethanol

100% Ethanol

182.27 ± 1.74

347.49 ± 0.66

28.18 ± 3.00

153.85 ± 4.35

272.47 ± 2.94

7.97 ± 3.42

494.4 ± 62.9

66.7

7.9

53443.8 ± 88.4

16240.0 ± 63.6

2865.0 ± 28.3

101387.5 ± 141.4

11.15 ± 1.02

345.34 ± 3.51

2.52 ± 0.10

7.10 ± 0.41

24.55 ± 1.10

1.62 ± 0.10

Hydroalcoholic extraction of antioxidant compounds in Japanese grape pseudofruits

Cad. Ciênc. Agrá., v. 15, p. 01–06, DOI: https://doi.org/10.35699/2447-6218.2023.45007

compounds such as acids (ascorbic, tartaric, and citric)

found in developing fruits, or their synergic action with

the phenolic compounds, may also have contributed to

the higher antioxidant activity of the pseudofruits at this

phase (Maieves et al., 2015).

Conclusions

The solvent system used for extraction and the

maturation stage of pseudofruits from the Japanese

grape influenced the total phenolic compound content

and the antioxidant activity of the extracts directly, with

50% ethanol (v/v) proving to be the best solvent for

extracting phenolic compounds and antioxidant activity

(evaluated through the ABTS radical capture method and

extract capable of reacting with 50% of the DPPH radical).

Pseudofruits in the development phase (DP) are richer

in phenolic compounds and present more considerable

antioxidant activity compared to those in the mature

phase (MP) when extracted with the solvents 100% water,

50% ethanol (v/v), and 100% ethanol. Moreover, the

pseudofruits of Hovenia dulcis T. present a high amount

of dietary fiber at the two maturation stages evaluated,

proving to be a promising source of this nutrient for use

in the food industry, along with its antioxidant potential.

Acknowledgements

The authors would like to thank ICL Food Spe-

cialties for supporting the research.

Funding

This study was financed in part by the Coorde-

nação de Aperfeiçoamento de Pessoal de Nível Superior

- Brazil (CAPES) - Finance Code 001 and by FAPESC

(Fundação de Amparo à Pesquisa e Inovação do Estado

de Santa Catarina) - Grant number: 2021TR854.

Full Disclosure

The authors also declare that there is no conflict

of interest in the research and publication of the manus-

cript.

Authors contribution

GCM

Conceptualization, Methodology, Valida-

tion, Formal analysis, Investigation, Writing - Original

Draft; SVS - Investigation; AMPA - Investigation;

ER - Funding acquisition, Methodology; GARS - Project

administration, Visualization, Writing - Review & Edi-

ting; Methodology, Conceptualization; DC - Supervision,

Visualization, Writing - Review & Editing; Methodology,

Conceptualization.

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