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. 2011 Jun 25;49(4):510–514. doi: 10.1007/s13197-011-0435-8

Influence of sonication treatments and extraction solvents on the phenolics and antioxidants in star fruits

H V Annegowda 1, Rajeev Bhat 2,✉, Liong Min-Tze 3, A A Karim 2, S M Mansor 1
PMCID: PMC3550895  PMID: 23904662

Abstract

The present study was conducted to examine the effects of sonication treatments (time intervals of 0, 15, 30, 45 and 60 min.) on phenolics and other antioxidant compounds in starfruits extracted in methanol and water. Overall, methanolic extracts exhibited significantly higher extractability, percentage inhibition of DPPH radicals, ferric reducing antioxidant property (FRAP) value, antioxidant capacity, flavonoids, total phenolics and tannins (p < 0.05) compared to control (0 min) and aqueous extracts. Methanolic extract obtained after 30 min of sonication proved to be the best treatment with regard to various parameters evaluated. Results of the present study clearly indicated sonication treatments to be effective in enhancing the antioxidant compounds in starfruit extracts and could be further explored for commercial purposes to benefit the consumers.

Keywords: Antioxidants, Extraction, Polyphenols, Sonication, Star fruits

Introduction

Incorporation of fresh fruits and vegetables in the normal diet is known to promote health and can overcome some of the degenerative diseases like cardiovascular disease, ageing, brain dysfunction and cancer (Alothman et al. 2009). The positive health effects are linked to the presence of phenolic compounds, which exhibits rich antioxidant activities (Oboh and Ademosun 2011). These facts has led the researchers all over the world to explore the potential of enhancing the bioactive compounds in fresh plant products (fruits and vegetables), especially antioxidants by application of various simple and reliable food processing techniques (Bhat et al. 2011a, b). Ultrasound (sonication) treatment is an emerging, food processing technology, which posses varied applications when applied alone or in combination with other food processing methods. Sonication treatments have been reported to be effective for microbial decontamination along with enhancing certain quality parameters of fruit juices (Rawson et al. 2010; Bhat et al. 2011b). This treatment is considered to be advantageous due to its reduced processing time with lower energy consumption and being environmental friendly (Mason et al. 2005; Tiwari et al. 2008). It has been reported (Wang and Weller 2006) that ultrasound treatments are inexpensive, simple, reliable, and can be an effective alternative to conventional extraction techniques. Furthermore, the same authors have also stated that the major benefits of using ultrasound treatments in solid-liquid extraction include the enhancement of extraction yield and faster kinetics and like Soxhlet extraction, a wide range of solvents can be used for extracting natural bioactive compounds.

Starfruit or carambola (Averrhoa carambola L.) is a popular juicy fruit grown widely in the tropical and subtropical regions of the world. Fresh fruits (matured with greenish-yellow tinge) are widely used as a basic raw material for preparation of clarified juices, jellies, confectionaries and jam (Bhat et al. 2011a). Star fruits is reported to be low in sugar, sodium and acid, and rich in phenolic compounds like (−) epicatechin, vitamin C, proanthocyanidins and carotenoids (Shui and Leong 2004). Nevertheless, no detailed studies have been reported on the antioxidant capacity and the effects of sonication treatments on star fruits. Therefore, the main objective of the present study was (i) to comparatively evaluate the efficacy and/or efficiency of different solvents (methanol and aqueous) on the extraction of polyphenols and antioxidant compounds and (ii) to study the effects of sonication treatment at different time intervals on these compounds in star fruits.

Materials and methods

Fruit samples and chemicals: Fresh star fruit samples were purchased from the local wet market in Penang, Malaysia. All the fruits used in the study were of eating quality with uniform shape, size, color and ripening stage. The selected fruits were devoid of any apparent physical or microbial damage. Further, before use, fruits were washed with water (tap water followed by distilled water, 2–3 times) to remove the surface dirt and adhering particles. Chemicals: 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tri (2-pyridyl)-s-triazine (TPTZ), Folin–Ciocalteu reagent, vanillin, sodium carbonate, aluminium chloride hexahydrate, ferric chloride, sodium acetate, ascorbic acid, gallic acid, catechin were purchased from Sigma Chemicals, Germany. All the solvents used in this study were of analytical grade and purchased from Fisher Scientific Sdn. Bhd, Malaysia.

Extraction and ultrasound treatment

Five individual groups (for each time interval including control sample) of fruit slices (25 g) were homogenized in a kitchen blender with methanol and distilled water (1:10, sample to solvent ratio) as extracting solvents. Further, the homogenized samples (juice) along with solvent were kept in a beaker and sonicated at different time intervals of 15, 30, 45 and 60 min. at room temperature (25 ± 1 °C). The ultrasonic treatment was performed using an ultrasonic cleaner (42 kHz, 135 W; Branson ultrasonic corporation, USA). The solvent surface in the beaker was kept at the same level of water in the ultrasonic bath, maintained at a constant room temperature (25 ± 1 °C) and was monitored by an oven thermometer. Whenever a slight change or increase in the temperature in the ultrasonic bath was observed, fresh water was circulated to maintain the stability of water temperature at 25 °C. In order to avoid and minimize the evaporative loss of methanol, the beaker was covered with the aluminium foil. Additionally, all the sonication treatments were performed in the dark to avoid interference of light. After the extraction, the sample residues were dissolved in a known quantity of the respective solvents (100 mL, water and methanol) and were repeatedly extracted until the extracts became clear. The obtained extract was then filtered through Whatman No. 1 filter paper and the filtrate was concentrated in a rotary evaporator (Buchi Rotavapor R-215, Switzerland) under controlled vacuum. The concentrated extract was then dried in a freeze dryer (Labconco, Free Zone 6 Liter, USA) and kept at 4 °C in air tight container until further analysis.

The percentage yield of the samples was calculated as:

graphic file with name M1.gif

Antioxidant activity

Measurement of free radical scavenging activity by DPPH assay

The DPPH radical (1,1-diphenyl-2-picrylhydrazyl) scavenging activity of the methanolic and aqueous extracts of star fruit was measured according to the method described by Blois (1958). The DPPH radical scavenging activity was calculated as:

graphic file with name M2.gif

where, Ao is the absorbance of the control and A1 is the absorbance of the extracts.

Phosphomolybdenum assay

The method described by Prieto et al. (1999) was adapted to measure the antioxidant capacity of the two extracts. A calibration curve was prepared using a standard solution of ascorbic acid (25–300 μg mL−1) and the antioxidant activity was expressed relative to that of ascorbic acid.

Ferric reducing antioxidant property (FRAP assay)

The ability of the sample extracts to reduce ferric ions was measured according to the modified method described by Benzie and Strain (1996). Ferrous sulphate solution with the concentration ranging from 0.1 to 1 μM was used for the preparation of standard calibration curve. The FRAP values were expressed as mM ferrous equivalents per gram of plant material.

Antioxidant compounds

Total phenolics, tannins and flavonoids

The total phenolics content of the extracts was determined using the Folin-Ciocalteu (FC) assay based on the method of Singleton and Rossi (1965) whereas vanillin-HCl method with slight modifications was used to measure the total tannins concentrations in the extracts (Bhat et al. 2007).

The total flavonoids in the extracts were determined based on the method described by Sakanaka et al. (2005). The results were expressed as mg of (+)- catechin equivalent per gram of extract.

Statistical analysis

The results of the present study are presented as mean values of three replicates ± standard deviation. A one way analysis of variance was performed (ANOVA) and the significant differences between mean values were determined by Tukey’s pair wise test at a level of significance of p < 0.05. The statistical analyses were carried out using SPSS 12.01 (SPSS Inc USA).

Results and discussion

In the present study, two different solvent systems: methanol and water were used for extraction of antioxidant compounds from starfruits at 4 different time intervals (15, 30, 45 and 60 min) of sonication with ‘0 min’ serving as control. Earlier, similar experimental setup as that of the present study on the influence of different time intervals of sonication on the extractability of bioactive compounds from different plants and their products have been reported with encouraging results (Li et al. 2007; Paniwnyk et al. 2009). Hence, this encouraged us to take up the present study on starfruits.

The results obtained in the present study are highlighted in Table 1. With regard to aqueous extracts, control samples (extract obtained without sonication or ‘0 min’) showed significantly higher phenolics and antioxidant activities compared to extracts obtained after sonication treatments. A significant (p < 0.05) time dependent decrease in the percentage inhibition of DPPH radicals, FRAP value, antioxidant capacity, total phenolics, total tannins, and total flavonoids were recorded at the prolonged sonication intervals (15, 30, 45 and 60 min). According to the available reports (Mason and Petrier 2004; Paniwnyk et al. 2009) addition of water to a system reduces the antioxidant levels due to poor solubility in the extraction solvent. Also, the generation of free radicals like hydroxyl and peroxyl during sonication process might possibly degrade and react with the antioxidants in the extract.

Table 1.

Effects of sonication treatments on antioxidants in aqueous and methanolic extracts of star fruit*

Extract Percentage yield Percentage inhibition1 FRAP value2 Phosphomoly-bdenum assay3 Total phenolics4 Total flavonoids5 Total tannins6
Aqueous extract
0 min 4.4 ± 0.10ab 40.5 ± 0.96c 1.14 ± 0.03c 128.9 ± 1.53d 60.5 ± 0.55c 28.8 ± 0.35c 12.4 ± 0.28c
15 min 4.8 ± 0.12c 39.6 ± 0.35bc 1.09 ± 0.01b 126.2 ± 0.69c 58.8 ± 0.60bc 27.6 ± 0.13b 11.7 ± 0.18b
30 min 4.7 ± 0.08c 38.1 ± 0.75ab 1.06 ± 0.00ab 122.7 ± 0.31b 58.5 ± 1.15bc 26.4 ± 0.35a 11.2 ± 0.14a
45 min 4.3 ± 0.10a 38.0 ± 0.43ab 1.04 ± 0.01a 120.2 ± 0.72a 55.3 ± 0.57a 26.8 ± 0.61ab 10.9 ± 0.19a
60 min 4.6 ± 0.10bc 37.6 ± 0.82a 1.08 ± 0.01ab 123.1 ± 0.81b 56.9 ± 0.71ab 27.2 ± 0.48ab 11.2 ± 0.14ab
Methanolic extract
0 min 4.8 ± 0.12b 68.6 ± 1.80a 2.1 ± 0.01a 174.1 ± 3.35a 120.8 ± 0.30a 63.8 ± 2.24a 26.9 ± 0.18a
15 min 4.8 ± 0.08b 77.2 ± 1.53b 2.3 ± 0.02b 179.3 ± 0.83ab 122.3 ± 1.55b 65.3 ± 2.32ab 26.1 ± 0.45a
30 min 4.7 ± 0.24b 87.4 ± 0.41d 2.4 ± 0.00c 192.7 ± 2.81c 142.0 ± 0.25e 79.7 ± 2.09c 31.8 ± 0.42c
45 min 4.3 ± 0.16a 83.3 ± 0.59c 2.3 ± 0.01b 182.9 ± 3.44b 127.0 ± 0.62d 68.4 ± 1.31ab 29.2 ± 0.19b
60 min 4.6 ± 0.10b 79.1 ± 1.14b 2.2 ± 0.05a 185.1 ± 3.78bc 124.8 ± 0.50c 69.3 ± 0.61b 27.0 ± 0.45a
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*Values are the means of three independent replicates ± standard deviation (S.D.). Means in the same column with different superscripts letters denotes significant differences from each other at p < 0.05

1Percentage inhibition of DPPH radicals by samples (100 μg/mL); 2mM ferric reduction to ferrous/g extract (FRAP, Ferric Reducing Antioxidant Power); 3mg ascorbic acid equivalent antioxidant activity/g extract; 4mg gallic acid equivalent/g extract; 5mg catechin equivalent/g extract; 6mg catechin equivalent/g extract

With regard to methanolic extracts, the evaluated parameters like percentage inhibition of DPPH radicals, antioxidant capacity, FRAP value, total phenolics, total tannins, and total flavonoids showed significant increase (p < 0.05) at the treatment time of 30 min, which later on decreased slightly (at 45 and 60 min), but the values were still significantly higher than the control samples (‘0 min’) (p < 0.05). The increase in the antioxidant activities in methanol extracts can be attributed to the differences in the polarity and viscosity, which is much higher compared to solvents with low viscosity, low density and high diffusivity. These features makes it possible for the solvent like methanol to easily diffuse into the pores of the plant materials (plant cell) and leach-out the bioactive compounds (like that of antioxidants) more effectively (Hemwimol et al. 2006; Naczk and Shahidi 2006). Whereas, the decrease in some of the parameters after the extended time duration (at 45 and 60 min) might be attributed to the lower concentration gradient of the solvent, prolonged interval of sonication as well as to the probable degradation that could have occurred due to the generation of free radicals, mainly the highly reactive hydroxyl radicals.

As indicated in Table 1, the methanolic extracts revealed higher antioxidant activities over aqueous extracts. The percentage inhibition of DPPH radicals, FRAP value, total phenolics, total tannins, and total flavonoids of methanolic extracts of starfruit were two fold higher than the aqueous extracts. Solvent extraction of phenolics and antioxidant compounds by using methanol, ethanol, acetone or ethyl acetate have been routinely employed for extraction from fresh fruits (Alothman et al. 2009; Wijekoon et al. 2011). The recovery of polyphenols and antioxidant from plant materials is known to be influenced by the solubility of these compounds in a particular solvent used for extraction process, and is dependent on the solvent polarity (Naczk and Shahidi 2006; Annegowda et al. 2011). In each case, the same plant material might exhibit varied results mainly due to the varied chemical characteristics of antioxidant compounds that necessitate the use of solvents with different polarities to obtain high yield of antioxidants. Sonication treatments using methanol as an extraction solvent has been reported to enhance the bioactive compounds in plants and their products, thus supporting our observations (Wang et al. 2008; Paniwnyk et al. 2009).

Sonication treatment induces microstreaming effect and can enhance the mass transfer produced on the cavitational bubble collapse. This in turn results in the cell wall destruction, thus providing better contact and interactions of solvents in and out of the plant materials. Additionally, sonication treatment has been reported to improve the solvents extractability of materials at low temperatures (Sališová et al. 1997; Vinatoru et al. 2001; Albu et al. 2004).

Generally, when using normal conventional extraction procedures, phenolic compounds including various antioxidants degrade when mild thermal treatments are used. Accordingly, this might not provide optimal yields or accurate results for the evaluated antioxidants. In this regard, ultrasound extraction has been reported to be much better wherein it can provide better optimal yield at lower temperatures (Wu et al. 2001; Xia et al. 2006; Paniwnyk et al. 2009).

Conclusions

In the present study, sonication treatment significantly enhanced various antioxidants in the methanolic extracts (up to 30 min) of the starfruit compared to aqueous extracts. The results also highlight the importance of the solvent system used for ultrasound mediated extractions. Overall, sonication treatments being a physical mode of food processing holds high potential to be explored for industrial applications as an effective environment-friendly method for enhancing the extractability of natural antioxidants, and thus could effectively play a significant role in preventing several physiological and degenerative diseases in consumers.

Acknowledgments

Conflict of interest

No conflict of interest exists in this manuscript.

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