Abstract
In this study, influence of the types of solvent and extraction time on the anthocyanins, polyphenols, and flavonoids content, and antioxidant activity of the extracts from sour cherry puree was investigated. Water, methanol, and ethanol, as well as those acidified solvents (with hydrochloric and acetic acid) were used. Extraction time was 1, 3, or 24 h at room temperature. Stability of the obtained extracts was monitored trough 42 days of storage at room temperature and at 4°C. The most efficient extraction solvent for anthocyanin, phenol, and flavonoid was methanol acidified with hydrochloric acid. The highest content of investigated compounds was observed after one hour extraction, except in the case of phenols, where prolongation of extraction time enhanced extraction of above mentioned compounds. During storage of samples, anthocyanins, total phenols, flavonoids contents, and antioxidant activity decreased, while the percentage of polymeric color increased.
INTRODUCTION
Polyphenols are secondary compounds widely distributed in the plant kingdom. They are divided into several classes, i.e., phenolic acids (hydroxybenzoic acids and hydroxycinnamic acids), flavonoids (flavonols, flavones, flavanols, flavanones, isoflavones, and proanthocyanidins) stilbenes, and lignans, which are distributed in plants and food of plant origin.[Citation1 − Citation3] They are an important constituent of fruit due to their contribution to taste, color, and nutritional properties.[Citation4] The beneficial effects of polyphenols are mainly attributed to their antioxidant properties, since they can act as chain breakers or radical scavengers depending on their chemical structures.[Citation3,Citation5] Phenolic compounds such as flavonoids, phenolic acid, and tannins possess various biological activities, such as anti-inflammatory, anticarcinogenic, and antiatherosclerotic activities. These activities might be related to their antioxidant activities.[Citation6]
Extraction of phenolic compounds from plant material depends on particle size of sample, extraction method, chemical structure of phenolic compounds, conditions during storage of the sample and presence of other compounds that could disturb the extraction procedure. Structure of phenolic compounds varies from simple phenols to polymerized ones. They can also be bounded to carbohydrates, proteins, and other compounds in plant material. Some phenolic compounds of higher molecular mass and their complexes could be insoluble thus hindering extraction from plant material. Obtained extracts of some plant material are mixture of phenolic compounds soluble in applied solvent. Sometimes, the additional step of removal of undesired phenols, lipids, wax, and terpens could be taken.[Citation7,Citation8] Solubility of phenolic compound depends on solvent polarity, degree of polymerization of phenols, interaction between phenols, and other components of food matrix and formation of insoluble complexes. There is no unique extraction procedure for all phenolic compounds. Different extraction procedures for studying phenolics in plant materials have been summarized by Naczk and Shahidi[Citation8] and they pointed out that water, methanol, ethanol, acetone, dichloromethane, diethyl ether, ethyl-acetate, and in lower extent propanol and dimethylformamid,[Citation8,Citation9] are used for extraction of phenolic compound. Acidified solvents were also wildly used for extraction of phenols, flavonoids, and anthocyanins.[Citation8,Citation9 − Citation19]
In this study efficiency of different solvents (methanol, ethanol and water) and acidified solvents on extraction of anthocyanins, phenols, flavonoids, and antioxidants from sour cherry puree was investigated. Solvents were acidified with hydrochloric acid and acetic acid. Extraction was conducted for 1, 3, or 24 h at room temperature. The objective of this study was to evaluate stability of extracts during storage at room temperature and at 4°C.
MATERIALS AND METHODS
Extraction of the Sour Cherry Puree
Sour cherry puree was prepared by mixing sour cherries which were bought at local market and kept at –20°C before analyses. Ten g of sour cherry puree was extracted with 100 mL of solvent. Solvent, water, methanol, and ethanol were used, as well as their acidified forms. Solvents were acidified with concentrated hydrochloric and acetic acid (0.5% of acid was added to solvents). Mixture of puree and solvent was well mixed and left for extraction at room temperature for 1, 3, or 24 h. Afterwards, the extraction mixture was filtrated and the extract was recovered and used for evaluation of anthocyanins, phenols, flavonoids, antioxidant activity, and polymeric color.
Monomeric Anthocyanin Content and Polymeric Color Determination
The monomeric anthocyanin pigment content of extracts was determined using the pH-differential method.[Citation20] Spectral measurements were conducted at 517 and 700 nm using spectrophotometer (Jenway 6300 Spectrophotometer). Total monomeric anthocyanins were expressed as mg cyanidin-3-glucoside/kg of fresh sour cherry (mg C-3-G/kg). Sample absorbance was read against a blank cell containing distilled water. The absorbance (A) of the diluted sample was then calculated according the following formula:
The monomeric anthocyanin pigment content in the original sample was calculated according to the following formula:
Polymeric color was determined using the bisulfite bleached sample using the following formula:
Total Phenol Content Determination
The total phenols content was determined by the Folin-Ciocalteu method.[Citation21] 0.1 mL of extract was diluted in 1.9 mL of deionized water, 10 mL of Folin-Ciocalteu reagent (1:10) was added and left to stand for 5 min, after which 8 mL of 7.5% of Na2CO3 solution was added. After 2 h, absorbance was read at 765 nm using a spectrophotometer. Total phenol content was determined using a gallic acid calibration curve. The results were expressed as mg gallic acid equivalent/kg of fresh sour cherry (mg GAE/kg).
Flavonoid Content Determination
Flavonoid content was determined by method described by Makris et al.[Citation22] One mL of extract diluted with methanol was mixed with 4 mL distilled water, than 0.3 mL 5% NaNO2 was added and allowed to react for 5 min. Following this, 0.3 mL 10% AlCl3 was added and the mixture was allowed to react for a further 5 min. At the end, 2 mL 1M Na2CO3 and 2.4 mL distilled water were added to the reaction mixture and the absorbance at 510 nm was read against a blank. Flavonoid content was calculated from a calibration curve using catechin as standard, and expressed as mg catechin equivalents/kg of fresh sour cherry (mg CTE/kg).
Antioxidant Activity
Antioxidant activity was determined using two methods, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-l-picryl-hydrazil (DPPH) method. The ABTS assay followed the method of Arnao et al.[Citation23] with some modifications. The results were expressed as μmol trolox equivalents (TE)/100 g of fresh sour cherry (μmol TE/100 g). Additional dilution was needed if the measured ABTS value was over the linear range of the standard curve. For DPPH assay, 0.2 mL of the sample was diluted with methanol, and 1 mL of DPPH solution was added. After 15 min, absorbance was read at 517 nm. The results were expressed as μmol trolox equivalents (TE)/100 g of fresh sour cherry (μmol TE/100 g). Additional dilution was needed if the measured DPPH value was over the linear range of the standard curve.
Storage of Sour Cherry Extracts
Obtained extracts (50 mL) were stored in glass jar at room temperature in dark and at 4°C. Evaluation of selected parameters was carried out after 7 and 42 days of storage.
RESULTS AND DISCUSSION
Anthocyanin Content and Polymeric Color
Results of anthocyanin content after 1, 3, or 24 h of extraction, with different solvents, are presented in . Anthocyanin content in extracts depended on both the solvent and time of extraction. Among the investigated extracts, anthocyanin content ranged from 40.82 to 64.12 mg C-3-G/kg. After 1 h extraction, the highest anthocyanin content had extract obtained with methanol/hydrochloric acid as solvent (64.12 mg C-3-G/kg) and lowest with water as a solvent (49.47 mg C-3-G/kg). With prolongation of the extraction time on 3 and 24 h, anthocyanin content in extracts didn't increase; in fact anthocyanin content was lower than after one hour extraction. Comparing solvents, anthocyanin content followed methanol > ethanol > water, meaning that methanol is most appropriate solvent for anthocyanin extraction from sour cherry puree. All samples which were obtained with extraction of puree with solvent and addition of hydrochloric acid had higher anthocyanin content. During storage degradation of anthocyanin occurred. At room temperature, anthocyanin content were lower than at 4°C, which was expected since it is well known that anthocyanins are susceptible to degradation at higher temperatures. Anthocyanin content followed the same tendency that was observed after extraction of puree. Retention of anthocyanins in extracts stored at room temperature for 42 days ranged from 61 to 80%, and in extracts stored at 4°C from 52 to 87%, depending on extraction time. Generally, the highest retention was observed in samples when extraction time was one hour. The highest retention (80%) of anthocyanins was observed in methanol, methanol/hydrochloric acid, methanol/acetic acid, and ethanol/hydrochloric acid extracts stored at room temperature. In extract stored at 4°C, the highest retention was observed in methanol extract (87%). Methanol/hydrochloric acid extracts at all extraction times had high retention of anthocyanins, 84 to 86%, in contrast to other extracts. Kajdžanoska et al.[Citation18] observed that methanol extracts of strawberry had higher anthocyanin yield than acidified extracts (acetic, hydrochloric, and formic acid). They reported that in extracts obtained with methanol acidified with formic acid and methanol/water/HCl mixture (80:18:2), acetylglucoside of pelargonidin was not detected, implying its hydrolysis. Rolle et al.[Citation24] reported that anthocyanin profile of Wine-Grape Skin extracts, obtained at different extraction times, didn't have significant differences among the distribution of different anthocyanins.
Table 1 Anthocyanin content (mg C-3-G/kg) in sour cherry puree extracts after 1, 3, or 24 h of extraction with different solvents and stability of extracts during 42 days of storage at room temperature and 4°C
The lowest polymer color percentage () was determined in samples obtained with water extraction, 8 to 17%, depending on time and solvent acidity, while the samples obtained with methanol and ethanol had the highest polymeric color (58 to 85%). During storage polymeric color increased, in higher extent at room temperature. Ito et al.[Citation25] conducted investigation with synthetic flavylium salts in solutions of different nature (acetonitrile/water, ethanol, propilenglycol, dioxane, and 2-butanone) and showed that changes on color depend on the solvent and the flavylium salts concentration. In protic solvents the flavylium salts exhibit red coloring, while in aprotic solvents the solutions were yellow. This fact has been explained by proposing that the red and yellow species correspond to a monomer and dimer respectively; therefore, when increasing flavylium salts concentration, the red coloring is favored. Also it was observed that, when increasing the water proportion, in acetonitrile/water mixtures, the monomer is transformed into a green color dimer (monomer with charge-transfer character). Thus, the water plays a fundamental role in the dimerisation of flavylium salts due to the fact that these molecules require neutralizing their own electrostatic repulsions with water molecules so that the dimerisation can be carried out.[Citation25,Citation26] The solubility of the solute into the solvent is expected to be different due polarity differences between solvents. Water, methanol, and ethanol are polar protic solvents of dielectric constants of 80, 33, and 24, respectively, while acetone and ethyl acetate are polar aprotic and non-polar solvents of dielectric constants of 21 and 6, respectively.[Citation27] It has been reported that extraction periods usually varying from 1 min to 24 h.[Citation8] Longer extraction times increase the chance of oxidation of phenolics unless reducing agents are added to the solvent system.[Citation28]
Table 2 Polymeric color (%) in sour cherry puree extracts after 1, 3, or 24 h of extraction with different solvents and stability of extracts during 42 days of storage at room temperature and 4°C
Table 3 Phenol content (mg GAE/kg) in sour cherry puree extracts after 1, 3, or 24 h of extraction with different solvents and stability of extracts during 42 days of storage at room temperature and 4°C
Table 4 Flavonoid content (mg CTE/kg) in sour cherry puree extracts after 1, 3, or 24 h of extraction with different solvents and stability of extracts during 42 days of storage at room temperature and 4°C
Total Phenol and Flavonoid Content
The total phenol content was estimated by the Folin-Ciocalteu colorimetric method, using gallic acid as a standard phenolic compound. The results of phenol content after extraction and during storage are presented in the . The highest value was obtained in methanol/hydrochloric acid extract (389.44 mg GAE/kg), while the lowest when water was used (186.96 mg GAE/kg) as a solvent. Comparing solvents, methanol was the most appropriate solvent, total phenol content followed methanol > ethanol > water order. Extraction time didn't have positive effect on total phenol content, except in case of methanol extracts were the highest phenol content was observed after 24 h of extraction. During storage, total phenol content decreased in higher extent at room temperature than at 4°C. Retention of phenols in extracts after 42 days of storage ranged from 75 to 95%, depending on storage temperature and extraction time. Retention of phenols was higher in extracts stored at 4°C. Interestingly, in the case of phenols, extraction time had high influence on their retention. Water/hydrochloric acid, water/acetic acid, and methanol/acetic acid extracts had the highest retention of phenols after 1 h of extraction at both investigated temperatures (90 and 93%, 83 and 85%, respectively). Methanol, methanol/hydrochloric acid, ethanol, ethanol/hydrochloric acid, and ethanol/acetic acid extracts had the highest retention of phenols in extracts obtained after 3 or 24 h of extraction. The highest retention of phenols was obtained in methanol/hydrochloric acid extracts obtained after 24 h of extraction (91% at room temperature and 95% at 4°C).
The total flavonoid content was estimated by a colorimetric method, using catechin as standard flavonoid. Results of flavonoid content are presented in the . The highest flavonoid content was obtained in methanol/hydrochloric acid extract (368.12 mg CTE/kg), and the lowest in water/acetic acid extract (143.75 mg CTE/kg). Comparison of solvent for extraction different tendency was observed than in a case anthocyanins and phenols. Flavonoid content followed ethanol > methanol > water order. Also, some differences in flavonoid content depending on extraction time were observed than in the case of anthocyanins and phenols. Extraction with water/hydrochloric acid and methanol after 3 h, water/acetic acid and ethanol/hydrochloric acid after 3 and 24 h were more efficient than extraction after 1 h. During storage, decrease of flavonoid content was observed. The highest retention of flavonoids was observed in methanol/hydrochloric acid extracts obtained after 24 h (87% at room temperature and 90% at 4°C). In all other cases, the highest retention was observed in samples after 3 h extraction. In ethanol extracts, determination of flavonoids couldn't be conducted probably due to some interactions between flavonoids and solvent.
The critical point in studying polyphenols in plant materials is the extraction procedure since it dictates the nature and quantity of polyphenols that will be transferred to the extract and further analyzed and characterized.[Citation18] Singh et al.[Citation29] investigated extraction of pomegranate peel and seed with methanol, ethyl acetate and water and found out that methanol extract of peel had the highest content of phenolic compounds, followed by water and ethyl acetate. The water extract of seeds had the highest phenolic content followed by the methanol and ethyl acetate extracts. They also concluded that the antioxidant activity may be directly correlated to the phenolic content of various peel extracts, but in the case of seed extracts, the phenolic content was quite low and there may not be any direct correlation between phenolic content and antioxidant activity. Negi et al.[Citation30] observed the highest phenol content in acetone extract, followed by methanol, ethyl acetate, and water extracts. Wang et al.[Citation27] investigated extraction of phenolics from pomegranate peels and observed that methanol gave the highest extract yield of the total phenolics, followed by water, ethanol, acetone, and ethyl acetate. The results of Amensour et al.[Citation31] showed the significant antioxidant activities of the methanol and water extracts, the overall strength being in the order of methanol > water > ethanol > ethyl acetate in leaf and berry myrtle extracts. They also found out that the amounts of phenolic compounds in the water extracts were highest, and lowest in the ethyl acetate extracts (in both leaves and berries). The extraction yields of phenolic compounds increased with increasing solvent polarity as reported by other authors.[Citation32,Citation33] In this case, different tendency was observed and methanol extracts had the highest phenol and anthocyanin content. In the case of flavonoid content the highest values showed methanolic extracts, followed by the aqueous extracts.[Citation31]
Antioxidant Activity
Antioxidant activity was determined using DPPH and ABTS method ( and ). After 1 h of extraction, values of antioxidant activity obtained by ABTS method were slightly higher when ethanol and methanol were used as extraction solvent in comparison to their acidified forms. After 3 h of extraction similar results were obtained, while after 24 h lower values were obtained. Results obtained with DPPH method showed similar results after 1 h of extraction regardless of used solvents. After 3 and 24 h of extraction lower values were obtained. During storage decrease of antioxidant activity occurred regardless of applied method.
Table 5 Antioxidant activity (ABTS method) (μmol TE/100 g) in sour cherry puree extracts after 1, 3, or 24 h of extraction with different solvents and stability of extracts during 42 days of storage at room temperature and 4°C
Table 6 Antioxidant activity (DPPH method) (μmol TE/100 g) in sour cherry puree extracts after 1, 3, or 24 h of extraction with different solvents and stability of extracts during 42 days of storage at room temperature and 4°C
CONCLUSION
Results of anthocyanins and total phenols obtained in this study showed that the most effective solvent for extraction was methanol, than ethanol and water. For the extraction of flavonoids, ethanol as a solvent would be better solution. Methanol acidified with hydrochloric acid showed the highest results in anthocyanin, total phenol, and flavonoid content. Selection of solvent and time of extraction highly influence on phenolic compounds yield in extracts but also stability of this compounds in solvent during storage. During storage degradation of phenols occurred, but the highest content of investigated compounds were determined in acidified methanol/hydrochloride acid extract. Calculating retention of investigated compounds during storage showed that the highest anthocyanin retention was observed in all methanol extracts and the highest retention of phenols and flavonoids in methanol/hydrochloric acid extracts.
ACKNOWLEDGMENT
This research is part of National Scientific Project financed by Croatian Ministry of Science, Education and Sports.
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