Angiotensin I-converting Enzyme (ACE) in Hibitory Activity of the Liquor
The ACE inhibitory activity of the liquor prepared from the berries was measured as described by Nagai et al. . The IC50 value was defined as the concentration of the liquor required to inhibit 50% of the ACE activity.
Hyaluronidase Inhibitory Activity of the Liquor
The hyaluronidase inhibitory activity of the liquor prepared from the berries was investigated by the Morgan-Elson method  with some modifications. A 0.02 ml of the extracts and 0.01 ml of hyaluronidase from Ovine testes (1,000U/ml; Wako Pure Chemicals Industries, Ltd., Osaka, Japan) were mixed in an Eppendorf tube and were pre-incubated at 37℃ for 20 min. The mixture was added 0.02 ml of compound 48/80 solution, and was incubated at the same condition. The enzyme reaction was started by the addition of 0.05 ml of hyaluronic acid solution. After incubation at 37℃ for 40 min, the reaction was stopped by addition of 0.02 ml of 1.77 N NaOH and 0.02 ml of 0.8 M boric acid-0.4 N NaOH. The mixture was boiled for 3 min, and then was cooled in water. The mixture was added 0.6 ml of p-dimethylaminobenzaldehyde solution. After incubation at 37℃ for 20 min, the absorbance of the mixture was measured at 585 nm. The inhibition rate was calculated by the measuring the amount of N-acetyl glucosamine released. Test samples were replaced by the buffer solution for the control. The enzyme solution was replaced by the buffer solution for the blank. Percent inhibition was calculated as the following equation.
Inhibition (%) = [(A - B) - (C - D)]/(A - B) x 100
A : control OD585; B : control blank OD585; C : sample OD585; D : sample blank OD585
Moreover, the IC50 value was defined as the concentration of the liquor required to inhibit 50% of the hyaluronidase activity.
Except for color analysis, each assay was repeated 3 times independently and the results were reported as means standard deviation (SD).
Results and Discussion
The liquor was prepared successfully from everbearing strawberry cultivar, 'Summertiara' berries (Figure 1). It gave a mellow rich and full-flavored. The liquor was sweet to the taste, because of addition of a large quantity of crystal sugar.
Chemical parameters of the liquor were investigated. The specific gravity and the pH at 20℃ were 1.047 and 3.65, respectively (Table 1). The Brix% at 20℃ was 25.7%. The titratable acidity of the liquor was estimated to 0.38% as citric acid equivalent; the sugar-acid ratio was calculated to 67.6. The sugar-acid ratio of the liquor was extremely high to add a large quantity of crystal sugar, although the Brix% of the berries as the ingredients was low (7.3%) and the titratable acidity was very high (1.19%) . The alcohol content was low about 6.1%, compared with the commercially available wine. Total vitamins B1, B2, and C contents were determined and were about 3.54 (μg/100 ml), 0.03 (μg/100 ml), and 344.8 (μg/100 ml), respectively (Table 1). Preservation and processing of the fruits cause significant changes of their nutritional properties and composition . Generally, it is known that the strawberry berries are the richest source of vitamin C . In fact, the content of total vitamin C of fresh 'Summertiara' berries was high (62.2 mg/100 g FW) . However, it suggested that not only vitamin C, but also vitamins B1 and B2 destroyed during brewing for over 4 years. It may be maintain the contents of vitamins B1, B2, and C by low temperature fermentation and under light-blocking condition. The contents of total phenols, total flavonoids, and total anthocyanins of the liquor were measured, and the results were as follows: 51.7 mg ellagic acid equivalent/100 ml, 17.0 mg (+)-catechin equivalent/100 ml, 2.0 mg P3G equivalent/100 ml, respectively (Table 1). Hakkinen et al.  reported the content of ellagic acid in strawberry cultivars, 'Jonsok' and 'Senga' berries in the storage conditions (a domestic freezer or refrigerator). The contents in these cultivars berries reduced to about 40% during nine months of storage at -20℃. They also reported the content in the jam maintained about 80% of that in fresh berries. In any case, the content of ellagic acid decreased in the storage condition or in the processing procedure .
The color of the liquor was Koji color tinged red color like fruit of evergreen shrubs in the rue family, Rutaceae (Figure 1). The color of the liquor was measured by a colorimeter, and the parameter was as follows: L* = 12.15, a* = 6.26, b* = 12.04 (Table 1). The characteristic of the berries is to have a predominately red color, either in the peels (L* = 26.57, a* = 22.14, b* = 15.51) or in the central portion (L* = 40.05, a* = 17.44, b* = 10.01) of the berries . After the red pigment of the berries was almost extracted to 24 hours from brewing start, the color gradually discolored during the brewing period for over 4 years.
Figure 1: The fruit liquor prepared from everbearing strawberry, cultivar, 'Summertiara' berries
Table 1: Chemical parameters of the fruit liquor prepared from everbearing strawberry cultivar, 'Summertiara' berries
*ellagic acid equivalent, **(+)-catechin equivalent, ***P3G equivalent
Antioxidative activity of the liquor prepared from the berries was determined to evaluate the inhibition effect at the initiation stage of linoleic acid peroxidation. The liquor showed the antioxidative activities and the activities increased with an increasing the concentration of the liquor (Table 2). The activities of 0.1 and 1.0% liquor were fairly low and the same as that of 1.0 mM ascorbic acid. The activity of 10% liquor was below the levels of 0.01 mM BHA, BHT, and trolox after 200 min. The activity of 100% liquor was almost equivalent to that of 5.0 mM ascorbic acid (Table 2). As the remaining total vitamin C content of the liquor was considerable low, it suggested that most of the effects were attributed to its high phenolic contents in the liquor.
Table 2: Antioxidative activity of the fruit liquor prepared from everbearing strawberry cultivar, 'Summertiara' berries
Superoxide Anion Radical Scavenging Activity
Superoxide anion radical scavenging activity of the liquor prepared from the berries was investigated. The activities tended to increase with an increasing degree of the concentration of the liquor, although 0.1% liquor was not detected the activity at all (Table 3). The 1.0% liquor was the same activity as 0.01% BHT, and the 10% liquor showed slightly lower activity than 1.0 mM BHA and α-tocopherol. On the contrary, the activity for 100% liquor was extremely high; it was higher than that of 1.0 mM trolox, but was not up to that of 5.0 mM ascorbic acid (Table 3). The IC50 value was calculated about 9.9% as the concentration of the liquor .Also, the TEAC against this radical was estimated to 7.47 x 104 μmol TE/kg. It has been widely known that vitamin C and bilirubin showed greater potential to scavenge superoxide anion radicals. The characteristics may be associated with total vitamin C contained in the liquor, even though the remaining total vitamin C content of the liquor was considerable low.
Hydroxyl Radical Scavenging activity
Hydroxyl radical scavenging activity of the liquor prepared from the berries was measured. The 0.1 and 1.0% liquor did not show the activity (Table 3). For 10% liquor, the activity was fairly low about 3.1% compared with 1.0 or 5.0 mM ascorbic acid. Even for 100% liquor the activity was not up to those of BHA, BHT, α-tocopherol, and trolox tested. The IC50 value was calculated to about 497.4% as the concentration of the liquor. The TEAC against the radical was 6.86 x 104 μmol TE/kg. Hydroxyl radical is scavenged by many species of compounds such as vitamin E, flavonoids, α- and β-carotene, cysteine, reduced glutathione, and so on. A great amount of total phenols, total flavonoids, and total anthocyanins were obtained in the liquor, nevertheless the scavenging activity against hydroxyl radicals were low.
Table 3: Superoxide anion radical, hydroxyl radical, and DPPH radical scavenging activities of the fruit liquor prepared from everbearing strawberry cultivar, 'Summertiara' berries
*0.1 mM ascorbic acid; **1.0 mM ascorbic acid
DPPH Radical Scavenging Activity
DPPH radical scavenging activity of the liquor prepared from the berries was determined. As a result, each sample exhibited the activity and these activities increased with increasing the concentration of the liquor (Table 3). The liquor for 0.1% did not show the activity. The 1.0% liquor showed the same activity as 1.0 mM ascorbic acid and 0.01 mM BHA and BHT. The activity for 10% liquor was slightly higher than those of 5 mM ascorbic acid and 1.0 mM BHT. On the other hand, 100% liquor possessed remarkable scavenging activity as well as 1.0 mM α-tocopherol and trolox (Table 3). The IC50 value was calculated to about 14.2% as the concentration of the liquor. The TEAC against the radical was 1.28 x 107 μmol TE/kg. The phenol compounds are reported to scavenge DPPH radicals, and its scavenging activity is correlated with the total phenol contents. As shown in Table 1, the liquor contained a great quantity of phenolic compounds. It seems that the higher scavenging activity of the liquor attributed to these phenolic compounds.
ACE Inhibitory Activity
ACE inhibitory activity of the liquor prepared from the berries was measured. Particularly, the liquor for 100% almost inhibited the activity about 94.1% (Table 4). The activity tended to increase the liquor concentration-dependently. High correlation was demonstrated between the concentration of the liquor and this activity, with R2 = 0.9953. The IC50 value against ACE activity was measured and was calculated to 51.7%.
Table 4: ACE and hyaluronidase inhibitory activities of the fruit liquor prepared from everbearing strawberry cultivar, 'Summertiara' berries
Hyaluronidase Inhibitory Activity
Hyaluronidase inhibitory activity of the liquor prepared from the berries was determined. As a result, it showed high dependence on the concentration of the liquor; the correlation coefficient against the activity was R2 = 0.9957. The liquor for 100% perfectly inhibited this activity (Table 4). The IC50 value against hyaluronidase activity was calculated to 48.6%.