ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF MENTHA ARVENSIS EXTRACT SDEPENDING ON ETHANOL CONCENTRATION

Sam Woong Kim and Il-Suk Kim. Department of Animal Resources Technology, Gyeongnam National University of Science and Technology, Gyeongnam, 660-758, South Korea. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 5 (5), 653-660 655 ABTS ·+ radical scavenging activity:-ABTS ·+ radical cation discoloration assay was examinedfor the radical scavenging activity of the mint extract according to method of Re et al. (1999). The ABTS ·+ stock solution (A9941-100TAB, SIGMA, USA)was diluted by 50% ethanol solution to prepare anABTS ·+ working solution. The ABTS ·+ working solution was adjusted to 0.700±0.05 at 732 nm in wavelength at room temperature. An aliquot of the extract (2 μL of 500 μg/mL) was mixed with 50 μL of ABTS ·+ working solution, reacted for 10 min, and then measured by a microplate reader (Multiscan GO, Thermo Scientific co. ltd., USA) at 734 nm in wavelength. Ascorbic acid and butylatedhydroxyanisole (BHA) were employed as controls. Cation radical scavenging was presented by absorbance ratio between sample and control, and calculated as follows: ABTS ·+ radical scavenging capacity (%) = (1 − (AS − AS0)/A0) × 100% Where A0 is the absorbance of the negative control group without sample, AS0 is the absorbance of the sample solution and AS is the absorbance of the treatment group with sample.
Determination of antibacterial activity:-Antibacterial activity was examined by 8 mm disc dispersion method (Barry 1976 (ATCC 19114), Bacillus cereus (ATCC 1178), Vibrio parahaemolyticus(ATCC 17802D-5), and Candida albicans(ATCC 1023) were allocated from KCTC (Korean Collection for Type Cultures). The allocated bacteria were subcultured in Nutrient broth (Difco, USA). The subcultured bacteria were smeared on Nutrient agar, and positioned by 8 mm disc accumulated with 0.4, 1, 2, and 4 mg of each extract, and then incubated for 24 h at 37℃. After incubation, antibacterial activity was calculated by size of clear zone around the disc.

Statistics analysis:-
Individual comparisons among least squares means (LSM) for significant differences were made according to the multiple range test of Duncan. All analyses were performed within the SAS statistical software package (version 9.1, SAS Inst., Inc., USA), and differences were considered significant at P<0.05.

Results and Discussion:-
Total polyphenol and flavonoid contents:-In order to examine total extract yield depending on ethanol concentration, yieldsfromM. arvensis were presented by weight ratio of dry extract/raw material. The yields extracted from 0%, 30%, 50%, 70% and 100% ethanol concentrations were detected by 15.80, 17.28, 17.18, 14.94 and7.38% (w/w), respectively (Fig. 1). As a result, the extract yield was the highest content at 30% ethanol extract. Especially, it is assumed that the extremely low extraction yield in 100% ethanol is totally reduced owing to the low extraction yield of hydrophilic substances.
It has been well-known that polyphenolic compounds and flavonoids are closely associated with antioxidant activity (Macheixet al. 1990; Lee et al. 2002;Chung 1999;Fidriannyet al. 2013). Therefore, to analyze antioxidant activity in this study, we examined total polyphenol and flavonoid contents fromthe mint extracts (Table 1). Total polyphenols from the 50% and 70% ethanol extracts were detected by relatively high contents, 74.97 and 77.86 mg gallic acid/g, respectively, whereas total flavonoid in 100% ethanol was extracted to the most content, 62.51 mg catechin/g.
Taken together, it is estimated that the 50, 70 and 100% ethanol extracts to maintain high polyphenol and flavonoid contents are related with high antioxidant activities.
656 DPPH radical scavenging activity:-DPPH activity was examined for analysis of free radical scavenging ability from the mint extracts. DPPH activity depending on ethanol concentration was showed at Fig. 2. DPPH activities of the extracts exhibited totally lower values than those of ascorbic acid and BHA, but the 70% ethanol extract to maintain the most total phenolic content exhibited the strongest DPPH radical scavenging activity among the extracts. This result is consistent with a report that the activity increases proportional to the polyphenol content from the correlation between antioxidant content and free radical scavenging activity (Chung 1999).
Furthermore, in this study, the 30% and 50% ethanol extracts to maintain comparative high polyphenol content were detected by comparative highDPPH radical scavenging activities among the extracts. However, although total polyphenol content in the 0% (cold-water) ethanol extract was detected by a value higher than that of the 100% ethanol extract, the 0% ethanol extract showed the lowest value of DPPH activity. It is assumed that the extracted materials in 0% ethanol concentration have differential chemical properties to exhibit differential DPPH activity with the other ethanol extracts. Therefore, we suggest that polyphenoliccompoundsin the mint extract is directly associated with DPPH radical scavenging activity.

ABTS ·+ radical scavenging activity:-
We examined ABTS ·+ activity to analyze anionic free radical scavenging activity. ABTS ·+ radical scavenging activities of the mint extracts depending on ethanol concentration were shown in Fig. 2. The highest activity was detected by the 100% ethanol extract with 9.65%, and the 70% ethanol extract was observed by relatively high value.
Flavonoidshave antioxidant abilities by various patterns according to extraction solvent or extract materials, and in particular the materials extractedfromn-hexane are associated with high ABTS ·+ radical scavenging activity (Fidriannyet al. 2013). In addition, the prenylated flavonoid reactsstrongly with ABTS ·+ radical, but does not react with DPPH radical (Lee et al. 2006). Since anionic radical scavenging ability in this study was detected by relatively high values from the 70% and 100% extracts amongthe M. arvensisethanol extracts,and it is predicted which the 70% and 100% ethanol extracts contain lower polarities than those of the other extracts in this study, we suggest that materials to have relatively low polarity in the extracted fractions maintain ABTS ·+ radical scavenging activity. When compared with ascorbic acid, very low activities were totally observed in the extracts, but the 70% and 100% extracts showed activities higher thanthat of BHA.
From these results, we suggest that ABTS ·+ activity is associated with flavonoid content because of maintaining relatively high flavonoid content in the 70% and 100% ethanol extracts when compared with the other extracts.However, since the 0% ethanol extract showed the lowest value of ABTS ·+ activity,in the same way as in the DPPH activity,it is assumed that the extracted materials in the 0% ethanol have differential chemical properties to show differential ABTS ·+ activity with the other ethanol extracts.

FRAP activity:-
We examined to analyze Ferric-reducing antioxidant power (FRAP; %) from the mint extracts. FRAP activities for the mint extract depending on ethanol concentration were presented at Fig. 4. FRAP activities of the extracts were totally detected by very lower levels when compared with ascorbic acid and BHA. However, among activities of the extracts, the 50% ethanol extract was showed by relatively high value.
Phenolic compound is a secondary metabolite to maintain the most abundance in fruit as hydrophilic antioxidants (Macheixet al. 1990). Antioxidant activities measured by DPPH and FRAP assays are highly associated with total polyphenol contents in nectarines, peaches and plums (Gil et al. 2002). Furthermore, the correlation between total polyphenol content and antioxidant activity is determined by FRAP or electron spin resonance spectroscopy in fruit juices (Gardner et al. 2000). Therefore, we suggest that the higher antioxidant activities of the 0, 30, 50 and 70% ethanol extracts than that of the 100% extract are owing to higher total polyphenol content.
Antibacterial activity:-In order to examine antibacterial activities from the extracts, we evaluated antibacterial activities by various bacterial strains including Gram positive, Gram negative and yeast. Antibacterial activities of the mint extracts depending on ethanol concentration was shown at Table 2. The results of clear zone assays surrounding disc appeared antibacterial activity against C. perfringensfrom all the extracts, except for the 0% extract. Especially, the 657 70% and 100% extracts showed strong activitiesagainst C. perfringens. On the other hand, the 70% and 100% extracts showed strong activities against E. coli. However, we didn't observe the activities against the other-applied strains.

Conclusions:-
The yield of extraction for M. arvensis appeared the most amount at the 30% ethanol extract. Total polyphenol content was detected by relatively high values at the 50 and 70% ethanol extracts, whereas total flavonoid content was showed by the most amount at the 100% ethanol extract. As a result consistent with these contents, DPPH, ABTS and FRAP activities were assayed by the highest values at the 70, 100 and 50% extracts, respectively. However, ABTS and FRAP activities, except for the activity of DPPH, were very low in comparison with ascorbic acid and BHA. Antibacterial activity was detected only for E. coli and C. perfringens.