Abstract: Antioxidant activity and total phenolic contents of methanolic and hydroalcoholic-acetone extracts of Iranian Mentha pulegium in two stages of maturity were investigated. The aim was to investigate the most suitable solvent for extraction of antioxidants and to find the correlation existed between plant growth stage and its antioxidant capacity. In vitro antioxidant properties of the extracts were examined by 1, 1 diphenyl-2-picrylhydrazyl (DPPH) and Ferric Reducing/Antioxidant Power (FRAP) methods. Moreover, total phenolic contents were determined by the Folin-Ciocalteu method. On the other hand, the phenolic compounds were analyzed by chromatographic methods, TLC and SPE-reversed phase-HPLC. The results revealed that the antioxidant capacities and total phenol contents of the extracts in flowering season were higher than pre-flowering season. A positive relationship was found between the results obtained from three different assay methods used, i.e. FRAP, DPPH and phenol contents. The TLC chromatogram of the two extracts showed differences in the number of separated compounds of extracts. HPLC results indicated that the fraction collected with washing buffer (pH = 6) had highest antioxidant activity.
INTRODUCTION
New evidence derived from literature review have shown that Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) are related to the pathogenesis of many neurodegenerative diseases including multiple sclerosis (Van Horssen et al., 2008), Alzheimer’s Disease (AD) (Smith et al., 2000), Parkinson’s Disease (PD) (Bolton et al., 2000), Mild Cognitive Impairment (MCI) (Guidi et al., 2006), cardiovascular disease and cancer (Kris-Etherton et al., 2002). The imbalance between production and breakdown of these species increases the risk of chronic diseases. The antioxidant defense system inhibits free radical damage in our body. Natural antioxidants in fruits, vegetables and herbs can play a great role in supporting our body's defense system (Ali et al., 2008).
In the recent years, plant-derived antioxidants have received extensive attention from both food preservation industries and pharmaceutical companies (Hai et al., 2012). The phytochemical components including phenolic acids, flavonoids, carotenoids and ascorbic acid are good alternatives for synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) whose side effects on human health have been frequently reported (Ito et al., 1983).
Mentha is an aromatic plant belonging to the Labiatae family. The genus of Mentha is comprised of 20 species that spread throughout the world. Mentha pulegium is one of the Mentha species known as pennyroyal, a native herb of Asia, near East, Europe and North Africa (Chalchat et al., 2000). In the flora of Iran, the Mentha genus is represented by 6 species (Mozaffarian 1996). Mentha pulegium L. has been traditionally used as antiseptic for treatment of cold, sinusitis, cholera, food poisoning, bronchitis and tuberculosis (Zargari, 1990). In traditional medicine, it has some possible effects as antiflatulent, carminative, expectorant, diuretic and antitussive. Its larvicidal, acaricidal and cytotoxic activities have also been investigated (Atmani et al., 2009). Mahboubi and Haghi (2008) have reported cytotoxic effect of the essential oil of M. pulegium against different human cell lines (Shirazi et al., 2004). Furthermore, it has been reported that the essential oil and extracts of M. pulegium can exerts antimicrobial and antioxidant effects (Mahboubi and Haghi, 2008; Shahmohamadi et al., 2011; Nickavar et al., 2008). Although M. pulegium is frequently used in Gilan folk medicine, a scientific research on the biological properties of the plant was not found from this region.
Therefore, the aim of present study was comparing the antioxidant activity of M. pulegium by two known antioxidant tests (FRAP and DPPH) as well as total phenol content in two different extracts of it during two stages of maturity. On the other hand, it was planned to determine the correlation between antioxidant properties and phenolic constituents of the extracts. Finally it was aimed to investigate its phenolic compounds by partial purification of the extracts using chromatographic techniques, including TLC and SPE-reverse phase-HPLC.
MATERIALS AND METHODS
Chemicals and reagents: The 1,1 diphenyl-2- picrylhydrazyl (DPPH), 2,4,6 tripyridyl-s- triazine (TPTZ), sodium acetate, ferric chloride, sodium carbonat, sodium hydroxide, gallic acid, Folin-Ciocalteu’s phenol reagent and ferrous sulphate were purchased from Sigma representative in Iran. Chromatographic standard was obtained from Sigma. All solvents, methanol, acetone, acetonitrile and ethyl acetate were of analytical grade and purchased from Merck.
Plant materials: Mentha pulegium L. samples were collected during the months of April (pre-flowering stage) and August (flowering stage) 2011 from Saravan. The place is a region located in about 40 km from the Caspian Sea in Guilan Province, North of Iran. The taxonomic identity was confirmed by comparing the specimen with those of known identities collected in the Herbarium of the Department of Biology, University of Guilan. Fresh leaves were transferred to the biochemistry research laboratory, washed, drained and air dried for two weeks in the absence of light at room temperature. The dried samples were weighed, marked and stored in opaque paper bags.
Preparation of the extracts: The samples were extracted by two solvent systems, pure methanol and water-methanol-acetone (50-30-20). Extraction procedure was similar to the method of Wojdylo et al. (2007) with slight modification. Briefly, 0.5 g of the dried ground plant samples and 10 mL of solvents were added to test tube and the suspensions were shaken softly. Suspensions were sonicated twice for 15 min and left at room temperature (25°C) for 24 h. The extracts were filtered using Whatman No. 1 paper. The filtrated were centrifuged for 10 min in 1500 g and supernatants were collected and stored in at 4°C.
Estimation of antioxidant activity
General free radical-scavenging ability
DPPH Assay: The free radical-scavenging activity was determined using
DPPH° radical described by Brand-Williams et al.
(1995). This method is based on the reduction of stable DPPH when it accepts
hydrogen from an antioxidant compound. Radical scavenging activity of methanol
and methanol-acetone-water extracts against stable DPPH° was determined
spectrophotometrically. The change in color (from deep-violet to light-yellow)
was measured at 515 nm using a uv/visible light spectrophotometer (Ultrospec
3000 from Pharmacia Biotech). Briefly, 100 μL of various concentrations
of each extracts was added to 1500 μL of fresh solution of DPPH° in
methanol (6x10-5 mM) and mixed. The samples were kept in the dark
for 30 min at room temperature and then the decrease in absorption was measured.
The absorption of a control containing the same amount of methanol and DPPH°
solution was then measured. Each experiment was carried out in triplicate. Radical
scavenging activity was calculated using the following relationship:
where, A0-absorption of control, A1-absorption of extract solution
Ferric reducing antioxidant power (FRAP) assay: The Antioxidant Activity (AOA) of extracts was determined using the ferric reducing ability of plasma (FRAP) assay as described by Benzie and Strain (1999). The FRAP reagent was prepared by mixing 10 volumes of 300 mmol L-1 acetate buffer, pH 3.6, with 1 volume of 10 mmol L-1 2,4,6 tripyridyltriazine (TPTZ) in 40 mmol L-1 hydrochloric acid and with 1 volume of 20 mmol L-1 ferric chloride. Freshly prepared FRAP reagent was shaken at 37°C for 10 min, then 3 mL of this reagent was added to each sample solution and mixed thoroughly. The absorbance was read at 595 nm after 30 min. A standard curve was prepared using different concentrations (0.1-1 mmol L-1) of FeSO4. 7H2O (1 mM). The results were corrected for dilution and expressed in mmol FeSO4 L-1. All solutions were used in the day of preparation. All determinations were performed in triplicate.
Determination of total phenol content: Total Phenolic Content (TPC) in extracts was determined according to the Folin-Ciocalteu procedure described by Gao et al. (2000). In short, an aliquot of 100 uL of extracts was mixed with 0.2 mL Folin-ciocalteu phenol reagent and 2 mL of H2O and incubated at room temperature for 3 min. A 1 mL of 20% sodium carbonate solution was then added and allowed to stand for 1 h at room temperature. The absorbance of the reaction mixture was read at 765 nm using a uv/visible light spectrophotometer (Ultrospec 3000 from Pharmacia Biotech). An aqueous gallic acid solution (100-1000 mg mL-1) was used for calibration. Total phenol content was expressed as miligrammes Gallic Acid Equivalents (GAE) per 1 g of dry weight (dw). All determinations were performed in triplicate.
Polyphenolic analysis by thin-layer chromatography: Thin-layer chromatography was used for initial isolation to detect the active components in plant extracts. About 5 μL of methanolic and ethanolic extracts were loaded on silica gel plate (10x10 cm, Merck F254) and developed in horizontal tanks using a solvent system consisted of ethyl acetone:methanol:water (77:13:10) (Carnat et al., 2005). The separated components were visualized under uv light.
Fractionation of phenolic compounds by SPE procedure: In order to partially concentrating the extracts, ion exchange chromatography and ethyl silica gel cartridge were used. Briefly, 500 μL of extract was applied to a column ethyl Silica Gel (100 mg, 1 mL) was from Supelco. The extracts were taken to a column which was previously balanced with washing buffer (buffer phosphate with pH 5). Then the buffer phosphate with different pH values (6, 7, 8.5, 10, 11.5) were used for washing. All fractions were collected and antioxidant activity in each fraction was examined using DPPH reagent.
HPLC-DAD system for analysis of phenolic compounds: A 20 μL extract samples were analyzed using an Agilent 1200 Series system (Agilent Technologies) composed of a G1379A degasser, a G1311A quaternary pump and a G1316A column oven set at 30°C, containing a ZORBAX Eclipse plus C18 (250x4.6 mm, 5 μm) column with a flow rate of 1 mL min-1, coupled to a G1315B Photodiode-Array Detector (PAD) set to scan from 190 to 400 nm. All samples and solvents were filtered through a 0.22 μm filter (Machery-Nagal memberafilter prorafil) before injection solvent A methanol: acetonitrile: water (5:5:95) and solvent B the same system in the proportion of (5:95:5). The gradient system was as follows: 5% B (0-6 min), 25-40% B (6-15), 40-60% B (15-16). Detection was carried out at 366 nm. Phenolic compounds were identified by comparing retention times and uv-vis spectra with those of standards. The calibration curve of rosmarinic acid was calculated at 366 nm. The results were expressed as mg rosmarinic acid per 100 g of dry weight (dw) samples.
Statistical analysis: All experiment were repeated at least three times and the data were expressed as Mean±SD and two-way ANOVA. Means were compared using Duncan’s Multiple Range Test with significant level of 0.05 and curves were drawn using Microsoft Office Excel program.
RESULTS AND DISCUSSION
Estimation of antioxidant activity
DPPH Assay: The stable organic radical, DPPH, has been widely used to investigate
the free radical scavenging activity of different plant extracts (Katalinic
et al., 2006). The radical scavenging activities of both extracts
obtained from M. pulegium leaves in pre-flowering and flowering stages
are presented in Fig. 1a. It was found that water-methanol-acetone
extract in both stages (pre-flowering and flowering) exhibited into HPLC column.
The mobile phase was composed of significantly (p<0.05) highest scavenging
activity, 84.03 and 87.9%, respectively.
| Fig. 1(a-c): | (a) DPPH radical scavenging activity, (b) FRAP assay, (c) TP content in the extracts of M.pulegium 1; Methanolic extract in flowering stage, 2; Water-methanol-acetone extract in flowering stages, 3; Methanol extract in pre-flowering stage, 4; Water-methanol-acetone extract in pre-flowering stages. Significant differences are shown with different letters (p<0.05) |
On the other hand, the DPPH scavenging activity by methanolic extracts in pre-flowering and flowering stages were 51.76% and 70.78%, respectively which was weaker than water-methanol-acetone extracts (Ali et al., 2008). It has been reported that polar solvents could extract portions with higher radical scavenging activity compared to non-polar solvents (Tepe et al., 2005). A subsequent study has shown that radical scavenging properties of the water extracts are higher than the methanolic extracts (Kamkar et al., 2010). This characteristic of the extracts can be ascribed to their phenolics, flavonoids and terpenoids contents. Antioxidant activity of water extracts are higher than organic extracts because active components such as flavonoid, terpenoid, polyphenol, carotenoid and sterol, exist in fiber section of plants and are more easily dissolved in water (Rahmat et al., 2003). Our results were supported by some similar investigations, for example, Spigno et al. (2007) reported that the solvent used for extraction as well as time and temperature affect antioxidant activity of the extracts. The results obtained from the present study also indicate that there was no statistically significant difference between the antioxidant activities of water-methanol-acetone extracts in both stages.
FRAP assay: In this study, the ability of extracts to reduce Fe+3 to Fe+2 was determined by FRAP assay. This is a simple method to estimate total antioxidant capacity as FRAP value. As can be seen in Fig. 1b, the highest total antioxidant capacity was significant (p<0.05) in flowering stage in water-methanol-acetone and in methanolic extracts, 0.557 and 0.515 mmol Fe mL-1, respectively. Moreover, the FRAP value was highest in water-methanol-acetone extracts at both stages (p<0.05). The obtained data in pre-flowering and flowering stages were 0.352 and 0.557, respectively. The antioxidant activity of Labiate family has also been evaluated previously by other authors. All plants from this family have shown great capacity in reducing ferric, Fe+3 to ferrous ion, Fe+2 (Wojdylo et al., 2007). The results obtained by Kumar and Patra (2012), on M. piperita have indicated that the FRAP value was poor in the leaves before flowering, it was then considerably increased during the flowering stages and the poorest reported value was after flowering stage. Szollosi has explained that the higher total antioxidant capacity of leaves in flowering period could be due to the great amount of phenolic compounds and reducing power of its OH-groups. He also reported that content of rosmarinic acid was great in leaves of M. piperita (Kumar and Patra, 2012). The FRAP value obtained in present study was similar to the results reported by Szollosi. Diversity in antioxidant activity of extracts could be due to the difference in polarity of solvent as well as the ability of solvent to extract antioxidant compounds differently.
Total phenol content (TPC): TPC for both extracts in pre-flowering and flowering period was determined using Folin-Ciocalteu method. The results were expressed as mg gallic acid per gr dry weight of plant sample as shown in Fig. 1c. The TPC determined by Folin-ciocalteu reagent doesn’t show the exact amount of phenolic antioxidants, as many non-phenolic compounds such as ascorbic acid, beta-carotene and Cu (I) can also react with this reagent (Sultana et al., 2007). The results showed that water-methanol-acetone extract in flowering stages have the highest total phenol content 20.76, whereas methanolic extracts in pre-flowering stages have the lowest content of total phenol 11.32 mg GAE/g dw. It has been suggested that the members of Labiate family are remarkably rich in polyphenols (Wojdylo et al., 2007; Kosar et al., 2005; Nickavar et al., 2008) have reported higher amounts of total phenol in ethanolic extract of M. pulegium than the values we obtained in this study. The difference between the results of different researches may be related to extraction method, polarity of solvent, environmental differences (climate, location and time harvesting of sample) and also the examination methods (Kim and Lee, 2004). The results obtained by the present research showed that more than type and polarity of solvent season of sample collection was effective in amount of polyphenol content and antioxidant activity.
Our findings were in agreement with some previous studies reported by other researchers. For example, Moure et al. (1999) reported that it is much better to use more polar solvents for extraction of polyphenols. Matkowski et al. (2008) has found that in extracts of Labiate family the polar fractions had greater amount of total phenol in comparison to non-polar fractions. This may be due to the higher concentration of hydroxyl cinamic acid derivatives in polar fractions. On the other hand, many studies have explained that time of harvesting, maturity and age of samples affected the amount of phenolic compounds (Ghasemnezhad et al., 2011; Wang et al., 2007) have investigated the influence of the maturation of peanuts on the total phenol content of the hulls and the antioxidant properties of their methanolic extracts. Their results indicated that amounts of polyphenols increased during maturity. The findings of present study is in agreement with previous researches and has shown that the time of harvesting of samples affected the amount of the total phenol content and antioxidant ability of extracts.
Relationship between total phenol and antioxidant activity: While some authors have reported a correlation between polyphenol contents and antioxidant properties (Wojdylo et al., 2007; Katalinic et al., 2006; Atmani et al., 2009), others have shown poor or no correlation between phenolic composition and antioxidant properties of some plants (Capecka et al., 2005; Ismail et al., 2004). Therefore, the antioxidant activity observed could be due to the presence of other compounds such as ascorbic acid, tocopherol and some pigments (Li et al., 2005). Our investigation indicated a relatively direct correlation between the total phenol content and radical scavenging activity of extracts (R2 = 0.81) on one hand and between the total phenol content and the total antioxidant capacity (R2 = 0.75) on the other. Wojdylo et al. (2007) have reported high correlation between antioxidant activity as measured DPPH and FRAP as well as TPC (R2 = 0.83, 0.91) in Labiatae family. These surveys propose that phenolic compounds with reducing properties associated with their OH-groups, are responsible for the reasonably high antioxidant capability of M. pulegium as medicinal plant.
Chromatographic analysis: The phenolic compounds in ethanolic and methanolic extracts of both stages (pre-flowering and flowering) of M. pulegium were analyzed by TLC and viewed under UV 366 nm (Fig. 2). The revelation with UV indicated that ethanolic and methanolic extracts have different bonds. The TLC chromatogram of the two extracts showed differences in the number of separated compounds of extracts.
| Fig. 2(a-b): | Revelation of TLC plate with UV of methanolic (a) and ethanolic (b) extracts in pre-flowering (a) and flowering (b) stages |
Moreover, exact analysis of phenolic compound performed by SPE-reversed phase-HPLC method. SPE clean-up using ethyl silica gel cartridge was applied in order to pre-concentrate, remove interfering compounds and increase the efficiency of HPLC. The water-methanol-acetone extract in flowering stage was fractionated with buffer phosphate (pH = 5, 6, 7, 8.5, 10, 11.5) as washing buffer. Antioxidant activities in collected fractions were determined with DPPH reagent. The fraction collected with washing buffer (pH = 6) showed the highest antioxidant activity. This is in agreement with the results reported by Matthaus (2007). In his review article, Matthaus has explained that washing buffer with pH = 6-7 is effective for maximum separation of phenolic compounds of resin. This fraction and the extract before SPE clean-up were then injected to HPLC (Fig. 3). Figure 3 is a typical representative of the HPLC chromatogram for only the flowering stages. These findings were compared with the results of the extracts which were directly injected into column.
On the other hand, Kim and Lee (2004) have reported that rosmarinic acid is the main antioxidant in the Labiatae family. Many other authors have evaluated the content of rosmarinic acid in peppermint, spearmint, rosemary, sage (Diandian, 2008). The results obtained by Diandian (2008) have indicated that rosmarinic acid is the major phenolic acid in the HPLC profile of 50% ethanolic extract from mint. We, therefore, decided to determine the concentration of rosmarinic acid in water-methanol-acetone extract from both stages. Using the serial dilutions of stoke standard solution (20 μg mL-1), the calibration curves for rosmarinic acid in the range of 0.5-20 μg mL-1 at five concentration levels was obtained. Calibration plot was obtained by reporting peak areas as a function of analyte concentrations (R2 = 0.998). It was found that the concentration of rosmarinic acid in the extract of pre-flowering and flowering period were 130 and 120 μg 100 g-1 dry weight, respectively. Our results showed that the level of rosmarinic acid in the samples in pre-flowering stage was higher than the samples in flowering stage. This result supported by similar studies about the effect of environment on accumulation of RA in M. piperita and M. spicata experienced by Fletcher et al. (2010). The author reported that concentration of RA in pre-flowering stage were greater than flowering stage. Their findings are in agreement with those reported by Papageorgiou et al. (2008) and both reports support our findings from this study. The physiological stages of the samples have the major role in accumulation of RA. The investigation done by Fletcher has shown that by entering the flowering stage, RA content began to decrease.
| Fig. 3(a-b): | Typical HPLC chromatogram of water-methanol-acetone extract in flowering stage (a) before and (b) after SPE clean-up |
They also found that the samples in the vegetative period had the highest amount RA. According to the results obtained by Fletcher et al. (2010), when the plant moves from the physiological state towards reproduction, it may reallocate resources from RA production to the reproductive phase. Most probably it draws phenolics towards the production of lignin and formation of suberin in order to strengthen stem for structural integrity and to flavonoid production for flowering. This must be a usual mechanism in the Lamiaceae family. Therefore, it can be recommended that in commercial purposes, it is more useful to harvest the plants before flowering stage to get the optimum RA of the leaves of M. pulegium.
CONCLUSION
The results obtained from this survey demonstrated that the extracts of M. pulegium in flowering season possess higher phenolic compounds and antioxidant capacity than the extracts in pre- flowering season. Moreover, the hydroalcholic solvents extracted the highest amount of polyphenols due to the polarity of phenolic compounds. It could be emphasized that the physiological stages of the plant possess a basic role in accumulation of phenolic acids. On the other hand, the positive relationship between phenol contents and antioxidant behavior shows the importance of the phenolic compounds in the antioxidant activity of M. pulegium extracts. The TLC chromatograms of extracts showed that using of solvents with different polarity leads to extraction of various secondary metabolites. Our finding approved that SPE clean-up is a suitable procedure for concentration of samples and removal of interfering compounds before injection to HPLC. It is concluded that M. pulegium obtained from Gilan province in northern Iran is potentially a rich natural source of antioxidant, especially during its flowering season. Furthermore, our investigations suggested that this herb could be used as an excellent natural preservative in food and pharmaceutical industries. However, more extensive studies are required for identification and isolation of their major antioxidant compounds.
ACKNOWLEDGMENTS
The financial support by University of Guilan is highly appreciated. We also express our sincere thanks to Professor Ali Akbar for his helpful scientific advices.