Phenolic composition and comparison of antioxidant ...

Phenolic composition and comparison of antioxidant activity of alcoholic extracts of Peppermint (Mentha piperita) Neda Farnad, Reza Heidari & Behnaz Aslanipour

Journal of Food Measurement and Characterization ISSN 2193-4126 Volume 8 Number 2 Food Measure (2014) 8:113-121 DOI 10.1007/s11694-014-9171-x

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Author's personal copy Food Measure (2014) 8:113–121 DOI 10.1007/s11694-014-9171-x

ORIGINAL PAPER

Phenolic composition and comparison of antioxidant activity of alcoholic extracts of Peppermint (Mentha piperita) Neda Farnad • Reza Heidari • Behnaz Aslanipour

Received: 12 September 2013 / Accepted: 22 January 2014 / Published online: 14 February 2014 Ó Springer Science+Business Media New York 2014

Abstract Peppermint (Mentha piperita) has long been regarded as a food and medicinal plant. At the present work, the antioxidant activity of the methanol, ethanol and methanol/ethanol (1:1) extracts of leaf fraction through various in vitro models was investigated in Iranian peppermints for the first time. Total phenol, flavonoid and anthocyanin contents were also determined. Our results showed the alcoholic extracts had different responses with different antioxidant methods. The methanol extract had maximum phenol content (3.57 ± 0.26 g Gallic acid/100 g Peppermint powder) and showed best superoxide radical (47.05 ± 0.85 %) and hydrogen peroxide (91.05 ± 1.50 %) scavenging activities. The methanol/ethanol (1:1) extract had maximum flavonoid (3.33 ± 0.12 g quercetin/ 100 g Peppermint powder) and anthocyanin contents (1.74 ± 0.21 g/100 g Peppermint powder) and showed best DPPH radical scavenging activity (82.82 ± 2.57 %, IC50 = 10.02 ± 0.63 mg/mL) as well as ferric reducing power (184.22 ± 14.10 lmol/100 g Peppermint powder). The ethanol extract only showed the highest nitric oxide radical scavenging activity (80.13 ± 7.12 %). Chlorogenic acid, rutin, and caffeic acid were found by HPLC analysis of the main phenolic components. These results show, Peppermint alcoholic extracts can be used as a natural antioxidants to reduce oxidative stress in human beings and as a possible food supplement or in pharmaceutical applications.

N. Farnad (&) R. Heidari B. Aslanipour Department of Biology, Faculty of Science, Urmia University, Urmia, Iran e-mail: [email protected]; [email protected]

Keywords Peppermint (Mentha piperita) Antioxidant activity Alcoholic extract Phenol Flavonoid Anthocyanin

Introduction Secondary metabolites such as phenolic acids and flavonoids are important products of medicinal plants which include: antioxidant, antibacterial, antitumor properties. Antioxidant compounds play significant roles in inhibiting formation of Reactive Oxygen Species (ROS) in organisms and decreasing the risk of chronic and degenerative diseases such as atherosclerosis, cancer, diabetes and alzheimer’s. Synthetic antioxidants are unsafe and in recent years because of their toxicity and carcinogenicity, considerable attention has been directed towards the identification of natural antioxidants from plants [1–7]. The genus Mentha (family Lamiaceae), comprises more than 25 species and represents about six species in the flora of Iran. One of the most important Mentha species is Mentha piperita known as Peppermint [8]. It is widely grown in temperate areas of the world but it is nowadays cultivated throughout all regions of the world for the production of essential oil [9]. The essential oil and extract from Peppermint have been used since ancient times as a folk medicine for the treatment of vomiting, flatulence, anorexia, bronchitis and also in cuisine [10, 11]. In recent years, a wide variety of applications Peppermint in perfume, cosmetic, pharmaceutical and food industries have been demonstrated. Many researchers have reported the antibacterial, antioxidant [12–14], anticongestive, antispasmodic, anti-inflammatory [15], antimutagenic [16], antiseptic, antipruritic, anticatarrhal [17], antifungal [18] and insecticidal properties [19]. Such multiple biological

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activities of Peppermint essential oil and extract might be ascribed to the presence of some components, such as flavonoids, phenols (caffeic acid, rosmarinic acid, eugenol and a-tocopherol), carotenes, betaine, choline, tannins and volatile oil composed primarily of menthol, menthone, menthofuran and menthyl acetate [20–23]. Several external factors including climate, cultivar, and geographic location affect in plant composition content; therefore, plant material collected from different parts of the world, may contain different novel compounds with other bioactivities [24]. Solvents differ in their extraction capabilities, depending on their own and chemical structure of the solute. The extractive efficiency of phenolic compounds from plant materials greatly depends on the solvent, therefore, the present study has been undertaken to evaluate the antioxidant activities of different leaf extractions of Peppermint cultivated in Iran (Sardasht in West Azerbaijan province), through various vitro models and also the extracts were further evaluated to determine the total amount of phenol, flavonoid and anthocyanin compounds. Moreover this study deals with determination of phenolic compounds in leaves of Peppermint with HPLC method.

Materials and methods Chemicals 1, 1-Diphenyl-2-picryl-hydrazyl (DPPH) was purchased from Sigma- Aldrich Company, Ltd. (Gillingham, UK). The solvents and all other chemicals and reagents were HPLC and analytical grade from Merck (Germany). Plant materials The fresh leaves of Peppermint collected from Sardasht mountains of West Azerbaijan province in May 2012. Mentha piperita species were identified by Dr Abbas Siami has been deposited in the herbarium of Science Faculty, Urmia University, Urmia, Iran. Preparation of extracts Fresh leaves of Peppermint were cut into small pieces and dried at room temperature. The dried plant materials were powdered with a grinder. Three extraction solvents were used: methanol, ethanol and methanol/ethanol (1:1). Extraction procedure involved the addition of 25 mL solvent to 2 g sample and shaking the samples for 60 min at low speed and then the extract was passed through Whatman filter paper No.1 (Whatman Ltd., England). Extraction was performed twice more with magnetic stirring for

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60 min. Finally, the three filtrates were combined, made up to 100 mL with solvent and stored at 4°°C in the dark. Light exposure was avoided during the extraction process. Determination of total phenolic contents (TPC) and total flavonoid contents (TFC) Total phenolic contents (TPC) of extracts were estimated with the Folin-Ciocalteu colorimetric method described previously [25] with a little modification. Folin Ciocalteu’s phenol reagent (1 mL) and 7.5 % w/v Na2CO3 (0.8 mL) were added to sample extract (20 lL) and the mixture reaction was incubated in the dark for 30 min. The absorbance of the reaction mixture was then measured at 765 nm. TPC were expressed in terms of g Gallic acid equivalents/ 100 g Peppermint powder (The calibration equation for Gallic acid: y = 0.042 x - 0.023, R2 = 0.998). Total flavonoid contents (TFC) of extracts were estimated with aluminum chloride colorimetric method described previously [26] with a little modification. 20 lL of extract was diluted with 1 mL of deionized water. Then 0.075 mL of 5 % NaNO2 was added to this mixture, which was allowed to stand for 5 min at room temperature, and 0.15 mL of 10 % AlCl36H2O was added. The mixture was allowed to stand for 6 min at room temperature, and 0.5 mL of 1 mol/L NaOH was added, and the total volume was made up to 3 mL with deionized water. The absorbance of the solution was measured immediately at 510 nm. TFC were expressed in terms of g quercetin equivalents/100 g Peppermint powder (The calibration equation for quercetin: y = 0.077 x - 0.013, R2 = 0.997). Determination of DPPH radical scavenging activity The free radical scavenging activity of plant extracts were determined by slight medications of the method described previously [27]. 40 lL of various concentrations (320 mg/mL) of the extracts in methanol (PM), ethanol (PE) and methanol/ethanol (1:1) (PME) was added to a 1 mL of DPPH(1,1-diphenyl 2-picryl hydrazyl) radical solution in ethanol (final concentration of DPPH was 0.2 mM). The solution was incubated for 30 min in the dark at room temperature. After the incubation, the mixture absorbance was measured at 520 nm. Gallic acid and ascorbic acid (0.125–20 mg/mL) were used as positive controls. The DPPH radical scavenging activity was calculated according to the following formula: % DPPH scavenging activity = ððA0 A1 Þ=A0 Þ 100: Where A0 is the absorbance of the control (blank, without extract) and A1 is absorbance in the presence of the extract, gallic acid and ascorbic acid. DPPH significantly decreases upon exposure to radical scavengers. The

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concentration of extract/positive control providing 50 % inhibition (IC50) was calculated from the graph plotted inhibition percentage against extract/positive control concentration.

measured at 540 nm against the corresponding blank solutions. The nitric oxide radical scavenging activity was calculated according to the following formula:

Determination of superoxide radical scavenging activity

Where A0 is the absorbance of the control (blank, without extract) and A1 is the absorbance of the present extract, Gallic acid and ascorbic acid.

For superoxide anion radical assay, the superoxide anion radicals were generated by a pyrogallol autoxidation system [28]. A volume of 9 mL of Tris–HCl buffer solution (50 mmol/L, pH 8.2) was added into a test tube, and the test tube was incubated in a water bath at 25 °C for 20 min. A volume of 40 lL of pyrogallol solution (45 mmol/L of pyrogallol in 10 mmol/L of HCl), which was also pre-incubated at 25 °C, was injected to the above test tube with a microlitre syringe and mixed up. The absorbance at 420 nm was measured 5 min later, and this denotes the speed of pyrogallol autoxidation. Ascorbic acid was used as positive control. The autoxidation speed was obtained by applying the above method and with the addition of a certain concentration of extract and positive control into the Tris–HCl buffer solution. Decreased absorbance of the reaction mixture indicates increased superoxide anion scavenging activity. The superoxide radical scavenging activity was calculated according to the following formula: % Superoxide scavenging activity = ððA0 A1 Þ=A0 Þ 100: Where A0 is the absorbance of the control (blank, without extract) and A1 is absorbance in the presence of the extract and ascorbic acid. Determination of nitric oxide radical scavenging activity Nitric oxide radical inhibition can be estimated by the use of Griess Ilosvay reaction [29]. In this investigation, Griess Ilosvay reagent is modified by using naphthyl ethylene diamine dihydrochloride (0.1 % w/v) instead of 1-naphthylamine (5 %). The reaction mixture (3 mL) containing sodium nitroprusside (10 mM, 2 mL), phosphate buffer saline (0.5 mL) and Peppermint leaves extracts (0.5 mL) was incubated at 25 °C for 150 min. After incubation, 0.5 mL of the reaction mixture was mixed with 1 mL of sulfanilic acid reagent (0.33 % in 20 % glacial acetic acid) and allowed to stand for 5 min to complete diazotization. Then, 1 mL of naphthyl ethylene diamine dihydrochloride was added, mixed and allowed to stand for 30 min at 25 °C. A pink colored chromophore was formed in diffused light. Gallic acid and ascorbic acid were used as positive controls. The absorbance of these solutions was

% Nitric oxide scavenging activity = ððA0 A1 Þ=A0 Þ 100:

Determination of hydrogen peroxide scavenging activity A modified version of the method described previously [30] was used to determine the hydrogen peroxide scavenging ability of Peppermint extracts. Peppermint extracts were dissolved in 3.4 mL of a 0.1 M phosphate buffer (pH 7.4) solution and mixed with 600 lL of a 43 mM solution of hydrogen peroxide (prepared in the same buffer). A blank solution was prepared the same way but without H2O2. The concentration of hydrogen peroxide was measured by reading the absorbance values at 230 nm of the reaction mixtures. Gallic acid was used as positive control. Scavenging activity of the hydrogen peroxide was calculated according to the following formula: % H2 O2 scavenging activity = ððA0 A1 Þ=A0 Þ 100: Where A0 is the absorbance of the control (blank, without extract) and A1 is the absorbance of the present extract and gallic acid. Determination of ferric reducing ability power (FRAP) The reducing power was determined by using FRAP assay described previously [31] with some modifications. Briefly, the FRAP reagent contained 2.5 mL of 10 mM tripyridyltriazine (TPTZ) solution in 40 mM HCl plus 2.5 mL of 20 mM FeCl3, and 25 mL of 0.3 M acetate buffer (pH 3.6) were freshly prepared. Peppermint extracts were dissolved in solvents at a concentration of 20 mg/mL. An aliquot of 10 lL of solution was mixed with 3 mL of FRAP reagent and the absorption of the reaction mixture was measured at k = 595 nm. Gallic acid and ascorbic acid were used as positive controls. Methanolic solutions of known Fe(II) concentration, in the range of 0.6–10 lmol/L (FeSO4), were used for obtaining the calibration curve. The FRAP value represents the ratio between the slope of the linear plot for reducing Fe3?-TPTZ reagent by Peppermint extract compared with the slope of the plot for FeSO4. Determination of total anthocyanin contents (TAC) The total anthocyanin contents (TAC) in the plant extracts were determined by using the pH-differential method

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previously described [32]. Anthocyanins demonstrate maximum of absorbance at 520 nm. The colored oxonium form predominates at pH 1.0, and the colorless hemiketal form at pH 4.5. The pH-differential method is based on reaction producing oxonium forms and permits accurate and rapid measurement of the total anthocyanins. Absorbances of the investigated extracts were calculated by:

Table 1 Total phenolic, flavonoid and anthocyanin contents of 20 mg/mL alcoholic extracts of Peppermint (Mentha piperita)

Ethanol

1.99 ± 0.32a

1.31 ± 0.20a

0.86 ± 0.07b

A ¼ ðA520 A700 Þ pH1:0 ðA520 A700 Þ pH4:5 :

Methanol/ ethanol

2.89 ± 0.30b

3.33 ± 0.12c

1.74 ± 0.21c

The concentration (mg/L) of each anthocyanin was calculated according to the following formula and expressed as Cyanidin-3-glucoside (Cy-3-glc) equivalents: concentration ðmg/LÞ of each anthocyanin A MW DF 10 ¼ : aL Where A is the absorbance = (A of pH 1.0- A of pH 4.5), MW is the molecular weight (g/mol) = 449.2 g/mol for Cy-3-glc, DF is the dilution factor, R is the extinction coefficients (L 9 cm-1 9 mol-1) = 26,900 for Cy3-glc, where L (path length in cm) = 1 for comparison the same extinction coefficient was used for other standard to calculate the concentration of each anthocyanins and thus reported result was expressed as Cy-3-glc equivalents. HPLC analysis For HPLC analysis, 1 g of dried and powdered plant material was extracted with 50 % methanol/water for 2 h at room temperature. The plant extract was hydrolyzed with 1.2 N HCl by refluxing in a water bath for 1 h [33]. All samples were filtered through a 0.45-lm pore size syringedriven filter before injection. A 20 lL aliquot of sample solution was separated using a HPLC Knauer system equipped with UV–Vis detector and a Eurospher 100-5 C-18 column (25 cm 9 4.6 mm; 5 lm). The mobile phase consisted of purified water with 2 % acetic acid (A) and acetonitrile (B) at a flow rate of 0.8 mL/min. Phenolic compounds were eluted under the following conditions: 0.8 mL/min flow rate and the temperature was set at 25 °C, isocratic conditions from 0 to 10 min with 0 % B, gradient conditions from 0 to 20 % B in 5 min, from 20 to 25 % B in 10 min, from 25 to 100 % B in 15 min, followed by washing and reconditioning the column. Phenolic compounds were detected at a wavelength of 280 nm and identified by comparing their relative retention times and UV spectra with authentic compounds and were detected using an external standard method. Statistical analysis All the assays were done in triplicate. Experimental data were expressed as mean ± standard deviation (SD). Data

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Extract

Total phenols content (g gallic acid/100 g)

Total flavonoids content (g quercetin/100 g)

Anthocyanin content (g/100 g)

Methanol

3.57 ± 0.26c

2.48 ± 0.10b

0.10 ± 0.04a

Each value is presented as the mean ± SD (n = 3)

analysis was done by using SPSS software, version 19.0. One-way analysis of variance was performed by ANOVA procedures. Significant differences between means were determined by Duncan’s multiple range tests. Values of p \ 0.05 were regarded as significant.

Results and discussion Total phenolic contents (TPC) and total flavonoid contents (TFC) Phenolic compounds are secondary metabolites of the plant which are widely spread throughout the plant. They are well known as highly effective free radical scavengers and antioxidant activity tests are positively correlated with the total phenol and flavonoid contents of the samples [34]. Free radicals are produced in higher amounts in a lot of pathological conditions and involved in the development of the most common chronic degenerative diseases, such as cardiovascular disease and cancer [35]. The antioxidant activity of phenolics is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers [36]. Phenolic compounds may contain one phenol subunit such as phenolic acids or more phenol subunits such as flavonoids and anthocyanins, etc. In this study, phenolic acids, flavonoids and anthocyanins are the major phenolics analyzed. Different types of compounds were extracted with organic and inorganic solvent because of their variable chemical nature but methanol was more selective for extracting phenolic compounds. In this study the TPC of the leaf extracts of PM, PE, and PME were noted to be 3.57 ± 0.26, 1.99 ± 0.32, and 2.88 ± 0.30 g Gallic acid/100 g Peppermint powder, respectively (Table 1). The results showed that the total phenolic contents in PM extract (3.57 ± 0.25 g Gallic acid/100 g Peppermint powder) were the highest as compared with the other extracts. The values of TFC varied from 1.31 ± 0.20 to 3.33 ± 0.12 g quercetin/100 g Peppermint powder (Table 1). The presence of methanol and ethanol together


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Table 2 DPPH, nitric oxide and superoxide radical scavenging activities values and IC50 values of 20 mg/mL alcoholic extracts of Peppermint (Mentha piperita) Extract

IC50 (mg/mL)

DPPH radical scavenging activity (%)

Nitric oxide radical scavenging activity (%)

Superoxide radical scavenging activity (%)

Methanol

14.77 ± 0.06d

66.98 ± 1.33a

53.33 ± 4.81a

47.05 ± 0.85c

Ethanol

11.32 ± 0.71c

80.74 ± 0.83b

80.09 ± 7.12c

15.12 ± 0.00b

Methanol/ethanol Gallic acid

10.02 ± 0.63b 0.15 ± 0.01a

82.82 ± 2.57b 93.00 ± 0.45c

51.67 ± 1.61a 61.16 ± 2.89b

11.20 ± 3.41a –

0.16 ± 0.01a

92.30 ± 0.26c

68.16 ± 0.90b

60.71 ± 1.78d

Ascorbic acid


with in the extraction solvent increased the total flavonoid contents yield as seen for PME extract (3.33 ± 0.12 g quercetin/100 g Peppermint powder). Comparison between PM and PE extracts shows that methanol is a better extracted phenol and flavonoid constitutes than ethanol. The difference in TPC and TFC could be the result of different polarity of the solvents and solubility of antioxidative compounds in these solvents. Oh and coworkers were evaluated the antioxidant and antimicrobial activities of various leafy herbal tea extracts [37]. Among the water extracts, the green tea (82.21 mg GAE/g herb tea), black tea (82.86 mg GAE/g herb tea), and peppermint tea (75.31 mg GAE/g herb tea) extracts had significantly (p \ 0.05) higher concentrations of phenolic compounds than the other tea extracts. In the ethanol extracts, green tea displayed the highest phenolic content (144.52 mg GAE/g herb tea), followed by mate tea (66.86 mg GAE/g herb tea) and persimmon leaf tea (46.42 mg GAE/g herb tea). Overall, the results indicate that ethanol extracts contain more phenolic compounds than water extracts, except for rooibos tea, black tea, mulberry leaf tea, and peppermint tea. Also, they found that total flavonoid contents in peppermint extract 19.75 mg CTE/g herb tea). The obtained results are more than those from our results. DPPH scavenging activity DPPH is a stable purple free radical, and accepts an electron or hydrogen radical to become a stable yellow compound (diphenyl-picrylhydrazine) at room temperature. Reductions in the DPPH radical induced by antioxidants are determined by the decrease in its absorbance at 520 nm [38]. DPPH is usually used as a reagent to evaluate free radical scavenging. Gallic acid and ascorbic acid, with well-known antiradical activity, were tested for comparison activity of antioxidants. The DPPH radical-scavenging activities of the reference substances and the extracts are shown in Table 2. Additionally, the concentrations of the extracts required to scavenge 50 % of the DPPH radicals, the IC50 values, were calculated. The IC50 value is negatively related to antioxidant activity.

In this investigation all the extracts of Peppermint are shown to possess significant DPPH radical scavenging activity. Both PME (82.82 ± 2.57 % IC50 = 10.02 ± 0.63 mg/mL) and PE (80.74 ± 0.83 % IC50 = 11.32 ± 0.71 mg/mL) extracts demonstrate significant DPPH scavenging activity; indicate their abilities to act as radical scavengers and also difference in scavenging activity is statistically insignificant. The PM extract shows the lowest DPPH scavenging activity (66.98 ± 1.33 %, IC50 = 14.77 ± 0.06 mg/mL) at a concentration of 20 mg/mL. The DPPH radical scavenging activity values for Gallic acid and ascorbic acid are 93.00 ± 0.45 % with IC50 = 0.15 ± 0.01 mg/mL and 92.30 ± 0.26 % with IC50 = 0.16 ± 0.01 mg/mL, respectively. The results indicate that the DPPH radical scavenging activities of the extracts are lower than standards and the DPPH radical scavenging activity of PME and PE is better than that of PM. Singh and coworkers were explored the essential oil and different extracts of M. piperita for antioxidant activity by evaluating their antioxidant capacity, DPPH free radical scavenging activity and reducing power [9]. Chloroform extract and peppermint oil showed almost equal DPPH antioxidant potency (about 90 %). Aqueous extract exhibited the least potency among all (70.3 ± 6.1). The rest of the leaves extracts showed DPPH scavenging in between those of chloroform and aqueous extracts. The IC50 (lg/mL) of peppermint oil by using DPPH scavenging method was found to be 15.2 ± 0.9. Nitric oxide radical scavenging activity Nitric oxide (NO) is a freely diffusible gaseous free radical. The interactions of NO with reactive oxygen species (ROS) such as H2O2 and O2- can be protective or cytotoxic [39]. At physiological level NO can limit oxidative injury but under high concentration of NO, a number of extremely reactive nitrogen oxide species, such as N2O3 and ONOOcan be produced, which cause toxic reactions including lipid peroxidation, DNA modification and SH- oxidation. Natural extracts with nitric oxide scavenging ability

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prevent the toxic effects of excessive NO generation in the human body [40, 41]. Nitric oxide radical inhibition can be estimated by the use of Griess Ilosvay reaction. Sodium nitroprusside in aqueous solution at a physiological pH, spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent. Scavengers of nitric oxide compete with oxygen, leads to reduce production of nitrite ions [42]. The maximum nitric oxide radical scavenging percentage was 80.13 ± 7.12 % for PE and the minimum nitric oxide radical scavenging percentage among extracts was 51.67 ± 1.61 % for PME (Table 2). The PE had more nitric oxide radical scavenging percentage than gallic acid (61.16 ± 2.89 %) and ascorbic acid (68.16 ± 0.90 %) and the others had lower nitric oxide radical scavenging percentage than standards. The scavenging effect of PE extract on the nitric oxide radical was more efficient than other extracts and the presence of methanol in the extraction solvent decreased nitric oxide radical scavenging activity of Peppermint. Superoxide radical scavenging activity The superoxide anion radical is generated via mitochondrial respiration [43], by NADPH oxidase [44], xanthine oxidase [45], cyclooxygenase and lipoxygenase [46], nitric oxidase synthetase (NOS) [47] and cytochrome P450 [48]. The formation of other reactive oxygen species such as hydrogen peroxide, hydroxyl radical and singlet oxygen occur in the presence of high concentration of superoxide anions. These anions affect the physiological function of cellular organelles through their reactivity’s (i.e. oxidative damage in lipids, proteins and DNA) [49]. It is therefore interesting to measure the ability of the antioxidant extracts to scavenge the superoxide radical. There are several methods for the generation of superoxide radicals in vitro conditions. In our study, superoxide radicals are generated by auto-oxidation of pyrogallol in the presence of Tris–HCl buffer. The decrease of absorbance at 420 nm with antioxidants indicates the consumption of superoxide anion in the reaction mixture [28]. Table 2 shows the super oxide radical scavenging activity with a 20 mg/mL concentration of PM, PE, PME, and ascorbic acid. As shown in Table 2 the inhibition percentage of superoxide generation by a 20 mg/mL concentration of PM, PE and PME are found to be 47.05 ± 0.85, 15.12 ± 0.00 and 11.20 ± 3.41 %. The scavenging effect of PM extract on the superoxide anion radical is more efficient than other extracts, but less efficient than ascorbic acid (60.71 ± 1.78 %) and the presence of ethanol in the extraction solvent decreases superoxide radical scavenging activity of Peppermint.

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N. Farnad et al. Table 3 H2O2 radical scavenging activities and FRAP values of 20 mg/mL alcoholic extracts of Peppermint (Mentha piperita) Extract

H2O2 radical scavenging activity (%)

FRAP value (lmol/100 g)

Methanol

91.05 ± 1.50da

175.91 ± 9.90a

Ethanol

63.25 ± 3.52c

181.45 ± 14.05a

Methanol/ethanol Gallic acid

31.86 ± 1.22b 16.91 ± 0.86a

184.22 ± 14.10a 707.68 ± 4.91c

Ascorbic acid

–

493.26 ± 10.94b

Each value is presented as the mean ± SD (n = 3) a

Values in the same column followed by different letters are significantly different at p \ 0.05

Hydrogen peroxide scavenging activity Hydrogen peroxide itself is not dangerous to cells but its toxicity derives from its conversion to other species of ROS such as hydroxyl radical which initiate lipid peroxidation can cause DNA damage [50]. In the body, H2O2 generates hydroxyl radicals by the Fenton reaction with iron or is rapidly decomposed into oxygen and water and this may produce ˙ ). Thus, removing hydrogen peroxide hydroxyl radicals (OH as well as other ROS is a very important protection to many cellular components such as protein and DNA [51]. The hydrogen peroxide scavenging activities of extracts are shown in Table 3. PM, PE and PME extracts show fairly high (91.05 ± 1.52 %), moderate (63.25 ± 3.52 %) and low (31.86 ± 1.22 %) scavenging capacity against H2O2, respectively. The PM extract has more hydrogen peroxide scavenging activity than Gallic acid (16.94 ± 2.86 %) at a concentration 20 mg/mL, strongly suggesting that this extract contains the necessary compounds for hydrogen peroxide elimination. Ferric reducing antioxidant power (FRAP) The FRAP method is used to evaluate the reducing potential Peppermint extracts. The principle of this method is based on the reduction of a ferric 2,4,6-tripyridyl-s-triazine complex (Fe3?-TPTZ) to its ferrous colored form (Fe2?-TPTZ) in the presence of antioxidants. In this research, we find that when we use 20 mg/mL of samples, the FRAP activity of the PME extract (184.22 ± 14.10 lmol/L) is the highest of other extracts but not significantly different which indicates the fact that the Peppermint extracts contain the highest levels of antioxidant compounds. The FRAP activities of extracts are lower than standards (Table 3). Total anthocyanin contents (TAC) Anthocyanins are a major class of flavonoid compounds that are existed in fruits and vegetables. Due to antioxidant


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Fig. 1 HPLC chromatograms of a extract of Peppermint and b mixture of standard phenolic compounds at 280 nm. Peak: 1 Ascorbic acid, 2 Rutin, 3 Gallic acid, 4 Chlorogenic acid, 5 Ferulic acid, 6 4-Hydroxy benzoic acid, 7 Vanillic acid, 8 Caffeic acid, and 9 1-Naphthol

activity, anthocyanins reduce the risk of several oxidative stress diseases. Table 1 shows the anthocyanin contents of all the extracts of which the PME possesses markedly higher anthocyanin contents (1.74 ± 0.21 g/100 g Peppermint powder) than others. The PE (0.86 ± 0.071 g/ 100 g Peppermint powder) also contains a good amount of anthocyanins in comparison with PM (0.10 ± 0.045 g/ 100 g peppermint powder).

Table 4 Content of phenolic compounds in extract of Peppermint

6

4-Hydroxy benzoic acid

6.23 ± 0.28

HPLC analysis

7

Vanillic acid

0.51 ± 0.07

8

Caffeic aicd

9.67 ± 0.06

After extraction and acid hydrolysis, the contents of phenolic substances were determined by quantitative HPLC analysis. A typical HPLC chromatogram of standard phenolic compounds and the leaf extract from Peppermint is presented in Fig. 1. The content of the individual phenolic acids are listed in Table 4 and expressed as mg/100 g Peppermint powder. It can be noticed that a satisfactory separation with good resolution can be achieved by the optimized HPLC condition. A total of eight phenolic compounds (ascorbic acid, rutin, chlorogenic acid, ferulic acid, 4-hydroxy benzoic acid, vanillic acid caffeic acid and 1-naphthol) are identified by comparison with the retention times and UV spectra of authentic standards analyzed under identical analytical conditions (Fig. 1). The quantitative data is calculated from their respective calibration curves (Table 5). The amount of phenolic compounds decreased in the order of: chlorogenic acid[ rutin[ caffeic

9

1-Naphtol

5.30 ± 0.07

No.

Phenolic compound

(mg/100 g Peppermint powder)

1

Ascorbic acid

1.20 ± 0.20b

2

Rutin

11.20 ± 0.20

3

Gallic acid

n.d.

4

Chlorogenic acid

21.49 ± 0.77

5

Ferulic acid

4.31 ± 0.29

Each value is presented as the mean ± SD (n = 3) n.d not detected

acid[ 4-hydroxy benzoic acid[ 1-naphthol[ ferulic acid[ ascorbic acid[ vanillic acid (Table 4). Three different polyphenol in the category of hydroxycinnamic acid derivatives in decreasing order are chlorogenic acid[ caffeic acid[ ferulic acid. From different polyphenols in the category of hydroxybenzoic acid derivatives the amount of 4-hydroxy benzoic acid is more than vanillic acid and Gallic acid is not detected. HPLC analysis reveals that Peppermint extract also contains rutin, ascorbic acid and 1-naphthol as other phenols. Lv and coworkers were investigated cinnamon and peppermint for their phenolic profile and antioxidant properties [52]. For peppermint, the most abundant

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Table 5 Linear calibration curves for the HPLC–UV analysis of the phenolic compounds of extract from Peppermint (Mentha piperita) No.

Phenolic compound

Retention time (min)a

Equation of linear regression (peak area-concentration)

R2

Linear range (mg/L) 0.005–5

1

Ascorbic acid

3.6

Y = 87834X - 58655

0.998

2

Rutin

5.4

Y = 2e ? 06X - 1e ? 06

0.998

0.5–10

3

Gallic acid

7.2

Y = 2E ? 06x - 2E ? 06

0.997

0.5–10

4

Chlorogenic acid

10.3

Y = 68759x ? 3509.5

0.989

0.5–10

5

Ferulic acid

11.0

Y = 165138x - 136553

0.988

0.5–10

6

4-Hydroxy benzoic acid

11.4

Y = 838185x - 1E ? 06

0.998

1–10

7

Vanillic acid

12.0

Y = 3E ? 06x - 29609

0.999

0.05–10

8

Caffeic aicd

14.7

Y = 1E ? 06x - 2E ? 06

0.988

1–10

9

1-Naphtol

22.3

Y = 1E ? 06x - 76169

0.998

1–10

a


phenolic compound was caffeic acid (2221.1 lg/g), but it was not detected in cinnamon. Catechin, ferulic acid, syringic acid, (-)-epigallocatechin gallate, gallic acid, vanillic acid and p-coumaric acid were also detected as 587.7, 507.9, 240.5, 259.6, 2.7, 85.2 and 318 lg/g, respectively.

Conclusion The present study shows that Peppermint (M. piperita) growing in Iran, which often used in Iranian food and folk medicine, is a strong radical scavenger. The extracts of various alcoholic solvents are found to have different levels of antioxidant activity in the systems tested. This could be due to the presence of an enormous amount of flavonoid and phenolic compounds. The study also reveals the possible antioxidant mechanism of the extracts such as electron-donating ability and direct free radical scavenging properties. The methanol extract showed the maximum phenol content with the best superoxide radical and hydrogen peroxide scavenging activities. While, the methanol/ethanol (1:1) extract had maximum flavonoid and anthocyanin contents and showed best DPPH radical scavenging activity as well as ferric reducing power. The ethanol extract only showed the highest nitric oxide radical scavenging activity. Also, the individual main phenolic components of studied extracts were determined by HPLC analysis. These extracts can be used as easily accessible source of natural antioxidants and as a possible food supplement or in pharmaceutical applications. It can also be used in stabilizing food against oxidative deterioration.

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