Antioxidant and lipoxygenase inhibitory activities of ...

Food Research International 42 (2009) 641–646

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Antioxidant and lipoxygenase inhibitory activities of pumpkin seed extracts Marianna N. Xanthopoulou, Tzortzis Nomikos *, Elizabeth Fragopoulou, Smaragdi Antonopoulou Department of Science of Nutrition-Dietetics, Harokopio University, 70 El. Venizelou Street, Athens 17671, Greece

a r t i c l e

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Article history: Received 13 November 2008 Accepted 2 February 2009

Keywords: Pumpkin seed Antioxidant activity Lipoxygenase DPPH Lipids

a b s t r a c t Pumpkin seeds have been implicated in providing health benefits. However their antioxidant or antiinflammatory activity of their extracts has never been studied. Therefore, four commercially available pumpkin seeds were treated with two different extraction methodologies in order to obtain fractions with different content. The extracts were screened for their antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay and for their inhibitory activity against lipid peroxidation catalyzed by soybean lipoxygenase. Most extracts tested have demonstrated radical scavenging activity, which depends on their total phenolic content, with fractions rich in phenolics showing the strongest activity. On the other hand, the phenolic content of extracts does not determine their activity against lipoxygenase, as acetone and polar lipid fractions are its strongest inhibitors. The presence of molecules being able to scavenge radicals and inhibit lipoxygenase in pumpkin seeds may in part explain the health benefits attributed to them. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Pumpkins belong to the family of Cucurbitaceae. They are classified to Cucurbita pepo, Cucurbita moschata, Cucurbita maxima and Cucurbita mixta, according to the texture and shape of their stems. Pumpkin seeds are a popular snack food in several countries among of which is Greece. They are consumed either raw or roasted (salted or not) and used in cooking and baking as an ingredient of bread, cereals, salads and cakes. Moreover, pumpkin seed oil nowadays gains wide acceptance not only as edible oil but as a nutraceutical, too. Pumpkin seed and seed oil have been implicated in providing many health benefits, which are attributed to their macro- and micro-constituent composition. They are a rich natural source of proteins, phytosterols (Phillips, Ruggio, & Ashraf-Khorassani, 2005; Ryan, Galvin, O’Connor, Maguire, & O’Brien, 2007), polyunsaturated fatty acids (Applequist, Avula, Schaneberg, Wang, & Khan, 2006; Sabudak, 2007), antioxidant vitamins, such as carotenoids and tocopherol (Stevenson et al., 2007) and trace elements, such as zinc (Glew et al., 2006). One of the most critical health benefits attributed to pumpkin seed oil is its activity against benign prostate hyperplasia. Styrian oil and phytosterol-rich pumpkin seed extracts are considered important phytotherapeutical agents for the treatment of benign prostate hyperplasia and have been used in the treatment of symptomatic micturition disorders (Fruhwirth & Hermetter, 2007). Re-

* Corresponding author. Tel.: +30 10 9549305; fax: +30 10 9577050. E-mail address: [email protected] (T. Nomikos). 0963-9969/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2009.02.003

duced bladder and urethral pressure and improved bladder compliance have been linked to pumpkin seed lipid components. Pumpkin seed snack supplementation inhibits the crystal formation or aggregation, which reduces the risk of bladderstone disease (Caili, Huan, & Quanhong, 2006). In a human trial, it was found that intake of a whole extract of Styrian oil pumpkin seeds is correlated to reduced benign prostate hyperplasia-associated symptoms (Fruhwirth & Hermetter, 2007). Animal studies have also shown that pumpkin seed oil can retard the progression of hypertension and reduce hypercholesterolemia (Al-Zuhair, Abdel-Fattah, & Abd el Latif, 1997) and arthritis (Fahim, Abdel-Fattah, Agha, & Gad, 1995). Treatment of spontaneously hypertensive rats with felodipine or captopril monotherapy or combined with pumpkin seed oil produced improvement in the measured free radical scavengers in the heart and kidney (AlZuhair, Abdel-Fattah, & El-Sayed, 2000). In addition, pumpkin seed oil has been found to alleviate diabetes by promoting hypoglycemic activity (Caili et al., 2006). Diets rich in pumpkin seeds have also been associated with lower levels of gastric, breast, lung, and colorectal cancer (Huang et al., 2004). Moreover, pumpkin seeds have been used in traditional medicine as vermifuges (Applequist et al., 2006) and were consumed fresh or roasted for the relief of abdominal cramps and distension due to intestinal worms (Caili et al., 2006). Oxidative stress and inflammation underlie the pathogenesis of the aforementioned diseases and the presence of antioxidant and anti-inflammatory molecules in pumpkin seeds may partly explain their beneficial actions against them. Lipoxygenases form a heterogeneous family of lipid-peroxidizing enzymes being involved in

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M.N. Xanthopoulou et al. / Food Research International 42 (2009) 641–646

the biosynthesis of inflammatory lipid mediators, such as leukotrienes, lipoxins, hepoxilins, and other hydroxylated fatty acid derivatives. They catalyze the stereospecific oxygenation of polyunsaturated fatty acids to the corresponding hydroperoxy derivatives and this reaction involves the formation of radical intermediates (Kühn, Chaitidis, Roffeis, & Walther, 2007). The importance of lipoxygenases at the progression of inflammation and cancer led researchers to the development of drugs targeting their activity. Lipoxygenases’ inhibitors possess antiproliferative effects against prostate cancer cells, therefore their presence in pumpkin seed may partly explain its protective action against prostate hyperplasia (Leone, Ottani, & Bertolini, 2007; Matsuyama et al., 2004). Until now, the antioxidant activities of pumpkin (Kwon, Apostolidis, Kim, & Shetty, 2007), its male and female flower extracts (Tarhan, Kayali, & Urek, 2007) and oil (Fruhwirth, Wenzl, El-Toukhy, Wagner, & Hermetter, 2003), but also of cold-pressed pumpkin seed flour (Parry, Cheng, Moore, & Yu, 2008) have been reported. However, a detailed study of the antioxidant and lipoxygenase (LOX) inhibitory activities of pumpkin seed extracts has not been conducted, yet. Therefore, the present study reports the antioxidant capacity and inhibitory activity of pumpkin seed extracts against DPPH free radical formation and soybean lipoxygenase, respectively. 2. Materials and methods 2.1. Reagents and chemicals Folin–Ciocalteau reagent and organic solvents were purchased from Merck (Darmstadt, Germany). Gallic acid, quercetin, resveratol, 2,2-Diphenyl-1-picryhydrazyl (DPPH), soybean lipoxygenase (type I-B), boric acid, linoleic acid and orcinol were purchased from Sigma–Aldrich (St. Louis, MO). All reagents and chemicals used were of analytical grade. 2.2. Samples Four commercially available pumpkin seeds were purchased from a local market. The pumpkin seed samples were of different origin and preparation process: PS1 (Bulgaria, roasted, salted), PS2 (China, roasted, salted), PS3 (Turkey, roasted, unsalted), PS4 (China, roasted, unsalted, organic farming). The seeds were hand selected to eliminate those that were cracked or otherwise damaged. PS1, PS2 and PS3 were manually peeled to obtain the kernels, while PS4 was purchased without husks. The kernels were sealed under nitrogen in plastic bags and stored at 20 °C. 2.3. Preparation of pumpkin seed extracts Two extraction techniques were utilized in order either to obtain the total lipid fraction of pumpkin seeds or to separate the pumpkin seed components according to their polarity. 2.3.1. Extraction No. 1 – extraction of pumpkin seeds with solvents of decreased polarity Ten grams of pumpkin seeds were homogenized in 50 mL acidified (2% acetic acid) deionised water by the aid of a homogeniser (Ultra-Turrax T25, JANKE & KUNKEL, IKA-Labortechnik). The suspension was left in room temperature for 10 min and centrifuged at 800 g for 10 min. The supernatant was collected in an Erlenmeyer flask and the pellet was treated with 50 mL of acidified water once more. The suspension was centrifuged at 800 g for 10 min and the resulting supernatant was combined with the supernatant of the first centrifugation, while the remaining pellet

was homogenized in 50 mL acidified (2% acetic acid) methanol and the aforementioned procedure was repeated. Two more solvents, namely acetone and ethylacetate, were consecutively used for the extraction of each extraction remaining pellet. Four pumpkin seed extracts were finally collected by the above extraction scheme, a water extract (W), a methanol extract (M), an acetone extract (Ac) and an ethylacetate extract (EtAc). All fractions were evaporated under reduced pressure and redissolved in a known volume of the solvent that was originally used for their extraction. The weight of each extract was determined gravimetrically. 2.3.2. Extraction No. 2 – extraction of pumpkin seed total lipids by the Folch method Total lipids (TL) were extracted according to method of Folch (Folch, Lees, & Sloane Stanley, 1957). Briefly, 10 g of pumpkin seeds was treated with 50 mL of chloroform:methanol (2:1, v/v) and the mixture was homogenized in a homogeniser as mentioned above. After 10 min in room temperature, the homogenate was centrifuged at 800 g for 10 min. The pellet was treated once more with 50 mL of chloroform:methanol (2:1, v/v) and centrifuged. Twenty millilitres of NaCl 0.9% w/v was added to the combined extracts (100 mL), the mixture was shaken vigorously in a separation funnel and left overnight at 4 °C for separating the two phases. The lower chloroform phase, containing the total lipids (TL) of pumpkin seeds, was collected in an Erlenmeyer flask and evaporated under reduced pressure in a flash evaporator. One tenth of the TL was weighed and stored at 20 °C, while the rest was further separated into polar and neutral lipid fractions. 2.4. Separation of total lipids to neutral and polar lipids by countercurrent distribution Total lipids were further separated into polar (PL) and neutral (NL) lipid fractions by counter-current distribution extraction procedure in a binary system formed by mixing three volumes of preequilibrated petroleum ether and one volume of pre-equilibrated 87% ethanol (Galanos & Kapoulas, 1962). After solvent evaporation, the two fractions were weighed and the ethanolic fraction containing the polar lipids was dissolved in chloroform:methanol (1:1, v/ v), while the petroleum ether fraction containing neutral lipids was dissolved in chloroform:methanol (2:1, v/v). All fractions were sealed under nitrogen and stored at 20 °C for further analysis. 2.5. Chemical determinations Sugar determination was carried out according to the method of Fisher, Hansen and Norton (Fisher, Hansen, & Norton, 1955), with some modifications. Briefly, samples were dried under a stream of nitrogen and dissolved in 1 mL of water. A volume of 2 mL of a solution of orcinol (5-methylresorcinol) (2 mg/mL of 70% sulfuric acid, v/v) was added followed by heating at 80 °C for 20 min. On cooling, the absorbance of the solution is measured at 505 nm. A blank sample is also analysed. The amount of sugar was estimated from a calibration curve prepared by performing the reaction on known amounts of glucose and the results are given as lmol Glucose per gram of extract. Total Phenol content was determined using a modified method of Singleton and Rossi (Singleton & Rossi, 1965). Briefly, samples were dried under a stream of nitrogen and dissolved in 3.5 mL of water. A volume of 0.1 mL of Folin–Ciocalteu reagent was added followed after 3 min by 0.4 mL of Na2CO3 35% w/v. The reaction mixture was rested for 1 h and the intensity of blue colour was measured at 725 nm. Standards of gallic acid were prepared similarly and the results are given as lmol Gallic acid per gram of extract.

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2.6. Determination of DPPH radical scavenging activity

sample was tested in triplicate. The % Inhibition for different concentrations of the extracts was determined.

The DPPH assay was used to measure the free radical scavenging capacity of the pumpkin seed extracts. This method was performed according to the method of Payet, Shum Cheong Sing and Smadja (Payet, Shum Cheong Sing, & Smadja, 2005), with modifications. After adjusting the blank to the appropriate solvent (ethyl acetate for NL and EtAc fractions, acetone for Ac fractions, H2O for W fractions, chloroform:methanol (1:1, v/v) for TL and ethanol for PL and M fractions), the proper volume of each sample was mixed with 0.035 mL of a freshly prepared ethanolic solution of 0.4 mg/mL DDPH in microplate wells. The total volume of the assay was 0.2 mL. The solutions were incubated at 37 °C for 30 min and the absorbance was measured at 492 nm with a microplate reader (Sunrise, TECAN). The percentage inhibition was calculated by the formula: % Inhibition = 100 (Acontrol Aextract)/Acontrol, where Acontrol is the absorbance of the control and Aextract is the absorbance of the sample at 492 nm. The EC50 values, which reflect the scavenging ability of the extracts expressed as equivalent concentration to give 50% loss of the DPPH activity, were estimated by the plot of % Inhibition towards concentration and were expressed in terms of mg sample weight/mL of the reaction mixture. Every sample was tested in triplicate. 2.7. Soybean lipoxygenase inhibition assay The bioassay was performed according to a previously described procedure (Axelrod, Cheesbrough, & Laakso, 1981), with some modifications. The incubation mixture consisted of the appropriate amount of the sample solution in the chosen solvent and 5 lL of the enzyme solution (100 units/ll in boric acid buffer 0.2 M, pH 9.0) in boric acid buffer 0.2 M, pH 9.0. After incubation at room temperature for 10 min in the dark, the reaction started by adding 2 lL of linoleic acid solution (450 mM in dimethylsulfoxide). The total volume of the reaction solution was 3 mL. The conversion of linoleic acid to 13-hydroperoxylinoleic acid was recorded at 234 nm (room temperature) and compared to the appropriate standard solution, which did not contain the extracts. Every

Table 1 Yield of pumpkin seed extracts. Extraction No. 1

PS1 PS2 PS3 PS4

Extraction No. 2

W

M

Ac

EtAc

TL

PL

NL

9.49 12.30 12.57 11.00

1.54 2.06 1.90 3.04

37.59 38.42 37.94 33.15

5.92 7.17 7.64 7.17

39.21 38.87 36.86 36.78

1.78 1.10 1.20 1.15

37.91 38.06 35.84 35.96

2.8. Statistical analysis Chemical and enzymatic analyses of individual samples were performed in triplicates. The results presented are the mean and standard deviations of the obtained values. Data manipulation was performed by means of Microsoft Excel (Microsoft Corp., Redmond, WA). 3. Results and discussion 3.1. Chemical composition of the extracts Four different pumpkin seed kernels were extracted using two different methods of extraction in order to obtain fractions with different content. Table 1 shows the yield of each fraction. Four fractions were obtained from the extraction method No. 1. This method distributes the constituents to the fractions according to their polarity and solubility to the extraction solvent. The majority (33–37%) of the extractable compounds in all pumpkin seed samples consist of acetone soluble constituents. Polar compounds soluble in water are 9.5–12%, whereas non-polar constituents (ethylacetate fractions) are 6.0–7.6%. The rest 1.5–3.0% consists of methanol soluble constituents. The extractable material by this method was the 54–60% of the fresh pumpkin seed weight. Extraction method No. 2 isolates the Total Lipid fraction (TL) of pumpkin seeds and the subsequent counter-current distribution separates them into polar (PL) and neutral (NL) lipids. TL comprises 36–39% of pumpkin seeds, which is slightly lower than the content reported by the USDA Data Bank (42–45%) (US Department of Agriculture (2005)). The 92–96% of TL content is neutral lipids, while the rest is polar lipids. The total phenolic content was determined in each extract since it is considered as major determinant of the antioxidant activity of nuts and plants (Kris-Etherton et al., 2002). The results are presented in Table 2. The first extraction scheme yielded 50–70 lmol gallic acid equivalents of total phenolics in all fractions among of which the water extracts were the richest in phenolic constituents (54–64 lmol gallic acid, 85–92% of total extractable phenolics). The high concentration of phenolics in the water extract can be attributed to the presence of proteins and other water soluble constituents that contain phenolic rings. Among the other three fractions methanol extracts contain the higher amounts of phenolics (5–11 lmol gallic acid, 7–15% of total extractable phenolics), while the phenolic content of the acetone and ethylacetate fractions is negligible.

Results are expressed as g of extract per 100 g of fresh pumpkin seeds.

Table 2 Total phenolic content of pumpkin seed extracts. Extraction No.1

PS1 PS2 PS3 PS4

Extraction No. 2

W

M

Ac

EtAc

TL

PL

NL

64.07 ± 9.20 (61.02 ± 8.76) 47.71 ± 5.70 (59.1810.87) 42.49 ± 6.30 (54.17 ± 8.03) 56.81 ± 5.80 (63.63 ± 6.50)

33.71 ± 3.70 (5.21 ± 0.57) 27.89 ± 4.80 (5.81 ± 1.00) 41.66 ± 5.20 (8.04 ± 1.00) 35.86 ± 3.4 (11.05 ± 1.05)

0.078 ± 0.010 (0.29 ± 0.038) 0.071 ± 0.020 (0.27 ± 0.078) 0.301 ± 0.035 (1.16 ± 0.134) nda

nda

0.50 ± 0.064 (1.99 ± 0.252) 0.47 ± 0.058 (1.85 ± 0.227) 0.59 ± 0.083 (2.18 ± 0.307) 0.59 ± 0.065 (2.18 ± 0.241)

7.26 ± 0.95 (1.29 ± 0.17) 6.50 ± 0.86 (0.72 ± 0.09) 11.30 ± 1.35 (1.36 ± 0.16) 10.61 ± 1.20 (1.22 ± 0.13)

nda

nda nda 0.236 ± 0.056 (0.17 ± 0.041)

nda nda nda

Results are expressed as lmol Gallic acid/g extract and as the total phenolic content of the extract expressed as lmol Gallic acid (in parentheses). Each value is expressed as mean ± standard deviation. a nd: not detected.

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The extraction of the total lipids by the Folch method yielded 1.9–2.2 lmol gallic acid equivalents. The determination of total phenolic content of NL fraction was not possible, due to solubility problems. PL fractions contain the 55–65% of total extractable phenolics by the extraction method No. 2, apart from the PL fraction of PS2, which contains only the 39% of total extractable phenolics. Recently, Parry et al. reported the total phenolic content of acetone/water fraction of flour (Parry et al., 2008) and methanol fraction of oil of cold-pressed pumpkin seed (Parry et al., 2006). The reported values (1.58 and 0.98 mg gallic acid equivalents per g of flour or oil, respectively) are an order of a value higher than the values of our methanol fractions (0.09–0.20 mg gallic acid equivalents per g of fresh weight). However, direct comparisons are difficult to be made since the initial sample and the extraction procedures are different from ours. The total carbohydrate contents of pumpkin seed extracts are presented in Table 3. In all cases, the carbohydrate content decreases as the polarity of the extraction solvent decreases. The carbohydrates are distributed mostly in water and then to methanol fractions of extraction No.1 and to polar lipid fractions of extraction No. 2. Water fractions contain the higher amounts of carbohydrates (78–93%), whereas all the other fractions contain negligible amounts. Additionally, the countercurrent separation distributes the carbohydrate content mainly to the PL fraction, although a significant amount of sugar residues are also found in the NL fractions. This is probably due to the presence of glycolipids, which are abundant in plant tissues and can be distributed in both polar and non-polar solvents (Demopoulos, Kyrili, Antonopoulou, & Andrikopoulos, 1996).

activity of extracts. Pumpkin seed extracts’ radical scavenging activities, expressed as EC50 values, are presented in Table 4. The results indicate that all pumpkin seed extracts possess phenol concentration-dependent antiradical activity. The lowest antioxidant activities were detected for ethyl-acetate fractions, whereas the highest were detected for water and methanol fractions in all cases. Also PL fractions appear to be more effective scavengers (an order of magnitude) against DPPH radical than NL fractions. It is worth commenting the relationship between the total phenolic content and scavenging activity against DPPH, since phenolics contribute to the antiradical activity. A plot correlating these two characteristics is presented in Fig. 1. Fractions rich in total phenolics (W, M and PL) are more effective scavengers of DPPH radicals than phenol poor fractions (Ac, EtAc and NL). However, the inhibition of DPPH radical scavenging by pumpkin seed fractions is not strictly proportional to the concentration of total phenolics, since water and methanol fractions possess similar antioxidant properties despite the higher concentration of phenolics in the water fraction. This may be attributed to the different quality of phenolics they contain, and, consequently, to the different antioxidant activity they posses, but also to the different content of other constituents (carbohydrates, phospholipids, fatty acids), that may contribute to the antioxidant activity as well. Similar correlations between the carbohydrate content and scavenging activity further support this suggestion. Water, methanol and PL fractions, which contain the higher amounts of carbohydrates, are more effective radical scavengers (data not shown). However, since the concentration of carbohydrates and phenolics of the fractions is proportional to each other (probably because the higher phenolic content corresponds to a higher phenolic glucoside content) the observed correlation between carbohydrate content and antiradical activity may be misleading. The EC50 values of gallic acid, resveratrol and quercetin were also determined in order to compare the antiradical activity of pumpkin seed extracts with the activity of standard phenolic com-

3.2. Antioxidant capacity of pumpkin seed extracts The antioxidant activities of pumpkin seed extracts were determined using the DPPH free radical assay. Scavenging activity on DPPH radicals assay provides information about the antiradical

Table 3 Total carbohydrate content of pumpkin seed extracts. Extraction No. 1

PS1 PS2 PS3 PS4

Extraction No. 2

W

M

Ac

EtAc

TL

PL

NL

2386 ± 293.2 (2272 ± 279.5) 2050 ± 257.5 (2543 ± 319.4) 1561 ± 178.6 (1990 ± 227.7) 897.9 ± 109.5 (1006 ± 122.6)

1070 ± 110.3 (165.4 ± 17.04) 1050 ± 121.2 (218.7 ± 25.24) 1133 ± 109.4 (218.7 ± 21.13) 861.7 ± 126.8 (265.7 ± 39.09)

1.606 ± 0.181 (6.059 ± 0.683) 1.642 ± 0.178 (6.367 ± 0.690) 1.587 ± 0.180 (6.107 ± 0.690) 2.717 ± 0.250 (9.132 ± 0.840)

nda

3.891 ± 0.276 (15.37 ± 1.090) 3.792 ± 0.387 (14.82 ± 1.513) 3.461 ± 0.394 (12.79 ± 1.456) 5.862 ± 0.635 (21.77 ± 2.359)

79.93 ± 8.650 (14.23 ± 1.540) 122.0 ± 14.30 (13.48 ± 1.579) 102.1±11.95 (12.30 ± 1.439) 107.1 ± 12.64 (12.35±1.458)

0.726 ± 0.086 (2.752 ± 0.326) 0.509 ± 0.055 (1.937 ± 0.209) 1.088 ± 0.135 (3.900 ± 0.484) 0.984 ± 0.102 (3.538 ± 0.367)

0.074 ± 0.029 (0.054 ± 0.021) 1.200 ± 0.150 (0.930 ± 0.120) 1.715 ± 0.161 (1.246 ± 0.117)

Results are expressed as lmol glucose/g extract and as the carbohydrate content of the extract expressed as lmol glucose (in parentheses). Each value is expressed as mean ± standard deviation. a nd: not detected.

Table 4 Radical scavenging activity of pumpkin seed extracts. Extraction No. 1

PS1 PS2 PS3 PS4

Extraction No. 2

W

M

Ac

EtAc

TL

PL

NL

4.63 ± 0.4 5.57 ± 0.5 4.51 ± 0.4 6.71 ± 0.7

4.40 ± 0.3 5.80 ± 0.4 4.90 ± 0.3 5.42 ± 0.3

26.5 ± 2.1 48.8 ± 3.5 35.2 ± 3.0 43.4 ± 3.0

48.76 ± 3 78.45 ± 4.5 >>50a 78.00 ± 5.0

14.97 ± 1.2 26.31 ± 2.0 26.62 ± 2.1 22.79 ± 2.0

5.5 ± 0.5 2.5 ± 0.2 4.9 ± 0.4 3.83 ± 0.4

34.7 ± 2.1 45.5 ± 3.9 61.6 ± 3.0 51.37 ± 4.2

Values are expressed as EC50 values (mg/mL). a Due to solubility problems, it was not able to determine the EC50 value of this fraction, and the highest concentration tested was 50 mg/mL. Each value is expressed as mean ± standard deviation.

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60

60

50

50 40 40 30

30

20

20

10

10

0

0 M

Ac

EtAc

TL

PL

60

70 50

60

40

50

30

40 30

20

20 10

10

0

0 W

NL

M

Ac

EtAc

90

60

EC50

80 70

50

60

40

50

30

40 30

20

20 10

10

0

0 Ac

EtAc

PL

NL

70

Phenolic content (µmol Gallic acid / g extract)

PS3

DPPH scavenging capacity (EC50, mg/ml)


70

M

TL

fraction

fraction

W

80

TL

PL

90 PS4

60

EC50

70 50

60

40

50

30

40 30

20

20 10

10

0

NL

80


W

PS2 EC50


70


70

80



PS1 EC50

90

70

90

80

0 W

M

Ac

EtAc

fraction

TL

PL

NL

fraction

Fig. 1. Concentration of total phenolic content and DPPH radical scavenging activity for all pumpkin seed samples.

pounds under the same assay conditions. The values obtained (0.0011 ± 0.0002, 0.0606 ± 0.0044 and 0.0086 ± 0.0013 mg/mL, respectively) were much lower than the EC50 values of pumpkin seed fractions, as expected, since our fractions are mixtures of different activity components. A direct comparison with the antioxidant activity of nut seed extracts of other studies cannot be made due to the difference in the extraction methods and assay methodologies utilized by them. However, it seems that the radical scavenging capacity of the pumpkin seed methanol extracts (Table 4) is slightly lower than the corresponding capacity of almond methanol extracts (EC50 0.7–7 mg/mL) (Barreira, Ferreira, Oliveira, & Pereira, 2008). Additionally, hazelnut kernel acetone extracts (0.098 mg/mL) (Alasalvar, Karamac, Amarowicz, & Shahidi, 2006) and walnut water extracts (0.15–0.22 mg/mL) (Pereira et al., 2008) seems to be more effective quenchers of DPPH radicals than the corresponding pumpkin seed extracts. 3.3. Determination of lipoxygenase inhibitory activity of pumpkin seed extracts Fractions’ anti-LOX activity was also measured as inhibition of linoleic acid’s peroxidation to hydroperoxylinoleic acid, a reaction,

which is catalyzed by soybean lipoxygenase. In several aspects (reaction mechanism, kinetic parameters, iron content etc.), soybean LOX-1 constitutes a suitable model for mammalian LOXs (Kühn, Römisch, & Belkner, 2005). Because of the non-linear correlation between % Inhibition and concentration, the IC50 values cannot be determined, and therefore the inhibitory activity of the extracts is given as % inhibition at final concentration of 0.4 mg/mL (Table 5), in which the solubility of all samples in the assay solution is efficient. Water extracts show a biphasic mode of action, promoting LOX activity at lower concentrations (0.4 mg/mL), while they inhibit lipid peroxidation at 1.5 mg/mL (34–60%). This pattern can be expected by an extract that may contain both activators and inhibitors of the enzyme. The methanol extracts inhibit 50% of LOX activity at concentration ranging from 0.3 mg/mL (PS4) to 1.02 mg/mL (PS1), while the respective concentrations of acetone extracts range from 0.23 (PS1) to 0.80 mg/mL (PS3) and those of PL fractions from 0.16 (PS4) to 0.48 mg/mL (PS1, PS2). The results indicate that, in contrast to the scavenging activity of extracts, where methanolic and water fractions were found more effective, acetone fractions are more effective inhibitors of li-

Table 5 Inhibitory activities of pumpkin seed extracts against soybean lipoxygenase at final concentration of 0.4 mg/mL. Extraction No. 1 W PS1 PS2 PS3 PS4

Extraction No. 2 M

a

33.80 ± 4.6 19.35 ± 2.5a 0.00 ± 1.7 34.00 ± 4.9a

15.00 ± 1.3 21.58 ± 1.9 18.49 ± 2.5a 74.23 ± 5.3

Ac

EtAc

100.00 67.86 ± 6.3 33.33 ± 4.2 32.61 ± 3.1

24.83 ± 3.1 15.00 ± 1.4 27.50 ± 3.5 2.53 ± 0.32a

Each value is expressed as mean ± standard deviation. a The negative values indicate activation of peroxidation product rather than inhibition.

TL 10.00 ± 1.39 20.00 ± 2.45a 10.00 ± 1.6 2.00 ± 0.58

PL

NL

47.00 ± 5.0 33.85 ± 3.25 43.33 ± 4.6 89.36 ± 9.6

14.00 ± 1.6 10.00 ± 1.23 12.00 ± 1.54 12.82 ± 1.32

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pid peroxidation catalyzed by lipoxygenase. On the other hand, PL fractions were found more effective inhibitors of lipoxygenase than NL and TL fractions, whose effect upon peroxidation reaction is almost negligible for the tested concentrations. In the case of lipoxygenase inhibition, the phenolic content and the inhibitory activity of the fractions are not correlated. The acetone fractions, even though their phenolic concentration is lower compared to methanol and water fractions, are more effective inhibitors of lipoxygenase. On the other hand, it is worth commenting that PL fractions contain the highest concentration of total phenolics and carbohydrates and, at the same time, are both the most effective radical scavengers and better lipoxygenase inhibitors among the extraction No. 2 fractions. This observation can be explained, considering the fact that PL fractions of extraction No. 2 contain the majority of the active constituents of methanol and acetone fractions of extraction No.1. Therefore it seems that the radical scavenging activity of the extracts does not necessarily correlate with their anti-LOX activity. This is probably due to the fact that radical scavenging is just one way by which a molecule can inhibit LOX activity. Moreover, the radical intermediates formed in the LOX reaction usually remain enzyme-bound and thus may not be readily accessible for radical scavengers (Kühn et al., 2007). The presence of allosteric regulators having no radical scavenging ability may also determine the LOX inhibitory potential of the extract. 4. Conclusions Pumpkin seed extracts of different polarity and phenolic content are able both to quench DPPH free radicals and to inhibit lipid peroxidation catalyzed by LOX. The fractions with higher amounts of phenolics (water, methanol and PL fractions) are better quenchers of DPPH free radical. However, there is no correlation between total phenolic content and anti-LOX activity. It seems that the relative concentration of each species of phenolic molecules rather than the total phenolic content determines the antioxidant potential of the extracts. The presence of molecules being able to scavenge radicals and inhibit LOX in pumpkin seeds may in part explain the health benefits attributed to them. References Al-Zuhair, H., Abdel-Fattah, A. A., & Abd el Latif, H. A. (1997). Efficacy of simvastatin and pumpkin-seed oil in the management of dietary-induced hypercholesterolemia. Pharmacological Research, 35(5), 403–408. Al-Zuhair, H., Abdel-Fattah, A. A., & El-Sayed, M. I. (2000). Pumpkin-seed oil modulates the effect of felodipine and captopril in spontaneously hypertensive rats. Pharmacological Research, 41(5), 555–563. Alasalvar, C., Karamac, M., Amarowicz, R., & Shahidi, F. (2006). Antioxidant and antiradical activities in extracts of Hazelnut Kernel (Corylus avellana L.) and Hazelnut green leafy cover. Journal of Agriculture and Food Chemistry, 54, 4826–4832. Applequist, W. L., Avula, B., Schaneberg, B. T., Wang, Y.-H., & Khan, I. A. (2006). Comparative fatty acid content of seeds of four Cucurbita species grown in a common (shared) garden. Journal of Food Composition and Analysis, 19, 606–611. Axelrod, B., Cheesbrough, T. M., & Laakso, S. (1981). Lipoxygenase from soybeans. Methods in Enzymology, 71, 441–451. Barreira, J. C. M., Ferreira, I. C. F. R., Oliveira, M. B. P. P., & Pereira, J. A. (2008). Antioxidant activity and bioactive compounds of ten Portuguese regional and commercial almond cultivars. Food and Chemical Toxicology, 46, 2230–2235. Caili, F., Huan, S., & Quanhong, L. (2006). A review on pharmacological activities and utilization technologies of pumpkin. Plant Foods for Human Nutrition, 61, 73–80. Demopoulos, C. A., Kyrili, M., Antonopoulou, S., & Andrikopoulos, N. K. (1996). Separation of several main glycolipids into classes and partially into species by

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