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Evaluation of the antioxidant activities and nutritional properties of ten edible plant extracts and their application to fresh ground beef
Meat Science 93 (2013) 715–722
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Evaluation of the antioxidant activities and nutritional properties of ten edible plant extracts and their application to fresh ground beef Sung-Jin Kim a,1, Sea C. Min b,1, Hyo-Jin Shin a, Yun-Jeong Lee a, Ah Reum Cho a, So Yeon Kim c, Jaejoon Han a,⁎ a b c
Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon 440-746, Republic of Korea Department of Food Science and Technology, Seoul Women's University, Seoul 139-774, Republic of Korea Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea
a r t i c l e
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Article history: Received 13 February 2012 Received in revised form 21 September 2012 Accepted 3 November 2012 Keywords: Antioxidant activity Nutritional property Edible plants Lipid oxidation Ground beef
a b s t r a c t In this study, we assessed the antioxidant efficacy and nutritional value of 10 leafy edible plants and evaluated their potential as natural antioxidants for meat preservation. We measured total phenolic content, 2,2-diphenyl1-picryl-hydrazil (DPPH) radical scavenging activity, and vitamin C, chlorophyll, and carotenoid contents of 70% ethanol and water extracts of the edible plants. Based on these results, we investigated the effects of butterbur and broccoli extracts on lipid oxidation in ground beef patties. Plant extracts and butylated hydroxytoluene (BHT) were individually added to patties at both 0.1% and 0.5% (w/w) concentrations. Thiobarbituric acid reactive substance (TBARS) values and color parameters were tested periodically during 12 days of refrigerated storage. TBARS levels were significantly lower (p≤0.05) in the samples containing plant extracts or BHT than the non-treated control. In addition, the beef patties formulated with the selected plant extracts showed significantly (p≤0.05) better color stability than those without antioxidants. These results indicate that edible plant extracts are promising sources of natural antioxidants and can potentially be used as functional preservatives in meat products. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Lipid oxidation is a major cause of quality deterioration in meat and meat products because it leads to color alteration, off-flavor, and loss of nutrients, all of which are major determinants of meat quality (Chan, Decker, & Means, 1993). Lipid oxidation is promoted by diverse factors such as heat, light, metal ions, heme (in meat), oxygen, free radicals, and oxidative enzymes (Barbut, Josephson, & Mauer, 1985; Buckley et al., 1989). Color is another important factor that influences the quality and acceptability of meat. Color is regarded as an indicator of perceived quality and freshness of meat and is the first limiting factor in the shelf-life of meat (Smith, Belk, Sofos, Tatum, & Williams, 2000). Discoloration of meat is closely related to oxidative denaturation of meat pigments (Kanner & Harel, 1985). One way to prevent oxidative deterioration is to use antioxidants in meat products (Lee et al., 2010). These antioxidants can retard lipid oxidation by acting as free radical scavengers, oxygen scavengers, and/or metal chelators (Teets & Were, 2008). Various synthetic antioxidants, such as BHT, butylated hydroxyanisole (BHA), and tertiary butyl hydroquinone (TBHQ), are used in the food industry to delay lipid oxidation (Mansour & Khalil, 2000). However, concerns have been
⁎ Corresponding author at: 300 Cheoncheon-dong, Jangan-gu, Suwon 440-746, Republic of Korea. Tel.: +82 31 290 7803; fax: +82 31 290 7882. E-mail address: [email protected] (J. Han). 1 These authors are considered as co-first authors. 0309-1740/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.meatsci.2012.11.029
raised about using synthetic antioxidants due to their possible side-effects, which has given impetus to finding alternative “natural” antioxidants (Sebranek, Sewalt, Robbins, & Houser, 2005). Most natural antioxidants are obtained from plant resources including culinary herbs, spices, fruits, vegetables, and oilseed products (Shahidi & Zhong, 2010). Many previous studies have focused on herbs and spices consumed in Europe, Southern Asia, and Southeast Asia. Several herbs and spices, such as clove (Naveena, Muthukumar, Sen, Babji, & Murthy, 2006), rosemary (Georgantelis, Blekas, Katikou, Ambrosiadis, & Fletouris, 2007), and oregano and sage (Fasseas, Mountzouris, Tarantilis, Polissiou, & Zervas, 2007) have been reported to significantly improve the keeping quality of meat products, and are effective in delaying lipid oxidation. However, their strong flavor is unfamiliar to East Asians, which limits their applications to foods. Therefore, in this study, we attempted to explore new natural antioxidant sources suitable for East Asians. We selected several leafy, green, and edible plants commonly consumed in Northeast Asian regions. These culinary plants have been eaten for centuries and their safety and edibility are recognized by the Korean Food and Drug Administration (KFDA). They have a milder flavor than herbs and spices and are reported to be good for health. They are often used in salads or as side dishes, either fresh or blanched. Phenolic compounds are the major constituents of plants that contribute to their antioxidant capacity. Phenolic compounds include phenolic acids, flavonoids, and tocopherols (Wong, Hashimoto, & Shibamoto, 1995). Several studies have demonstrated that phenolic compounds scavenge free radicals. Therefore, the total amount of
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phenolic compounds is one of the most important factors affecting antioxidant activity (Møller, Madsen, Aaltonen, & Skibsted, 1999). There are also many other compounds that have functional and nutritional value in edible plants, such as ascorbic acid, nitrogen compounds (amino acids, amines, alkaloids, and chlorophyll derivatives), and carotenoids (Hall & Cuppett, 1997; Hudson 1990). These compounds play a role as nutrients, bioactive substances, as well as antioxidants. Ascorbic acid, also known as vitamin C, is a strong antioxidant found mainly in fresh fruits and vegetables (Gardner, White, Mcphail, & Duthie, 2000). Vitamin C retards free radical-induced cellular damage, and acts as a cofactor of several biosynthesis reactions in the human body (Chan, 1993). Chlorophylls and carotenoids are the most abundant plant pigments in nature, and have antioxidant activity due their singlet oxygen quenching properties (Hirayama, Nakamura, Hamada, & Kobsyasi, 1994; Tanielian & Wolff, 1988). Some carotenoids, such as α- and β-carotene, are precursors of vitamin A (Harrison, 2012). Chlorophyll and its derivatives may prevent certain types of cancers, aid in wound healing, and reduce inflammation in some cases (Morgan, Jackson, Zheng, Pandey, & Pandey, 2010; Smith & Livingston, 1945; Subramoniam et al., 2012). The antioxidant activities of edible plants have been explored by several research groups. However, it is difficult to compare the antioxidant capacity of these plant materials between studies because of different methods of extraction and antioxidant activity determination. Moreover, the nutritional values of these plants were rarely evaluated in these previous studies. Our objectives in this study were therefore to (1) evaluate the antioxidant activities and nutritional properties of 70% ethanol and water extracts of 10 edible plants; and (2) to determine the effectiveness of these extracts in preventing or reducing lipid oxidation as well as color changes in ground beef patties during storage at a chilled temperature (4 °C). 2. Materials and methods 2.1. Edible plants Fresh leaves of crown daisy (Chrysanthemum coronarium var. spatiosum), pumpkin (Cucurbita moschata Duch.), chamnamul (Pimpinella brachycarpa (Kom.) Nakai), fatsia (Aralia elata), leek (Allium tuberosum), bok choy (Brassica campestris var. chinensis), acanthopanax (Acanthopanax sessiliflorum Seeman), butterbur (Petasites japonicus), soybean (Glycine max L. Merr), and the flower heads of broccoli (Brassica oleracea L. var. italica Plenck) were purchased from local farms during the harvest season in April. Although broccoli is not a leafy edible plant, it was used for comparison with other plant materials because of its well-documented antioxidant activities (Aldrich et al., 2011). The edible plants were washed and dehydrated using an electric food dehydrator (Lequip Co., Hwaseong, Korea) at 65 °C for 24 h. Each edible plant was finely pulverized using an electric grinder (Daesung Artron Co., LTD., Seoul, Korea), weighed, and then stored at −20 °C until extraction. 2.2. Preparation of edible plant extracts Extracts were prepared using 70% (v/v) ethanol and water as solvents. Ethanol 70% (v/v) was added to the finely ground plant powders at a ratio of 1:20 (w/v) and the mixture was stirred at 200 rpm using an overhead stirrer (Mtops Co., Seoul, Korea) for 24 h at room temperature. Water extracts were also prepared using distilled–deionized water (1:20, w/v) with stirring for 1 h at 95−100 °C. The extracts were then separated from the residue by filtration through Whatman no.1 filter paper (Whatman International Ltd., Maidstone, U.K.) and the filtrate was concentrated under reduced pressure at 55 °C using a rotary evaporator (Büchi Laboratory Equipment, Postfach, Switzerland). The extra solvent was removed by freeze-drying and the dried extract powder was stored at − 20 °C until further analyses.
2.3. Antioxidant content and antioxidant activity 2.3.1. Total phenolic content The content of total phenolics was measured spectrophotometrically using the Folin–Ciocalteu colorimetric method (Dewanto, Wu, Adom, & Liu, 2002). All plant extracts were diluted with extraction solvent (70% ethanol or distilled–deionized water) to obtain readings within the standard curve range of 0.0−0.8 mg gallic acid/mL. Briefly, 100 μL of diluted plant extract or gallic acid standard solution was mixed well with 2 mL of 2% (w/v) Na2CO3 solution. The mixture was then left to stand for 3 min, after which 100 μL of Folin–Ciocalteu reagent was added. After letting the mixture sit for 30 min at room temperature for color development, the absorbance was measured at 750 nm using a UV–visible spectrophotometer. Results are expressed as mg gallic acid equivalent (GAE)/g dried plant. 2.3.2. DPPH radical scavenging activity The free radical scavenging activity of extracts was measured by the DPPH method as proposed by Brand-Williams et al. (1995). A solution of DPPH in methanol (0.078 mg/mL) was prepared and 0.25 mL of this radical solution was added to 0.05 mL of sample solution. The mixture was incubated for 30 min in the dark at room temperature and then the absorbance was measured at 517 nm with a spectrophotometer. Ascorbic acid solutions in the concentration range of 0.02−0.08 mg/ml were used to establish a standard curve. DPPH radical scavenging activity was expressed as mg ascorbic acid equivalent (AAE)/g dried plant. 2.4. Nutritional value 2.4.1. Vitamin C content Vitamin C content was determined by the titrimetric method as described in the Association of Official Analytical Chemists (AOAC, 1995) method no. 967.21 (1995). Dried plant powder (2 g) was magnetically stirred in 25 mL of extract solution (metaphosphoric acid: acetic acid = 1:5). The homogenate was centrifuged at 12,000 rpm for 5 min, and the supernatant was filtered through Whatman no. 4 filter paper. Aliquots (2 mL) were then placed in test tubes, after which 200 μL of indophenol and 2 mL of thiourea metaphosphoric solution were added. After that, 1 mL of 2,4-dinitrophenyl hydrazine (DNP) solution was mixed with the sample solution and the mixture was allowed to stand at 37 °C for 3 h and then cooled on ice. Next, 5 mL of 85% H2SO4 solution was added, and the resulting mixture was incubated in the dark for 30 min at room temperature. Absorbance was measured at 540 nm and vitamin C content was expressed as mg ascorbic acid (AAE) equivalent/g of plant on a dry weight basis. 2.4.2. Total chlorophyll and total carotenoid contents The content of chlorophylls a and b, as well as that of total carotenoids, was spectrophotometrically determined using the method of Lichtenthaler (1987). Dried edible plant samples (1 g) were stirred in 50 mL of 80% acetone (v/v) solution in the dark for 24 h at room temperature. After filtration (Whatman no. 4 filter paper), the filtrate volume was adjusted to 100 mL with 80% acetone (v/v). Absorbance was read at 662, 644, and 470 nm to measure the content of chlorophyll a, chlorophyll b, and carotenoids, respectively. Total chlorophyll was calculated as the sum of chlorophylls a and b. Total chlorophyll and total carotenoid contents were expressed as mg/100 g of plant on a dry weight basis. 2.5. Effects on lipid oxidation and color of beef patties 2.5.1. Determination of fat content in ground beef Fresh raw beef was obtained from a local supermarket and prepared using separable lean from top round roasts. On the day of purchase, the roasts were trimmed of all separable fat and connective
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tissue, and then ground twice through a 1.27 cm plate, followed by a 0.32 cm plate. Before patty preparation, the fat content of the meat sample was analyzed according to the method of Folch et al. (1957). A 5 g portion of meat sample was homogenized with 75 mL of chloroform: methanol (2:1, v/v) using a stirrer for 3 min. After filtering, the meat residue was re-homogenized for 3 min with 5 mL of chloroform: methanol (2:1), and then re-filtered. The collected filtrate was transferred to a 500 mL separatory funnel, and then 25 mL of distilled water was added and the phases were allowed to separate. The lower phase in the funnel (organic phase) was drained from the funnel into a 100 mL volumetric flask. The organic phase was brought to a volume of 100 mL with pure chloroform. The solvent was removed under reduced pressure at below 40 °C using a rotary evaporator, and the samples were dried at 100 °C for 1 h. The mean fat content was determined by averaging the triplicates.
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replicates, two samples/replicate, two types of extraction solvent, 10 edible plants). Experiments to determine nutritional values (vitamin C, chlorophyll, carotenoid contents) were carried out using a 3 × 2 × 10 factorial design (three replicates, two samples/replicate, 10 edible plants). Preservation studies (TBARS and color measurements) were performed using a 3 × 2 × 2 × 4 × 4 factorial design (three replicates, two samples/replicate, two concentrations of preservative, four types of preservative including control, four storage periods). Data analyses were performed using Statistical Analysis System (SAS) software, version 8.1 (SAS Institute, Cary, NC, USA). The General Linear Models procedure was used for analysis of variance, with main effect means separated by the Student–Newman–Keuls test. Significance was defined as p ≤ 0.05.
3. Results and discussion 2.5.2. Preparation of ground beef patties Ground beef patties were prepared for four different treatments. The negative control group was formulated without antioxidants. Considering the overall association between phenolic content and antioxidant capacity that we found after performing the experiments described above, 70% ethanol extract from butterbur leaf was selected as the best material due to its antioxidant content and activity. Ethanolic broccoli extract was also chosen as a natural antioxidant for meat preservation because broccoli is a vegetable commonly consumed worldwide and its nutritional value and antioxidant properties have been well characterized (Aldrich et al., 2011). Ground beef patties with plant extract were prepared by adding two concentrations (0.1% and 0.5%, w/w) of ethanolic butterbur and broccoli extracts. Positive controls were prepared by treating the patties with BHT solution (0.1% and 0.5%, w/w). A 20 g portion of each meat sample was made into patties using a patty mold (6 cm diameter, 1.2 cm height) and wrapped with LLDPE film (Clean Wrap, Kimhae, Korea). Prepared beef patties were stored in a refrigerator at 4 °C for 12 days and the TBARS values and color parameters were determined at 0, 3, 6, 9, and 12 days of storage. The preservation experiments were replicated three times on different days, with two patties stored for each storage time within each treatment replication. 2.5.3. TBA value The lipid oxidation potential of meat was evaluated by measuring TBARS. A 20 g portion of each meat sample was homogenized with 50 mL of distilled–deionized water and 10 mL of trichloroacetic acid (15%, final concentration) using a stomacher for 2 min. The homogenate was centrifuged at 20,000 rpm for 5 min, and the supernatant was filtered through Whatman no. 1 filter paper. A 2 mL aliquot of 0.06 M thiobarbituric acid was added to 8 mL of the filtrate. The solution was vortexed for 15 s, placed in a 80 °C water bath for 90 min, and then cooled on ice. Absorption was measured at 532 nm using a UV–visible spectrophotometer. Results were expressed as mg malondialdehyde (MDA) equivalent/kg of meat sample.
3.1. Antioxidant content and antioxidant activity 3.1.1. Total phenolic content Phenolic compounds, containing one or more acidic hydroxyl residues attached to an aromatic arene (phenyl) ring, are one of the most active constituents that contribute to the antioxidant activities of plant foods (Velioglu, Mazza, Gao, & Oomah, 1998). Therefore, it is important to quantify phenolic content and to assess its contribution to the antioxidant activity of plant materials. The total phenolic contents of 70% ethanol and hot water extracts of 10 edible plants are depicted in Fig. 1. The amount of total phenolics varied widely in the edible plant extracts, ranging from 3.13 to 72.30 mg GAE/g extract. The seventy percent ethanol extract of fatsia had the highest polyphenol content, followed by the butterbur and chamnamul ethanol extracts. Among hot water extracts, butterbur, acanthopanax, and fatsia extracts had significantly (p ≤ 0.05) higher concentrations of phenolic compounds than the hot water extracts of the other plants. Hot water and 70% ethanol extracts of fatsia and butterbur had relatively high total phenolic content (p ≤ 0.05). The antioxidant effects of fatsia extract have been studied previously by Bol'shakova et al. (1997). Cha et al. (2009) also reported that ethanol extracts from fatsia exhibited strong antioxidant activity against lipid oxidation as measured by the TBA method. In addition, Bae and Noh (2002) reported that butterbur fractions protected biological membranes from benzoyl peroxide-induced peroxidation. Takenaka et al. (2000) found that the phenolic compounds that made a major contribution to the antioxidant capacity of butterbur were caffeic acid and chlorogenic acid.
2.5.4. Color measurement The color of raw beef patties was evaluated using a Minolta Chroma Meter CR-400 equipped with a DP-400 data processor (Minolta, Inc, Japan). Color was described as L* (lightness), a* (redness), and b* (yellowness) color space values. A Chroma meter was standardized with a standard white plate (L* = 93.80, a* = −0.3157 and b* = 0.3319). Measurements were made perpendicular to the patty surface at five different locations per patty, and mean values (L*, a*, and b*) from each patty were analyzed. 2.6. Statistical analysis Experiments to evaluate antioxidant content and antioxidant activity were carried out using a 3 × 2 × 2 × 10 factorial design (three
Fig. 1. Total phenolic content of 70% ethanol and distilled water edible plant extracts. (A) Crown daisy leaf; (B) pumpkin leaf; (C) chamnamul; (D) fatsia; (E) leek leaf; (F) bok choy; (G) acanthopanax; (H) butterbur leaf; (I) soybean leaf; (J) broccoli.
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In the present study, two different extraction solvents, namely 70% ethanol and hot water, were used. Overall, the 70% ethanol extracts contained more phenolic compounds than the hot water extracts. Therefore, in this study, ethanol was more efficient than water at extracting phenolic compounds from edible plants. Although the extraction solvents affected the total phenolic content of the extracts, there was no relationship in phenolic content between the 70% ethanolic and hot water extracts. Total phenolic amounts of the ethanolic extracts, in decreasing order, were fatsia, butterbur, chamnamul, leeks, broccoli, bok choy, acanthopanax, crown daisy, pumpkin leaf, and soybean leaf. Meanwhile, those of the aqueous extracts were butterbur, acanthopanax, fatsia, soybean leaf, leeks, broccoli, chamnamul, bok choy, pumpkin leaf, and crown daisy, in contrast to the ethanolic extract results. These differences are most likely due to many different compounds with distinct polarities in the edible plant extracts.
and antioxidant activity in 30 aqueous plant extracts. Similar results were obtained by Velioglu et al. (1998) and Deighton et al. (2000). However, no correlation between phenolic content and antioxidant activity was found by Gazzani et al. (1998) or Heionnen et al. (1998), similar to our findings in this study. For example, the ethanol extract of soybean leaf had the lowest total phenolic content, whereas it had higher DPPH radical scavenging activity than fatsia ethanolic extract, which had a higher total phenolic content. There are many kinds of phenolic compounds and they vary widely in their antioxidant capacity. The antioxidant capacity of phenolic compounds is determined not just by their quantity, but also by their chemical structure. Thus, the antioxidant activity of an extract cannot be explained simply on the basis of phenolic content, but requires proper characterization (Heionnen et al., 1998).
3.1.2. Antioxidant activity Generally, the antioxidant activity of phenolic compounds is due to their radical scavenging effects. The DPPH assay is one of the most widely employed and preferred methods for measuring the radical scavenging activity of plant extracts. This assay is rapid, relatively simple, and easily standardized (Nanjo et al. 1996). DPPH is a stable nitrogen-centered free radical that produces a violet color in methanol solution. When DPPH radicals react with suitable reducing agents (for example, antioxidants), the solution loses its color depending on the number of electrons taken up (Umamaheswari and Chatterjee, 2008). The DPPH radical scavenging activities of 70% ethanolic and hot water extracts of the 10 edible plants are shown in Fig. 2. The antioxidant activities of 70% ethanol extracts of crown daisy, soybean, leek, and butterbur leaves were relatively stronger (p ≤ 0.05) than other plant extracts. Water extracts of chamnamul, bok choy, and butterbur leaves exhibited the strongest radical scavenging activity, whereas crown daisy, leeks leaf, and broccoli had the lowest radical scavenging activity. To examine the relationship between total content and DPPH radical scavenging activity, correlations were calculated; the results are presented in Fig. 3. There was very little correlation (p > 0.05) between total phenolics and DPPH radical scavenging activity. For 70% ethanolic and hot water extracts, the correlation factors (r 2) were 0.0338 and 0.0011, respectively. Several studies have investigated the relationship between phenolic content and antioxidant activity. Some groups found a correlation between the phenolic content and the antioxidant activity, whereas others found no such relationship. Dudonné et al. (2009) demonstrated a high correlation between total phenolics
3.2.1. Vitamin C content Vitamin C or ascorbic acid is a water-soluble vitamin that is ubiquitous in fresh fruits and vegetables. Vitamin C has many nutritional and clinical benefits for human health. As a bioactive constituent, it is involved in wound healing, tyrosine metabolism, conversion of folic acid to folinic acid, carbohydrate metabolism, synthesis of lipids and proteins, iron metabolism, resistance to infections, and cellular respiration. In addition, vitamin C is also a strong antioxidant due to its preventative effect on the oxidation of other compounds upon donation of its electrons. Under certain conditions, it can protect against oxidativelyinduced DNA damage in human cells (Suntornsuk, Gritsanapun, Nilkamhank, & Paochom, 2002). Data obtained from the AOAC method for analyzing vitamin C content is shown in Fig. 4. The vitamin C content of the edible plants varied from 29.85 mg to 107.14 mg AAE/g of plant on a dry weight
Fig. 2. Antioxidant activity of 70% ethanol and distilled water edible plant extracts. (A) Crown daisy leaf; (B) pumpkin leaf; (C) chamnamul; (D) fatsia; (E) leek leaf; (F) bok choy; (G) acanthopanax; (H) butterbur leaf; (I) soybean leaf; (J) broccoli.
3.2. Nutritional values
Fig. 3. Correlation between total polyphenol content and DPPH radical scavenging activity in (A) 70% ethanolic extracts, (B) aqueous extracts.
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basis. Considering the large variation in the vitamin C content, the edible plants could be divided into three groups, namely a high vitamin C group (>80 mg AAE/g plant), a medium vitamin C group (40−80 mg AAE/g plant), and a low vitamin C group (b40 mg AAE/g plant). Interestingly, acanthopanax, bok choy, pumpkin leaf, and soybean leaf had the highest concentrations of vitamin C followed by butterbur, leeks leaf, broccoli, and crown daisy leaf. The low group was represented by chamnamul and fatsia (p≤0.05). As mentioned above, vitamin C is water-soluble and has antioxidant activity. Thus we expected that aqueous extracts of samples containing large amounts of vitamin C would also exhibit high levels of DPPH radical scavenging activity. Gardner et al. (2000) and Larson (1988) reported that samples that contained the highest levels of vitamin C also had the highest antioxidant activity. However, we did not observe this tendency in our study. For example, though the aqueous extract of acanthopanax had the highest vitamin C content, it only had the sixth highest activity in DPPH assays. These results suggest that vitamin C is not the main contributor to the antioxidant activity of plant extracts. It also indicates that the extracts contain many other substances that have antioxidant activity besides vitamin C. Thermal degradation of vitamin C can be a possible explanation for the lack of contribution of vitamin C content to antioxidant activity observed in our study. Vitamin C may be partially degraded during the drying process used for sample preparation (samples were dehydrated at 65 °C for 24 h). According to Kaya et al. (2010), increasing the drying air temperature affects the vitamin C content because of its low stability to heat treatment. Goula and Adamopoulos (2006) also provided information in support of this hypothesis.
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Fig. 5. Chlorophyll contents of edible plant solutions. (A) Crown daisy leaf; (B) pumpkin leaf; (C) chamnamul; (D) fatsia; (E) leek leaf; (F) bok choy; (G) acanthopanax; (H) butterbur leaf; (I) soybean leaf; (J) broccoli.
3.2.2. Total chlorophyll and carotenoid contents Total chlorophyll and carotenoid contents are presented in Figs. 5 and 6, respectively. The chlorophyll a content of the edible plants ranged from 5.06 mg to 44.21 mg/100 g of plant on a dry weight basis (Fig. 5). Among the edible plants, the highest content of chlorophyll a was found in leek leaf, whereas broccoli had the lowest chlorophyll a content. Crown daisy leaf contained the largest amount of chlorophyll b, while fatsia leaf had the lowest content. However, chamnamul leaf had the highest (p≤0.05) total chlorophyll content of the 10 edible plants. The total carotenoid content of the edible plants varied from 3.0 mg to 17.7 mg/100 g of plant (Fig. 6). Chamnamul leaf had the highest carotenoid content while broccoli had the lowest. Butterbur, crown daisy, and pumpkin leaves also had high amounts of total carotenoids. Chlorophyll is the single most critical substance in plants that allows them to absorb light from the sun and convert that light into
usable energy. Therefore, the chlorophyll content of agricultural products is regarded as an indicator of their photosynthetic activity. Moreover, chlorophyll has antioxidant activity under dark conditions. All green plants contain at least one type of chlorophyll (chlorophyll a) and some also contain a second type of chlorophyll (chlorophyll b). The general ratio of chlorophyll a to chlorophyll b is 3:1. Although these values are affected by the cultivar of plant and various environmental factors, plants typically contain more chlorophyll a than chlorophyll b. However, our results are not consistent with those from several previous studies (Bojović & Stojanović, 2005; Endo, Usuki, & Kaneka, 1985). Carotenoids have many physiological functions. Carotenoids are not only precursors of vitamin A, but also contribute to the prevention of cancer and cardiovascular diseases. Epidemiological studies have shown that people with high β-carotene intake and high levels of high β-carotene have a significantly reduced risk of lung cancer. In addition, carotenoids are efficient free radical scavengers, and most carotenoids have antioxidant activity (Machlin, 1995). Despite their documented antioxidant activity, the chlorophyll and carotenoid contents of the plant extracts had no direct relationship to the antioxidant activity of the plant extracts. This implies that these compounds may have been degraded at the high drying temperatures. Another possibility is that chlorophyll and carotenoids might be not the main antioxidant constituents in these edible plants, although the antioxidant effects of these compounds have been demonstrated, as mentioned earlier.
Fig. 4. Vitamin C contents of the edible plant solutions. (A) Crown daisy leaf; (B) pumpkin leaf; (C) chamnamul; (D) fatsia; (E) leek leaf; (F) bok choy; (G) acanthopanax; (H) butterbur leaf; (I) soybean leaf; (J) broccoli.
Fig. 6. Total carotenoid content of the edible plant solutions. (A) Crown daisy leaf; (B) pumpkin leaf; (C) chamnamul; (D) fatsia; (E) leek leaf; (F) bok choy; (G) acanthopanax; (H) butterbur leaf; (I) soybean leaf; (J) broccoli.
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3.3. Effect on lipid oxidation and the color of beef patties
reported that ginger rhizomes and fenugreek extracts at 0.05 and 0.1% (w/w) were effective at controlling lipid oxidation of refrigerated beef patties for up to 12 days. Han and Rhee (2005) showed that 0.25% (w/w) extracts of rosemary, sappanwood, and red or white peony almost completely inhibited lipid oxidation in raw beef patties. We found that a relatively high concentration (0.5%, w/w) of ethanolic butterbur extract showed strong antioxidant activity against lipid oxidation in ground beef patties. Interestingly, a diet with added butterbur effectively prevented lipid peroxidation, protein oxidation, and oxidative DNA damage in an in vivo experiment in mice (Oh, Yang, Kwon, & Kim, 2006).
3.3.1. TBA value The fat content of ground beef was 12±0.7% (w/w). The antioxidative effects of butterbur leaf and broccoli extracts and the synthetic antioxidant BHT in ground beef patties (0.1% and 0.5%, w/w) are shown in Fig. 7. All extracts showed effective antioxidant activity against lipid oxidation, although the TBARS contents of the patties treated with edible plant extracts were higher than those of the patties treated with BHT. As expected, the TBARS values of the control sample increased steadily by 5.7-fold after 12 days, whereas the TBARS values of patties with 0.1% and 0.5% butterbur extract increased by 3.3-fold and 1.8-fold, respectively, after 12 days; significantly less than the control (p ≤ 0.05). The ethanolic extract of broccoli was moderately antioxidative at both 0.1 and 0.5% in beef patties, with significantly lower (p ≤ 0.05) TBARS values than the control, but it was less antioxidative than butterbur extract or BHT treatment. Moreover, 0.5% butterbur extract inhibited lipid oxidation as effectively as 0.5% BHT in beef patties. There was no significant (p >0.05) difference between the butterbur extract and the BHT treatments at 0.5% for up to 12 days. We concluded that a 0.5% butterbur leaf extract is as capable as BHT of maintaining lipid stability and efficiently delaying lipid oxidation in refrigerated beef patties. Our results are in agreement with various other studies, all of which reported that natural antioxidants from culinary herbs and edible plants were effective at controlling lipid oxidation and extending the shelf life of meat products. However, the amounts of extract as well as the extraction solvents, methods, or test systems differed across studies. McCarthy et al. (2001) demonstrated that extracts of tea catechins (0.25%), rosemary (0.10%), and sage (0.05%) were effective at reducing lipid oxidation in pork patties. Similarly, Mansour and Khalil (2000)
3.3.2. Color changes Color changes in raw ground beef were significantly affected by the two edible plant extracts (butterbur and broccoli) and BHT (Fig. 8). All treatments significantly decreased lightness (L*) and yellowness (b*) during the storage period (p ≤0.05; data not shown), and there was no difference (p > 0.05) between the 0.1% or 0.5% extracts (Fig. 8). The a* value (redness) is the most important color parameter in evaluating meat oxidation, as a decrease in redness makes the meat product unacceptable to consumers (Renerre, 2000). In all samples, the redness (a* value) decreased as storage time progressed, but the redness of the control samples faded very rapidly. At the end of storage, the a* value of the control sample was significantly lower (p ≤0.05) than that of the antioxidant-containing meat products. Therefore, the natural plant extracts affected meat color, specifically red, and are therefore potentially useful in prolonging the shelf life of the meat product. Several researchers have examined the effects of different antioxidants on the color of meat and meat products, and have shown that meat oxidation decreases a* values (Higgins, Kerry, Buckley, & Morrisey, 1998; Lee, Hendricks & Cornforth, 1998). We observed a decrease in the
Fig. 7. Effect of butterbur and broccoli extracts, and BHT added at concentrations of 0.1% (A) and 0.5% (B) to lipid oxidation of ground beef compared to control patties. Treatment means within the same storage time that do not share a common letter are significantly different (p≤ 0.05).
Fig. 8. Effect of butterbur and broccoli extract and BHT at concentrations of 0.1% (A) and 0.5% (B) on the a* color value of ground beef as compared to control samples. Treatment means within the same storage time that do not share a common letter are significantly different (p≤0.05).
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a* value (due to myoglobin oxidation) after extract treatment despite the protective effects of the extracts against lipid oxidation (Fig. 7). Meat color deterioration (or oxidation of oxymyoglobin) and lipid oxidation may be dependent on the test system, and whether the sample is stored frozen or not. Studies on meat color have often concentrated on a* values because the redness of meat is an important factor that impacts visual attraction in consumers. Akamittath et al. (1990) indicated that color discoloration or oxidized pigments might promote lipid oxidation. O'Grady et al. (2000), however, concluded that oxymyoglobin oxidation followed lipid oxidation in minced beef. Nevertheless, many researchers have tried to establish a correlation between lipid oxidation and discoloration in meat products (Akamittath, Brekke, & Schanus, 1990; Gray, Gomaa, & Buckley, 1996). Meanwhile, other studies have supported a lack of interaction between lipid oxidation and myoglobin oxidation, which means that the addition of certain natural extracts with polyphenolic compounds may retard lipid oxidation but show no efficacy against meat discoloration (McBride, Hogan, & Kerry, 2007). This is in accordance with our results; although the natural antioxidants inhibited lipid oxidation of ground beef, their ability to stabilize color was unremarkable. 4. Conclusions We evaluated the antioxidant activities and nutritional properties of 10 edible plant extracts. Crown daisy leaf extract had the highest antioxidant activity. However, considering the overall association between phenolic content and antioxidant activity, we decided to further investigate alcohol extract from butterbur leaf. As expected, butterbur leaf extract (0.5%), which showed remarkable antioxidant activity in vitro, exhibited a large inhibitory effect on lipid oxidation in ground beef patties. In addition, butterbur extract was capable of retarding the discoloration of the patties. Furthermore, butterbur extract was equally as effective as the synthetic antioxidant BHT at preventing lipid oxidation of ground beef patties, which indicates that it could potentially be substituted for BHT. Our results indicate that butterbur leaf extract can be used to prevent or minimize lipid oxidation in meat products, and add nutritional quality. Sensory tests need to be conducted to determine the effect of adding edible plant extracts to meat on consumers' perceptions and acceptance of the meat product. Acknowledgments This research was supported by the High Value-added Food Technology Development Program of the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (No. 111138-03-1-SB010). References Akamittath, J. G., Brekke, C. J., & Schanus, E. G. (1990). Lipid oxidation and color stability in restructured meat systems during frozen storage. Journal of Food Science, 55, 1513–1517. Aldrich, H. T., Kendall, P., Bunning, M., Stonaker, F., Kulen, O., & Stushnoff, C. (2011). Environmental temperatures influence antioxidant properties and mineral content in broccoli cultivars grown organically and conventionally. Journal of Agronomy and Crop Science, 2, 1–10. AOAC (1995). Official methods for the analysis (16th ed.). Washington, DC: Association of Official Analytical Chemists. Bae, S. J., & Noh, O. J. (2002). Antioxidant activities of Chrysanthemum coronarium L. fractions on the liposomal phospholipid membrane. Journal of Life Science, 12, 144–150. Barbut, S., Josephson, D. B., & Mauer, A. J. (1985). Antioxidant properties of rosemary oleoresin in turkey sausage. Journal of Food Science, 50, 1356–1359. Bojović, B. M., & Stojanović, J. (2005). Chlorophyll and carotenoid content in wheat cultivars as a function of mineral nutrition. Archives of Biological Sciences, 57, 283–290. Bol'shakova, I. V., Lozovskaia, E. L., & Sapezhinskiĭ, I. I. (1997). Antioxidant properties of a series of extracts from medicinal plants. Biofizika, 42, 480–483. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT, 28, 25–30. Buckley, D. J., Gray, J. I., Asghar, A., Price, J. F., Crackel, R. L., Booren, A. M., Pearson, A. M., & Miller, E. R. (1989). Effects of dietary antioxidants and oxidized oil in membranal lipid stability and pork product quality. Journal of Food Science, 54, 1193–1197.
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Meat Science journal homepage: www.elsevier.com/locate/meatsci
Evaluation of the antioxidant activities and nutritional properties of ten edible plant extracts and their application to fresh ground beef Sung-Jin Kim a,1, Sea C. Min b,1, Hyo-Jin Shin a, Yun-Jeong Lee a, Ah Reum Cho a, So Yeon Kim c, Jaejoon Han a,⁎ a b c
Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon 440-746, Republic of Korea Department of Food Science and Technology, Seoul Women's University, Seoul 139-774, Republic of Korea Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea
a r t i c l e
i n f o
Article history: Received 13 February 2012 Received in revised form 21 September 2012 Accepted 3 November 2012 Keywords: Antioxidant activity Nutritional property Edible plants Lipid oxidation Ground beef
a b s t r a c t In this study, we assessed the antioxidant efficacy and nutritional value of 10 leafy edible plants and evaluated their potential as natural antioxidants for meat preservation. We measured total phenolic content, 2,2-diphenyl1-picryl-hydrazil (DPPH) radical scavenging activity, and vitamin C, chlorophyll, and carotenoid contents of 70% ethanol and water extracts of the edible plants. Based on these results, we investigated the effects of butterbur and broccoli extracts on lipid oxidation in ground beef patties. Plant extracts and butylated hydroxytoluene (BHT) were individually added to patties at both 0.1% and 0.5% (w/w) concentrations. Thiobarbituric acid reactive substance (TBARS) values and color parameters were tested periodically during 12 days of refrigerated storage. TBARS levels were significantly lower (p≤0.05) in the samples containing plant extracts or BHT than the non-treated control. In addition, the beef patties formulated with the selected plant extracts showed significantly (p≤0.05) better color stability than those without antioxidants. These results indicate that edible plant extracts are promising sources of natural antioxidants and can potentially be used as functional preservatives in meat products. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Lipid oxidation is a major cause of quality deterioration in meat and meat products because it leads to color alteration, off-flavor, and loss of nutrients, all of which are major determinants of meat quality (Chan, Decker, & Means, 1993). Lipid oxidation is promoted by diverse factors such as heat, light, metal ions, heme (in meat), oxygen, free radicals, and oxidative enzymes (Barbut, Josephson, & Mauer, 1985; Buckley et al., 1989). Color is another important factor that influences the quality and acceptability of meat. Color is regarded as an indicator of perceived quality and freshness of meat and is the first limiting factor in the shelf-life of meat (Smith, Belk, Sofos, Tatum, & Williams, 2000). Discoloration of meat is closely related to oxidative denaturation of meat pigments (Kanner & Harel, 1985). One way to prevent oxidative deterioration is to use antioxidants in meat products (Lee et al., 2010). These antioxidants can retard lipid oxidation by acting as free radical scavengers, oxygen scavengers, and/or metal chelators (Teets & Were, 2008). Various synthetic antioxidants, such as BHT, butylated hydroxyanisole (BHA), and tertiary butyl hydroquinone (TBHQ), are used in the food industry to delay lipid oxidation (Mansour & Khalil, 2000). However, concerns have been
⁎ Corresponding author at: 300 Cheoncheon-dong, Jangan-gu, Suwon 440-746, Republic of Korea. Tel.: +82 31 290 7803; fax: +82 31 290 7882. E-mail address: [email protected] (J. Han). 1 These authors are considered as co-first authors. 0309-1740/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.meatsci.2012.11.029
raised about using synthetic antioxidants due to their possible side-effects, which has given impetus to finding alternative “natural” antioxidants (Sebranek, Sewalt, Robbins, & Houser, 2005). Most natural antioxidants are obtained from plant resources including culinary herbs, spices, fruits, vegetables, and oilseed products (Shahidi & Zhong, 2010). Many previous studies have focused on herbs and spices consumed in Europe, Southern Asia, and Southeast Asia. Several herbs and spices, such as clove (Naveena, Muthukumar, Sen, Babji, & Murthy, 2006), rosemary (Georgantelis, Blekas, Katikou, Ambrosiadis, & Fletouris, 2007), and oregano and sage (Fasseas, Mountzouris, Tarantilis, Polissiou, & Zervas, 2007) have been reported to significantly improve the keeping quality of meat products, and are effective in delaying lipid oxidation. However, their strong flavor is unfamiliar to East Asians, which limits their applications to foods. Therefore, in this study, we attempted to explore new natural antioxidant sources suitable for East Asians. We selected several leafy, green, and edible plants commonly consumed in Northeast Asian regions. These culinary plants have been eaten for centuries and their safety and edibility are recognized by the Korean Food and Drug Administration (KFDA). They have a milder flavor than herbs and spices and are reported to be good for health. They are often used in salads or as side dishes, either fresh or blanched. Phenolic compounds are the major constituents of plants that contribute to their antioxidant capacity. Phenolic compounds include phenolic acids, flavonoids, and tocopherols (Wong, Hashimoto, & Shibamoto, 1995). Several studies have demonstrated that phenolic compounds scavenge free radicals. Therefore, the total amount of
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phenolic compounds is one of the most important factors affecting antioxidant activity (Møller, Madsen, Aaltonen, & Skibsted, 1999). There are also many other compounds that have functional and nutritional value in edible plants, such as ascorbic acid, nitrogen compounds (amino acids, amines, alkaloids, and chlorophyll derivatives), and carotenoids (Hall & Cuppett, 1997; Hudson 1990). These compounds play a role as nutrients, bioactive substances, as well as antioxidants. Ascorbic acid, also known as vitamin C, is a strong antioxidant found mainly in fresh fruits and vegetables (Gardner, White, Mcphail, & Duthie, 2000). Vitamin C retards free radical-induced cellular damage, and acts as a cofactor of several biosynthesis reactions in the human body (Chan, 1993). Chlorophylls and carotenoids are the most abundant plant pigments in nature, and have antioxidant activity due their singlet oxygen quenching properties (Hirayama, Nakamura, Hamada, & Kobsyasi, 1994; Tanielian & Wolff, 1988). Some carotenoids, such as α- and β-carotene, are precursors of vitamin A (Harrison, 2012). Chlorophyll and its derivatives may prevent certain types of cancers, aid in wound healing, and reduce inflammation in some cases (Morgan, Jackson, Zheng, Pandey, & Pandey, 2010; Smith & Livingston, 1945; Subramoniam et al., 2012). The antioxidant activities of edible plants have been explored by several research groups. However, it is difficult to compare the antioxidant capacity of these plant materials between studies because of different methods of extraction and antioxidant activity determination. Moreover, the nutritional values of these plants were rarely evaluated in these previous studies. Our objectives in this study were therefore to (1) evaluate the antioxidant activities and nutritional properties of 70% ethanol and water extracts of 10 edible plants; and (2) to determine the effectiveness of these extracts in preventing or reducing lipid oxidation as well as color changes in ground beef patties during storage at a chilled temperature (4 °C). 2. Materials and methods 2.1. Edible plants Fresh leaves of crown daisy (Chrysanthemum coronarium var. spatiosum), pumpkin (Cucurbita moschata Duch.), chamnamul (Pimpinella brachycarpa (Kom.) Nakai), fatsia (Aralia elata), leek (Allium tuberosum), bok choy (Brassica campestris var. chinensis), acanthopanax (Acanthopanax sessiliflorum Seeman), butterbur (Petasites japonicus), soybean (Glycine max L. Merr), and the flower heads of broccoli (Brassica oleracea L. var. italica Plenck) were purchased from local farms during the harvest season in April. Although broccoli is not a leafy edible plant, it was used for comparison with other plant materials because of its well-documented antioxidant activities (Aldrich et al., 2011). The edible plants were washed and dehydrated using an electric food dehydrator (Lequip Co., Hwaseong, Korea) at 65 °C for 24 h. Each edible plant was finely pulverized using an electric grinder (Daesung Artron Co., LTD., Seoul, Korea), weighed, and then stored at −20 °C until extraction. 2.2. Preparation of edible plant extracts Extracts were prepared using 70% (v/v) ethanol and water as solvents. Ethanol 70% (v/v) was added to the finely ground plant powders at a ratio of 1:20 (w/v) and the mixture was stirred at 200 rpm using an overhead stirrer (Mtops Co., Seoul, Korea) for 24 h at room temperature. Water extracts were also prepared using distilled–deionized water (1:20, w/v) with stirring for 1 h at 95−100 °C. The extracts were then separated from the residue by filtration through Whatman no.1 filter paper (Whatman International Ltd., Maidstone, U.K.) and the filtrate was concentrated under reduced pressure at 55 °C using a rotary evaporator (Büchi Laboratory Equipment, Postfach, Switzerland). The extra solvent was removed by freeze-drying and the dried extract powder was stored at − 20 °C until further analyses.
2.3. Antioxidant content and antioxidant activity 2.3.1. Total phenolic content The content of total phenolics was measured spectrophotometrically using the Folin–Ciocalteu colorimetric method (Dewanto, Wu, Adom, & Liu, 2002). All plant extracts were diluted with extraction solvent (70% ethanol or distilled–deionized water) to obtain readings within the standard curve range of 0.0−0.8 mg gallic acid/mL. Briefly, 100 μL of diluted plant extract or gallic acid standard solution was mixed well with 2 mL of 2% (w/v) Na2CO3 solution. The mixture was then left to stand for 3 min, after which 100 μL of Folin–Ciocalteu reagent was added. After letting the mixture sit for 30 min at room temperature for color development, the absorbance was measured at 750 nm using a UV–visible spectrophotometer. Results are expressed as mg gallic acid equivalent (GAE)/g dried plant. 2.3.2. DPPH radical scavenging activity The free radical scavenging activity of extracts was measured by the DPPH method as proposed by Brand-Williams et al. (1995). A solution of DPPH in methanol (0.078 mg/mL) was prepared and 0.25 mL of this radical solution was added to 0.05 mL of sample solution. The mixture was incubated for 30 min in the dark at room temperature and then the absorbance was measured at 517 nm with a spectrophotometer. Ascorbic acid solutions in the concentration range of 0.02−0.08 mg/ml were used to establish a standard curve. DPPH radical scavenging activity was expressed as mg ascorbic acid equivalent (AAE)/g dried plant. 2.4. Nutritional value 2.4.1. Vitamin C content Vitamin C content was determined by the titrimetric method as described in the Association of Official Analytical Chemists (AOAC, 1995) method no. 967.21 (1995). Dried plant powder (2 g) was magnetically stirred in 25 mL of extract solution (metaphosphoric acid: acetic acid = 1:5). The homogenate was centrifuged at 12,000 rpm for 5 min, and the supernatant was filtered through Whatman no. 4 filter paper. Aliquots (2 mL) were then placed in test tubes, after which 200 μL of indophenol and 2 mL of thiourea metaphosphoric solution were added. After that, 1 mL of 2,4-dinitrophenyl hydrazine (DNP) solution was mixed with the sample solution and the mixture was allowed to stand at 37 °C for 3 h and then cooled on ice. Next, 5 mL of 85% H2SO4 solution was added, and the resulting mixture was incubated in the dark for 30 min at room temperature. Absorbance was measured at 540 nm and vitamin C content was expressed as mg ascorbic acid (AAE) equivalent/g of plant on a dry weight basis. 2.4.2. Total chlorophyll and total carotenoid contents The content of chlorophylls a and b, as well as that of total carotenoids, was spectrophotometrically determined using the method of Lichtenthaler (1987). Dried edible plant samples (1 g) were stirred in 50 mL of 80% acetone (v/v) solution in the dark for 24 h at room temperature. After filtration (Whatman no. 4 filter paper), the filtrate volume was adjusted to 100 mL with 80% acetone (v/v). Absorbance was read at 662, 644, and 470 nm to measure the content of chlorophyll a, chlorophyll b, and carotenoids, respectively. Total chlorophyll was calculated as the sum of chlorophylls a and b. Total chlorophyll and total carotenoid contents were expressed as mg/100 g of plant on a dry weight basis. 2.5. Effects on lipid oxidation and color of beef patties 2.5.1. Determination of fat content in ground beef Fresh raw beef was obtained from a local supermarket and prepared using separable lean from top round roasts. On the day of purchase, the roasts were trimmed of all separable fat and connective
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tissue, and then ground twice through a 1.27 cm plate, followed by a 0.32 cm plate. Before patty preparation, the fat content of the meat sample was analyzed according to the method of Folch et al. (1957). A 5 g portion of meat sample was homogenized with 75 mL of chloroform: methanol (2:1, v/v) using a stirrer for 3 min. After filtering, the meat residue was re-homogenized for 3 min with 5 mL of chloroform: methanol (2:1), and then re-filtered. The collected filtrate was transferred to a 500 mL separatory funnel, and then 25 mL of distilled water was added and the phases were allowed to separate. The lower phase in the funnel (organic phase) was drained from the funnel into a 100 mL volumetric flask. The organic phase was brought to a volume of 100 mL with pure chloroform. The solvent was removed under reduced pressure at below 40 °C using a rotary evaporator, and the samples were dried at 100 °C for 1 h. The mean fat content was determined by averaging the triplicates.
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replicates, two samples/replicate, two types of extraction solvent, 10 edible plants). Experiments to determine nutritional values (vitamin C, chlorophyll, carotenoid contents) were carried out using a 3 × 2 × 10 factorial design (three replicates, two samples/replicate, 10 edible plants). Preservation studies (TBARS and color measurements) were performed using a 3 × 2 × 2 × 4 × 4 factorial design (three replicates, two samples/replicate, two concentrations of preservative, four types of preservative including control, four storage periods). Data analyses were performed using Statistical Analysis System (SAS) software, version 8.1 (SAS Institute, Cary, NC, USA). The General Linear Models procedure was used for analysis of variance, with main effect means separated by the Student–Newman–Keuls test. Significance was defined as p ≤ 0.05.
3. Results and discussion 2.5.2. Preparation of ground beef patties Ground beef patties were prepared for four different treatments. The negative control group was formulated without antioxidants. Considering the overall association between phenolic content and antioxidant capacity that we found after performing the experiments described above, 70% ethanol extract from butterbur leaf was selected as the best material due to its antioxidant content and activity. Ethanolic broccoli extract was also chosen as a natural antioxidant for meat preservation because broccoli is a vegetable commonly consumed worldwide and its nutritional value and antioxidant properties have been well characterized (Aldrich et al., 2011). Ground beef patties with plant extract were prepared by adding two concentrations (0.1% and 0.5%, w/w) of ethanolic butterbur and broccoli extracts. Positive controls were prepared by treating the patties with BHT solution (0.1% and 0.5%, w/w). A 20 g portion of each meat sample was made into patties using a patty mold (6 cm diameter, 1.2 cm height) and wrapped with LLDPE film (Clean Wrap, Kimhae, Korea). Prepared beef patties were stored in a refrigerator at 4 °C for 12 days and the TBARS values and color parameters were determined at 0, 3, 6, 9, and 12 days of storage. The preservation experiments were replicated three times on different days, with two patties stored for each storage time within each treatment replication. 2.5.3. TBA value The lipid oxidation potential of meat was evaluated by measuring TBARS. A 20 g portion of each meat sample was homogenized with 50 mL of distilled–deionized water and 10 mL of trichloroacetic acid (15%, final concentration) using a stomacher for 2 min. The homogenate was centrifuged at 20,000 rpm for 5 min, and the supernatant was filtered through Whatman no. 1 filter paper. A 2 mL aliquot of 0.06 M thiobarbituric acid was added to 8 mL of the filtrate. The solution was vortexed for 15 s, placed in a 80 °C water bath for 90 min, and then cooled on ice. Absorption was measured at 532 nm using a UV–visible spectrophotometer. Results were expressed as mg malondialdehyde (MDA) equivalent/kg of meat sample.
3.1. Antioxidant content and antioxidant activity 3.1.1. Total phenolic content Phenolic compounds, containing one or more acidic hydroxyl residues attached to an aromatic arene (phenyl) ring, are one of the most active constituents that contribute to the antioxidant activities of plant foods (Velioglu, Mazza, Gao, & Oomah, 1998). Therefore, it is important to quantify phenolic content and to assess its contribution to the antioxidant activity of plant materials. The total phenolic contents of 70% ethanol and hot water extracts of 10 edible plants are depicted in Fig. 1. The amount of total phenolics varied widely in the edible plant extracts, ranging from 3.13 to 72.30 mg GAE/g extract. The seventy percent ethanol extract of fatsia had the highest polyphenol content, followed by the butterbur and chamnamul ethanol extracts. Among hot water extracts, butterbur, acanthopanax, and fatsia extracts had significantly (p ≤ 0.05) higher concentrations of phenolic compounds than the hot water extracts of the other plants. Hot water and 70% ethanol extracts of fatsia and butterbur had relatively high total phenolic content (p ≤ 0.05). The antioxidant effects of fatsia extract have been studied previously by Bol'shakova et al. (1997). Cha et al. (2009) also reported that ethanol extracts from fatsia exhibited strong antioxidant activity against lipid oxidation as measured by the TBA method. In addition, Bae and Noh (2002) reported that butterbur fractions protected biological membranes from benzoyl peroxide-induced peroxidation. Takenaka et al. (2000) found that the phenolic compounds that made a major contribution to the antioxidant capacity of butterbur were caffeic acid and chlorogenic acid.
2.5.4. Color measurement The color of raw beef patties was evaluated using a Minolta Chroma Meter CR-400 equipped with a DP-400 data processor (Minolta, Inc, Japan). Color was described as L* (lightness), a* (redness), and b* (yellowness) color space values. A Chroma meter was standardized with a standard white plate (L* = 93.80, a* = −0.3157 and b* = 0.3319). Measurements were made perpendicular to the patty surface at five different locations per patty, and mean values (L*, a*, and b*) from each patty were analyzed. 2.6. Statistical analysis Experiments to evaluate antioxidant content and antioxidant activity were carried out using a 3 × 2 × 2 × 10 factorial design (three
Fig. 1. Total phenolic content of 70% ethanol and distilled water edible plant extracts. (A) Crown daisy leaf; (B) pumpkin leaf; (C) chamnamul; (D) fatsia; (E) leek leaf; (F) bok choy; (G) acanthopanax; (H) butterbur leaf; (I) soybean leaf; (J) broccoli.
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In the present study, two different extraction solvents, namely 70% ethanol and hot water, were used. Overall, the 70% ethanol extracts contained more phenolic compounds than the hot water extracts. Therefore, in this study, ethanol was more efficient than water at extracting phenolic compounds from edible plants. Although the extraction solvents affected the total phenolic content of the extracts, there was no relationship in phenolic content between the 70% ethanolic and hot water extracts. Total phenolic amounts of the ethanolic extracts, in decreasing order, were fatsia, butterbur, chamnamul, leeks, broccoli, bok choy, acanthopanax, crown daisy, pumpkin leaf, and soybean leaf. Meanwhile, those of the aqueous extracts were butterbur, acanthopanax, fatsia, soybean leaf, leeks, broccoli, chamnamul, bok choy, pumpkin leaf, and crown daisy, in contrast to the ethanolic extract results. These differences are most likely due to many different compounds with distinct polarities in the edible plant extracts.
and antioxidant activity in 30 aqueous plant extracts. Similar results were obtained by Velioglu et al. (1998) and Deighton et al. (2000). However, no correlation between phenolic content and antioxidant activity was found by Gazzani et al. (1998) or Heionnen et al. (1998), similar to our findings in this study. For example, the ethanol extract of soybean leaf had the lowest total phenolic content, whereas it had higher DPPH radical scavenging activity than fatsia ethanolic extract, which had a higher total phenolic content. There are many kinds of phenolic compounds and they vary widely in their antioxidant capacity. The antioxidant capacity of phenolic compounds is determined not just by their quantity, but also by their chemical structure. Thus, the antioxidant activity of an extract cannot be explained simply on the basis of phenolic content, but requires proper characterization (Heionnen et al., 1998).
3.1.2. Antioxidant activity Generally, the antioxidant activity of phenolic compounds is due to their radical scavenging effects. The DPPH assay is one of the most widely employed and preferred methods for measuring the radical scavenging activity of plant extracts. This assay is rapid, relatively simple, and easily standardized (Nanjo et al. 1996). DPPH is a stable nitrogen-centered free radical that produces a violet color in methanol solution. When DPPH radicals react with suitable reducing agents (for example, antioxidants), the solution loses its color depending on the number of electrons taken up (Umamaheswari and Chatterjee, 2008). The DPPH radical scavenging activities of 70% ethanolic and hot water extracts of the 10 edible plants are shown in Fig. 2. The antioxidant activities of 70% ethanol extracts of crown daisy, soybean, leek, and butterbur leaves were relatively stronger (p ≤ 0.05) than other plant extracts. Water extracts of chamnamul, bok choy, and butterbur leaves exhibited the strongest radical scavenging activity, whereas crown daisy, leeks leaf, and broccoli had the lowest radical scavenging activity. To examine the relationship between total content and DPPH radical scavenging activity, correlations were calculated; the results are presented in Fig. 3. There was very little correlation (p > 0.05) between total phenolics and DPPH radical scavenging activity. For 70% ethanolic and hot water extracts, the correlation factors (r 2) were 0.0338 and 0.0011, respectively. Several studies have investigated the relationship between phenolic content and antioxidant activity. Some groups found a correlation between the phenolic content and the antioxidant activity, whereas others found no such relationship. Dudonné et al. (2009) demonstrated a high correlation between total phenolics
3.2.1. Vitamin C content Vitamin C or ascorbic acid is a water-soluble vitamin that is ubiquitous in fresh fruits and vegetables. Vitamin C has many nutritional and clinical benefits for human health. As a bioactive constituent, it is involved in wound healing, tyrosine metabolism, conversion of folic acid to folinic acid, carbohydrate metabolism, synthesis of lipids and proteins, iron metabolism, resistance to infections, and cellular respiration. In addition, vitamin C is also a strong antioxidant due to its preventative effect on the oxidation of other compounds upon donation of its electrons. Under certain conditions, it can protect against oxidativelyinduced DNA damage in human cells (Suntornsuk, Gritsanapun, Nilkamhank, & Paochom, 2002). Data obtained from the AOAC method for analyzing vitamin C content is shown in Fig. 4. The vitamin C content of the edible plants varied from 29.85 mg to 107.14 mg AAE/g of plant on a dry weight
Fig. 2. Antioxidant activity of 70% ethanol and distilled water edible plant extracts. (A) Crown daisy leaf; (B) pumpkin leaf; (C) chamnamul; (D) fatsia; (E) leek leaf; (F) bok choy; (G) acanthopanax; (H) butterbur leaf; (I) soybean leaf; (J) broccoli.
3.2. Nutritional values
Fig. 3. Correlation between total polyphenol content and DPPH radical scavenging activity in (A) 70% ethanolic extracts, (B) aqueous extracts.
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basis. Considering the large variation in the vitamin C content, the edible plants could be divided into three groups, namely a high vitamin C group (>80 mg AAE/g plant), a medium vitamin C group (40−80 mg AAE/g plant), and a low vitamin C group (b40 mg AAE/g plant). Interestingly, acanthopanax, bok choy, pumpkin leaf, and soybean leaf had the highest concentrations of vitamin C followed by butterbur, leeks leaf, broccoli, and crown daisy leaf. The low group was represented by chamnamul and fatsia (p≤0.05). As mentioned above, vitamin C is water-soluble and has antioxidant activity. Thus we expected that aqueous extracts of samples containing large amounts of vitamin C would also exhibit high levels of DPPH radical scavenging activity. Gardner et al. (2000) and Larson (1988) reported that samples that contained the highest levels of vitamin C also had the highest antioxidant activity. However, we did not observe this tendency in our study. For example, though the aqueous extract of acanthopanax had the highest vitamin C content, it only had the sixth highest activity in DPPH assays. These results suggest that vitamin C is not the main contributor to the antioxidant activity of plant extracts. It also indicates that the extracts contain many other substances that have antioxidant activity besides vitamin C. Thermal degradation of vitamin C can be a possible explanation for the lack of contribution of vitamin C content to antioxidant activity observed in our study. Vitamin C may be partially degraded during the drying process used for sample preparation (samples were dehydrated at 65 °C for 24 h). According to Kaya et al. (2010), increasing the drying air temperature affects the vitamin C content because of its low stability to heat treatment. Goula and Adamopoulos (2006) also provided information in support of this hypothesis.
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Fig. 5. Chlorophyll contents of edible plant solutions. (A) Crown daisy leaf; (B) pumpkin leaf; (C) chamnamul; (D) fatsia; (E) leek leaf; (F) bok choy; (G) acanthopanax; (H) butterbur leaf; (I) soybean leaf; (J) broccoli.
3.2.2. Total chlorophyll and carotenoid contents Total chlorophyll and carotenoid contents are presented in Figs. 5 and 6, respectively. The chlorophyll a content of the edible plants ranged from 5.06 mg to 44.21 mg/100 g of plant on a dry weight basis (Fig. 5). Among the edible plants, the highest content of chlorophyll a was found in leek leaf, whereas broccoli had the lowest chlorophyll a content. Crown daisy leaf contained the largest amount of chlorophyll b, while fatsia leaf had the lowest content. However, chamnamul leaf had the highest (p≤0.05) total chlorophyll content of the 10 edible plants. The total carotenoid content of the edible plants varied from 3.0 mg to 17.7 mg/100 g of plant (Fig. 6). Chamnamul leaf had the highest carotenoid content while broccoli had the lowest. Butterbur, crown daisy, and pumpkin leaves also had high amounts of total carotenoids. Chlorophyll is the single most critical substance in plants that allows them to absorb light from the sun and convert that light into
usable energy. Therefore, the chlorophyll content of agricultural products is regarded as an indicator of their photosynthetic activity. Moreover, chlorophyll has antioxidant activity under dark conditions. All green plants contain at least one type of chlorophyll (chlorophyll a) and some also contain a second type of chlorophyll (chlorophyll b). The general ratio of chlorophyll a to chlorophyll b is 3:1. Although these values are affected by the cultivar of plant and various environmental factors, plants typically contain more chlorophyll a than chlorophyll b. However, our results are not consistent with those from several previous studies (Bojović & Stojanović, 2005; Endo, Usuki, & Kaneka, 1985). Carotenoids have many physiological functions. Carotenoids are not only precursors of vitamin A, but also contribute to the prevention of cancer and cardiovascular diseases. Epidemiological studies have shown that people with high β-carotene intake and high levels of high β-carotene have a significantly reduced risk of lung cancer. In addition, carotenoids are efficient free radical scavengers, and most carotenoids have antioxidant activity (Machlin, 1995). Despite their documented antioxidant activity, the chlorophyll and carotenoid contents of the plant extracts had no direct relationship to the antioxidant activity of the plant extracts. This implies that these compounds may have been degraded at the high drying temperatures. Another possibility is that chlorophyll and carotenoids might be not the main antioxidant constituents in these edible plants, although the antioxidant effects of these compounds have been demonstrated, as mentioned earlier.
Fig. 4. Vitamin C contents of the edible plant solutions. (A) Crown daisy leaf; (B) pumpkin leaf; (C) chamnamul; (D) fatsia; (E) leek leaf; (F) bok choy; (G) acanthopanax; (H) butterbur leaf; (I) soybean leaf; (J) broccoli.
Fig. 6. Total carotenoid content of the edible plant solutions. (A) Crown daisy leaf; (B) pumpkin leaf; (C) chamnamul; (D) fatsia; (E) leek leaf; (F) bok choy; (G) acanthopanax; (H) butterbur leaf; (I) soybean leaf; (J) broccoli.
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3.3. Effect on lipid oxidation and the color of beef patties
reported that ginger rhizomes and fenugreek extracts at 0.05 and 0.1% (w/w) were effective at controlling lipid oxidation of refrigerated beef patties for up to 12 days. Han and Rhee (2005) showed that 0.25% (w/w) extracts of rosemary, sappanwood, and red or white peony almost completely inhibited lipid oxidation in raw beef patties. We found that a relatively high concentration (0.5%, w/w) of ethanolic butterbur extract showed strong antioxidant activity against lipid oxidation in ground beef patties. Interestingly, a diet with added butterbur effectively prevented lipid peroxidation, protein oxidation, and oxidative DNA damage in an in vivo experiment in mice (Oh, Yang, Kwon, & Kim, 2006).
3.3.1. TBA value The fat content of ground beef was 12±0.7% (w/w). The antioxidative effects of butterbur leaf and broccoli extracts and the synthetic antioxidant BHT in ground beef patties (0.1% and 0.5%, w/w) are shown in Fig. 7. All extracts showed effective antioxidant activity against lipid oxidation, although the TBARS contents of the patties treated with edible plant extracts were higher than those of the patties treated with BHT. As expected, the TBARS values of the control sample increased steadily by 5.7-fold after 12 days, whereas the TBARS values of patties with 0.1% and 0.5% butterbur extract increased by 3.3-fold and 1.8-fold, respectively, after 12 days; significantly less than the control (p ≤ 0.05). The ethanolic extract of broccoli was moderately antioxidative at both 0.1 and 0.5% in beef patties, with significantly lower (p ≤ 0.05) TBARS values than the control, but it was less antioxidative than butterbur extract or BHT treatment. Moreover, 0.5% butterbur extract inhibited lipid oxidation as effectively as 0.5% BHT in beef patties. There was no significant (p >0.05) difference between the butterbur extract and the BHT treatments at 0.5% for up to 12 days. We concluded that a 0.5% butterbur leaf extract is as capable as BHT of maintaining lipid stability and efficiently delaying lipid oxidation in refrigerated beef patties. Our results are in agreement with various other studies, all of which reported that natural antioxidants from culinary herbs and edible plants were effective at controlling lipid oxidation and extending the shelf life of meat products. However, the amounts of extract as well as the extraction solvents, methods, or test systems differed across studies. McCarthy et al. (2001) demonstrated that extracts of tea catechins (0.25%), rosemary (0.10%), and sage (0.05%) were effective at reducing lipid oxidation in pork patties. Similarly, Mansour and Khalil (2000)
3.3.2. Color changes Color changes in raw ground beef were significantly affected by the two edible plant extracts (butterbur and broccoli) and BHT (Fig. 8). All treatments significantly decreased lightness (L*) and yellowness (b*) during the storage period (p ≤0.05; data not shown), and there was no difference (p > 0.05) between the 0.1% or 0.5% extracts (Fig. 8). The a* value (redness) is the most important color parameter in evaluating meat oxidation, as a decrease in redness makes the meat product unacceptable to consumers (Renerre, 2000). In all samples, the redness (a* value) decreased as storage time progressed, but the redness of the control samples faded very rapidly. At the end of storage, the a* value of the control sample was significantly lower (p ≤0.05) than that of the antioxidant-containing meat products. Therefore, the natural plant extracts affected meat color, specifically red, and are therefore potentially useful in prolonging the shelf life of the meat product. Several researchers have examined the effects of different antioxidants on the color of meat and meat products, and have shown that meat oxidation decreases a* values (Higgins, Kerry, Buckley, & Morrisey, 1998; Lee, Hendricks & Cornforth, 1998). We observed a decrease in the
Fig. 7. Effect of butterbur and broccoli extracts, and BHT added at concentrations of 0.1% (A) and 0.5% (B) to lipid oxidation of ground beef compared to control patties. Treatment means within the same storage time that do not share a common letter are significantly different (p≤ 0.05).
Fig. 8. Effect of butterbur and broccoli extract and BHT at concentrations of 0.1% (A) and 0.5% (B) on the a* color value of ground beef as compared to control samples. Treatment means within the same storage time that do not share a common letter are significantly different (p≤0.05).
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a* value (due to myoglobin oxidation) after extract treatment despite the protective effects of the extracts against lipid oxidation (Fig. 7). Meat color deterioration (or oxidation of oxymyoglobin) and lipid oxidation may be dependent on the test system, and whether the sample is stored frozen or not. Studies on meat color have often concentrated on a* values because the redness of meat is an important factor that impacts visual attraction in consumers. Akamittath et al. (1990) indicated that color discoloration or oxidized pigments might promote lipid oxidation. O'Grady et al. (2000), however, concluded that oxymyoglobin oxidation followed lipid oxidation in minced beef. Nevertheless, many researchers have tried to establish a correlation between lipid oxidation and discoloration in meat products (Akamittath, Brekke, & Schanus, 1990; Gray, Gomaa, & Buckley, 1996). Meanwhile, other studies have supported a lack of interaction between lipid oxidation and myoglobin oxidation, which means that the addition of certain natural extracts with polyphenolic compounds may retard lipid oxidation but show no efficacy against meat discoloration (McBride, Hogan, & Kerry, 2007). This is in accordance with our results; although the natural antioxidants inhibited lipid oxidation of ground beef, their ability to stabilize color was unremarkable. 4. Conclusions We evaluated the antioxidant activities and nutritional properties of 10 edible plant extracts. Crown daisy leaf extract had the highest antioxidant activity. However, considering the overall association between phenolic content and antioxidant activity, we decided to further investigate alcohol extract from butterbur leaf. As expected, butterbur leaf extract (0.5%), which showed remarkable antioxidant activity in vitro, exhibited a large inhibitory effect on lipid oxidation in ground beef patties. In addition, butterbur extract was capable of retarding the discoloration of the patties. Furthermore, butterbur extract was equally as effective as the synthetic antioxidant BHT at preventing lipid oxidation of ground beef patties, which indicates that it could potentially be substituted for BHT. Our results indicate that butterbur leaf extract can be used to prevent or minimize lipid oxidation in meat products, and add nutritional quality. Sensory tests need to be conducted to determine the effect of adding edible plant extracts to meat on consumers' perceptions and acceptance of the meat product. Acknowledgments This research was supported by the High Value-added Food Technology Development Program of the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (No. 111138-03-1-SB010). References Akamittath, J. G., Brekke, C. J., & Schanus, E. G. (1990). Lipid oxidation and color stability in restructured meat systems during frozen storage. Journal of Food Science, 55, 1513–1517. Aldrich, H. T., Kendall, P., Bunning, M., Stonaker, F., Kulen, O., & Stushnoff, C. (2011). Environmental temperatures influence antioxidant properties and mineral content in broccoli cultivars grown organically and conventionally. Journal of Agronomy and Crop Science, 2, 1–10. AOAC (1995). Official methods for the analysis (16th ed.). Washington, DC: Association of Official Analytical Chemists. Bae, S. J., & Noh, O. J. (2002). Antioxidant activities of Chrysanthemum coronarium L. fractions on the liposomal phospholipid membrane. Journal of Life Science, 12, 144–150. Barbut, S., Josephson, D. B., & Mauer, A. J. (1985). Antioxidant properties of rosemary oleoresin in turkey sausage. Journal of Food Science, 50, 1356–1359. Bojović, B. M., & Stojanović, J. (2005). Chlorophyll and carotenoid content in wheat cultivars as a function of mineral nutrition. Archives of Biological Sciences, 57, 283–290. Bol'shakova, I. V., Lozovskaia, E. L., & Sapezhinskiĭ, I. I. (1997). Antioxidant properties of a series of extracts from medicinal plants. Biofizika, 42, 480–483. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT, 28, 25–30. Buckley, D. J., Gray, J. I., Asghar, A., Price, J. F., Crackel, R. L., Booren, A. M., Pearson, A. M., & Miller, E. R. (1989). Effects of dietary antioxidants and oxidized oil in membranal lipid stability and pork product quality. Journal of Food Science, 54, 1193–1197.
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