Food Sci. Technol. Res., 16 (6), 549–556, 2010
Antioxidant and DNA Damage Prevention Activities of the Edible Parts of Gnetum gnemon and Their Changes upon Heat Treatment
1 1 2 3 4 Martha Santoso , Yuko Naka , Clement Angkawidjaja , Tomoko Yamaguchi , Teruyoshi Matoba and 5,6* Hitoshi Takamura
1 Graduate School of Humanities and Sciences, Nara Women’s University, Nara 630-8506, Japan 2 Department of Material and Life Science, Osaka University, Osaka 565-0871, Japan 3 Faculty of Education, Niigata University, Niigata 950-2181, Japan 4 Department of Nutritional Sciences for Well-being, Kansai University of Welfare Sciences, Kashiwara, Osaka 582-0026, Japan 5 Department of Food Science and Nutrition, Nara Women’s University, Nara 630-8506, Japan 6 KYOUSEI Science Center for Life and Nature, Nara Women’s University, Nara 630-8506, Japan
Received December 2, 2009; Accepted March 25, 2010
In this paper we report on the antioxidant components and activities of the edible parts of Gnetum gnemon (young and mature leaves, seed skin and endosperm), as well as their ability to inhibit DNA dam- age. Since Gnetum gnemon is usually consumed daily as a boiled vegetable, we also measured the effect of boiling on the antioxidant components and activities. The mature leaves contain the greatest levels of anti- oxidant components, followed by the young leaves. Accordingly, the mature and young leaves possess the highest antioxidant component and DNA damage prevention activities. However, the endosperm also has significant activity in ORAC, peroxyl radical-scavenging activity and DNA damage prevention activity at higher concentrations. Boiling decreased the amount of antioxidant components as well as the antioxidant activities in all edible parts examined, except for the seed skin. The results presented here indicate that the edible parts of G. gnemon are a potential source of natural antioxidants and can be beneficial, consumed as a daily vegetable or processed into an extract for use as a food additive.
Keywords: Gnetum gnemon, melinjo, antioxidant, DNA damage
Introduction to the deterioration of food’s nutritional and sensory qualities A significant number of plant species possesses large during processing and storage (Pegg and Shahidi, 2007; Hal- amounts of bioactive compounds that are naturally im- liwell, 1999). portant for their normal growth development and defense Gnetum gnemon (melinjo in Indonesian) belongs to the against infection and injury. Many of these compounds are Gymnophyta, a group of tropical plants that are widely dis- flavonoids or other polyphenolic compounds with antioxida- tributed in Southeast Asia and Melanesia (Manner and Ele- tive properties. These have been shown to exhibit multiple vitch, 2006). Its leaves and seeds are commonly consumed beneficial effects in humans, such as protection against the boiled, particularly in Indonesia. The endosperm is also a development of cancer, and heart and degenerative diseases, raw material in the production of the regionally popular which is why the consumption of the edible parts of plants melinjo chips. The roots and seeds of G. gnemon have been rich in antioxidants is highly recommended (Sun, 1990; Di reported to contain stilbenoids that, as shown by various rad- Carlo et al., 1999). In the food industry, these compounds ical scavenging activity assays (Wallace and Morris, 1978; are increasingly attracting attention for their great potential Iliya et al., 2003a; Iliya et al., 2003b; Kato et al., 2009), con- in the inhibition of lipid oxidation, a process which can lead tribute to their high antioxidant activity. In addition, the seed extract also possesses antimicrobial activity as well as lipase *To whom correspondence should be addressed. and amylase inhibition activities (Kato et al., 2009). The an- E-mail: [email protected] timicrobial activity may be attributable to the stilbene oligo- 550 M. Santoso et al. mers that are present in relatively abundant amounts in the leaves and whole seeds were boiled with purified water for seeds of the Gnetum family (Sakagami et al., 2007). Despite 10 min, immediately removed from the water and dried at the importance of G. gnemon for human consumption, there room temperature. The fresh and boiled whole seeds were have been no reports on the distribution and activity of anti- then peeled to obtain the endosperm and seed skin. The oxidant components in the other edible parts of G. gnemon samples were then immediately frozen using liquid nitrogen, and their stability against heat treatment. freeze-dried for 48 h and kept at -20℃. This study explores the distribution and activity of anti- The freeze-dried samples were extracted with 90% meth- oxidant components, as well as the DNA damage prevention anol contained 5% acetic acid at 10℃ for 30 min and used as activity, of the edible parts of G. gnemon (young and mature methanolic extracts. For the oxygen radical absorbance ca- leaves, endosperm, and seed skin). The effect of heat treat- pacity (ORAC) assay, prior to analysis, the hydrophilic and ment (boiling) on these properties was also examined. Heat lipophilic parts of the samples were separated accordingly. treatment was limited to boiling, since boiling is the most The sample was first extracted with hexane to obtain the li- widely used method to prepare the edible parts of G. gnemon pophilic portion. The upper layer was collected and dried un- for human consumption as a daily vegetable. der nitrogen flow and dissolved in acetone. The sample was diluted with 7% RMCD solution in acetone/water (1:1, v/v). Materials and Methods The defatted fraction (hexane insoluble) was then extracted Sample and materials The fresh edible parts (leaves with 90% methanol containing 5% acetic acid. The upper and seeds) of G. gnemon, planted in Java, Indonesia, were layer was collected and diluted with 75 mM phosphate buffer exported to Japan under low temperature by Hosoda SHC (pH 7.0) and used directly as the hydrophilic fraction. Co., Ltd. (Fukui, Japan) on July 2007 and kept at -20℃ until Total phenolic content The total phenolic content was use. Leaves light green in appearance and less than or equal determined by the Folin-Ciocalteu method (Singleton et al., to 10 cm in blade length were classified as young leaves and 1965) using the methanolic extract. Gallic acid was used as those dark green in appearance and more than 10 cm in blade the standard. The absorbance values of the samples and stan- length were classified as mature leaves. dard were measured at 765 nm using a UV-2100 PC UV-VIS l-ascorbate standard, gallic acid standard, Folin-Ciocal- spectrophotometer (Shimadzu, Kyoto, Japan). Total phenolic teu reagent, buffer components, 1,1-diphenyl-2-picrylhy- content was calculated as µmol gallic acid equivalent per 100 drazyl (DPPH), 2,4-dinitrophenylhydrazine, 1,4-dioxane, g fresh or boiled weight. sodium chloride, and DMSO were purchased from Nacalai Tocopherol content For the analysis of tocopherol con- Tesque (Kyoto, Japan). Acetic acid, 2,6-dichloroindophenol, tent, samples were subjected to saponification using 60% 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), potassium hydroxide containing 6% pyrogallol in methanol, 2′-deoxyguanosine, stannous chloride, pyrogallol, potassium incubated at 70℃ for 30 min and then extracted with 10% hydroxide, flavone standard, disodium ethylene diamine- ethyl acetate in hexane. The tocopherol (α, β, γ, and δ) con- N,N,N′,N′-tetraacetic acid (EDTA·2Na), ferrous sulfate, tents were determined according to the method of Ueda and hydrogen peroxide, metaphosphoric acid, trimethylamine, Igarashi (1990) by HPLC coupled with a fluorescence detec- ethyl acetate, acetone, ethanol, HPLC grade-methanol, tor. The HPLC system consists of a Cosmosil 5SL-11 col- acetonitrile, hexane, 2-propanol and other analytical grade umn (4.6 × 250 mm, Nacalai Tesque Inc., Kyoto, Japan), an chemicals were obtained from Wako Pure Chemical Indus- L-6000 pump and an F-1000 fluorescence detector (Hitachi, tries, Ltd. (Osaka, Japan). Vitamin E reference standards and Tokyo, Japan). Excitation and emission wavelengths were 2,2,5,7,8-pentamethyl-6-chromanol (PMC) were purchased set at 295 and 325 nm, respectively. The mobile phase was from Eisai (Tokyo, Japan). Fluorescein (sodium salt) and hexane/1,4-dioxane/2-propanol (98.5/1.0/0.5, by vol.) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid the flow rate was set at 1.0 mL/min. 2,2,5,7,8-pentamethyl- (Trolox) standard were purchased from Sigma-Aldrich (St. 6-chromanol (PMC) was used as an internal standard. The Louis, MO, USA). Randomly methylated β-cyclodextrin tocopherol content was expressed as mg of tocopherol per (RMCD) (Trappsol) was purchased from Cyclodextrin Tech- 100 g fresh or boiled weight. nologies Development Inc. (High Springs, FL, USA). The Ascorbic acid content Ascorbic acid content was de- gnemonoside D standard was provided by Hosoda SHC Co., termined by HPLC as described previously (Kishida et al., Ltd. The water used for HPLC was purified with Milli-Q 1992). The samples were first extracted with a 5% meta- Labo equipment (Millipore Japan, Tokyo, Japan). phosphoric acid solution and incubated at 10℃ for 30 min. Sample preparation All samples were defrosted for 1 h The extracts were filtered and directly used for analysis. at 10℃, washed with water and drained. The young, mature HPLC analysis was carried out on a Cosmosil 5C18-AR-2 Antioxidant activity of Gnetum gnemon 551 column (4.6 × 250 mm, Nacalai Tesque, Kyoto, Japan) with of 485 nm and an emission wavelength of 520 nm from the a Shimadzu SPD-10AV UV-VIS detector set at 505 nm, a bottom of the plate and programmed to read every min for Shimadzu LC-10ADVP pump and a Rheodyne injector fitted 60 min at 37℃. Plates were shaken for 8 s following each with a 20-µL loop. The mobile phase was acetonitrile/water reading. The ORAC values were calculated based on a re- (50/50, v/v) with 0.1% trimethylamine, adjusted to pH 3.5 gression equation between Trolox concentration and the net with phosphoric acid, at a flow rate of 1 mL/min. The data area under the fluorescence decay curve and were expressed were expressed as mg ascorbic acid per 100 g fresh or boiled as µmol Trolox equivalent per 100 g fresh or boiled weight. weight. Peroxyl radical-scavenging activity The peroxyl Total polyphenol and gnemonoside derivatives Total radical-scavenging activity assay was performed using the polyphenol and gnemonoside derivatives were measured ac- 2’-deoxyguanosine oxidation prevention method described cording to the method of Sakakibara et al. (2003). The meth- by Sakakibara et al. (2002). The methanolic extracts were anolic extracts were dried under nitrogen flow and dissolved dried under nitrogen flow and then dissolved with DMSO. in DMSO. The DMSO extracts were injected into an HPLC The analysis was performed with a Capcell pak C18 UG120 (Hitachi HPLC series D-7000) equipped with chromatog- column (4.6 × 250 mm, Shiseido, Tokyo, Japan), a Shimadzu raphy data station software, autosampler (D-7200), column SPD-10AV UV-VIS detector set at 254 nm and a Hitachi oven (D-7300), and diode array detection system (D-7450) L-6200 pump. The mobile phase was 20 mM potassium to monitor the absorbance at all wavelengths from 200-600 phosphate buffer (pH 4.5) containing 6.5% methanol with nm. A Capcell pak C18 UG120 (250 × 4.6 mm i.d., S-5, 5 the addition of 0.1 M EDTA·2Na at a flow rate 1 mL/min. μm, Shiseido Co., Ltd., Tokyo, Japan), joined with a guard Trolox in DMSO was used as a standard to make a standard column (10 × 40 mm i.d.) was used at 35℃. Gradient elution curve. The data were expressed as µmol Trolox equivalent was performed with solution A, composed of 50 mM sodium per 100 g fresh or boiled weight. phosphate (pH 3.3) and 10% methanol, and solution B, com- DNA damage prevention activity DNA damage preven- prising 70% methanol, delivered at a flow rate of 1.0 mL/min tion activity was measured according to a previously de- as follows: initially 100% of solution A; next 15 min, 70% A; scribed method (Luo et al., 1994) with several modifications. next 30 min, 65% A; next 20 min, 60% A; next 5 min, 50% Plasmid pBlueScript II SK(+) was propagated in E. coli JM A; and finally 0% A for 25 min. The injection volume for the 109 and purified using a Wizard DNA purification kit (Pro- extract was 10 μL. Flavone was used as an internal standard. mega, Madison, MI, USA). DNA (0.7 µg) was incubated in The gnemonoside derivatives were identified by comparing 50 mM phosphate buffer (pH 7.4) with various concentra- the spectra of the peaks with the spectrum of gnemonoside D tions of the methanolic extracts or Trolox as a standard. The standard. The amount of gnemonoside derivatives was cal- extracts and Trolox were diluted with 90% methanol contain- culated based on the peak area at λ maxima of 280 nm and ing 5% acetic acid. Freshly prepared FeSO4 and H2O2 solu- was expressed as µmol gnemonoside D equivalent per 100 tions were added at final concentrations of 50 µM and 6.25 g fresh or boiled weight. Total polyphenol was expressed as mM, respectively. The reaction mixture (20 µL) was incubat- total peak area at 280 nm (λmax) per g fresh or boiled weight. ed at 37℃ for 1 h in the dark. After incubation, 2 µL of 10 DPPH radical-scavenging activity The methanolic ex- × agarose loading buffer (Takara, Kyoto, Japan) was added tracts were used for the measurement of DPPH radical-scav- and the mixture was subjected to 1% agarose gel electropho- enging activity as previously described by Blois (1958). The resis for 1 h at 100 V. The gel was unloaded, stained with 1 absorbance of reaction was measured by a UV-2100PC UV- µg/mL ethidium bromide for 5 min and destained with 0.5x VIS spectrophotometer (Shimadzu, Kyoto, Japan) at 517 nm TAE buffer for 20 min. The bands were visualized under UV after incubation for 20 min at room temperature in the dark. light, and the gel was photographed using Printgraph (Atto The DPPH radical-scavenging activity was calculated from Bioinstrument, Tokyo, Japan). The images were scanned and the difference in absorbance between blank and samples with analyzed using Scion Image (Scion Corporation, Frederick, Trolox as a standard. The DPPH radical-scavenging activity MD, USA). Calculation of the amount of nicked (damaged) was expressed as μmol Trolox equivalent per 100 g fresh or and supercoiled (undamaged) DNA were adjusted accord- boiled weight. ingly. The data were presented as percentage of undamaged ORAC assay All extracts were evaluated for their (supercoiled) DNA according to the following equation: ORAC value according to the lipophilic and hydrophilic Undamaged DNA(%) = ORAC methods of Prior et al. (2003), respectively. The fluo- Intensity of supercoiled DNA #100% rescence microplate reader Gemini XPS (Molecular Devices, Intensity of supercoiled + nicked DNA Sunnyvale, CA, USA) was set with an excitation wavelength The percentage of undamaged DNA was plotted against 552 M. Santoso et al. the concentrations of extracts or Trolox using SigmaPlot (Sy- (Sakakibara et al., 2003). The result shows that the greatest stat Software, Inc., San Jose, CA, USA). IC50 was determined amount of total polyphenol was found in the young leaf, by fitting the plots to a hyperbolic regression curve using the followed by endosperm or mature leaf (not significant), and following equation: seed skin (Fig. 1B). The greatest amount of gnemonoside, ax one of the main stilbenoids of G. gnemon, was found in the Y = Y 0 + b+ x endosperm. Young and mature leaves contain much smaller
The IC50 values were expressed as ng fresh or boiled weight amounts of gnemonoside than the endosperm, and the seed per mL reaction solution. skin does not contain any (Fig. 1C). As shown in Fig. 2, the Statistical analysis All values are presented as the main polyphenol compounds present in young and mature means of triplicate analyses. Data analysis was performed leaves (peak A) are not gnemonoside derivatives (peaks B using Microsoft Excel. Two-sample t-test was used to calcu- and C) and have yet to be identified. The mature leaf con- late statistical significance. The values are reported as means tained the greatest amount of ascorbic acid, followed by ± SD in all the results tables and graphs unless otherwise young leaf, seed skin and endosperm (Fig. 1D). The differ- specified. A p value < 0.05 is considered as significant. ences in total ascorbic acid are significant among the samples (p < 0.05). Results Boiling significantly decreased the total phenolic and Antioxidant components The amounts of antioxidant polyphenol contents in all samples except the seed skin (Fig. components (total phenolic content, ascorbic acid, gnemono- 1A and 1B). There was a 37.2, 25.3 and 22.8% decrease in side, and total polyphenol) in the edible parts of G. gnemon the total phenolic content of young leaf, mature leaf and en- are shown in Fig. 1. The greatest total phenolic content was dosperm, respectively, and a 28.1, 14.4 and 26.2% decrease found in the mature leaf, followed by young leaf, endosperm in the total polyphenol content of the corresponding samples. and seed skin (Fig. 1A). The differences in total phenolic The amount of gnemonoside also decreased in the boiled content among samples are significant (p < 0.05), except be- young leaf and endosperm, but did not significantly de- tween the young leaf and endosperm. Because the presence creased in the boiled mature leaf, when compared with that of various molecules, such as proteins and peptides, often in- of the unprocessed samples (Fig. 1C). Total gnemonoside terfere with the measurement of total phenolic content using decreased by 36.1, 15.9 and 20.6% in the young leaf, mature the Folin-Ciocalteu method, we also measured total polyphe- leaf and endosperm, respectively. Boiling also decreased the nol content using a polyphenol-specific HPLC measurement total ascorbic acid in young and mature leaves, while that of
Fig. 1. Antioxidant component levels of the unprocessed (dark gray) and boiled (light gray) edible parts of Gnetum gnemon. A, total phenolic content; B, total polyphenol; C, total gnemonoside; D, total ascorbic acid. Antioxidant activity of Gnetum gnemon 553
Fig. 2. HPLC profile of the unprocessed edible parts of Gnetum gnemon monitored at λ maxima of 280 nm. A, unidentified compound; B and C, gnemonoside derivatives.
Table 1. The tocopherol contents (mg/100 g sample) of the edible parts of Gnetum gnemon with or without boiling treatment.
Sample Treatment α-tocopherol β-tocopherol γ-tocopherol δ-tocopherol
Young leaf Unprocessed 16.36 ± 0.54 1.60 ± 0.13 0.26 ± 0.03 0.53 ± 0.02 Boiled 22.82 ± 0.51 1.46 ± 0.07 0.78 ± 0.04 0.57 ± 0.01 Unprocessed 181.57 ± 13.23 5.21 ± 0.99 8.60 ± 0.47 0.69 ± 0.01 Mature leaf Boiled 196.62 ± 1.46 12.44 ± 1.61 7.31 ± 0.75 2.19 ± 0.26 Unprocessed 0.53 ± 0.02 9.76 ± 0.25 182.29 ± 10.80 8.02 ± 0.44 Seed skin Boiled 0.61 ± 0.06 13.84 ± 0.50 206.99 ± 9.81 7.47 ± 0.74 Unprocessed 0.19 ± 0.00 3.49 ± 0.01 0.10 ± 0.01 0.00 ± 0.00 Endosperm Boiled 0.02 ± 0.00 3.76 ± 0.00 0.11 ± 0.00 0.10 ± 0.01 seed skin and endosperm was not significantly affected (Fig. tivities of the samples were measured using three methods, 1D). DPPH radical-scavenging activity, hydrophilic ORAC and The tocopherol contents of the edible parts of G. gnemon peroxyl radical-scavenging activity. As shown in Fig. 3A-B, are shown in Table 1. The most abundant tocopherol isomer the greatest DPPH radical-scavenging activity and hydro- present in the young and mature leaves is α-tocopherol, fol- philic ORAC values were found in young and mature leaves lowed by β-, δ- and γ-tocopherol. Mature leaf contains much (no significant difference), followed by endosperm and seed greater α-, β- and γ-tocopherol levels than young leaf. The skin. The young and mature leaves and endosperm have seed skin contains γ-tocopherol as the most abundant tocoph- similar peroxyl radical-scavenging activity and their values erol isomer, followed by β-, γ- and α-tocopherols. The endo- are greater than that of seed skin (Fig. 3C). In contrast, the li- sperm contains much less tocopherol than the other edible pophilic ORAC value was found to be greatest in the mature parts, with the most abundant isomer being β-tocopherol. leaf and endosperm, followed by young leaf and seed skin Boiling did not affect the tocopherol content significantly (p (Fig. 3D). The lipophilic ORAC value is much lower than > 0.05 for all data presented). the hydrophilic ORAC for all samples, suggesting that the Antioxidant activity The hydrophilic antioxidant ac- antioxidant components in the edible parts of G. gnemon are 554 M. Santoso et al.
Fig. 3. Antioxidant activities of the unprocessed (dark gray) and boiled (light gray) edible parts of Gnetum gnemon. A, DPPH radical-scavenging activity; B, hydrophilic ORAC; C peroxyl radical-scavenging activity; D, lipophilic ORAC. mostly water-soluble. leaf, but is significant in the mature leaf and endosperm. The Boiling caused a decrease in the antioxidant activities of lipophilic ORAC value of the endosperm decreased most all samples except the seed skin (Fig. 3A-D). This result is in dramatically, by 60%, while that of mature leaf decreased by accordance with the effect of boiling on the amount of anti- 20.5%. oxidant components (Fig. 1). In the seed skin, its hydrophilic DNA damage prevention activity In this experiment, antioxidant activities (DPPH radical-scavenging activity, we measured the capacity of the G. gnemon extracts to in- hydrophilic ORAC and peroxyl radical-scavenging activity) hibit Fenton reaction-mediated DNA damage. Fenton reac- increased dramatically upon boiling such that they are not tion between transition metal ions, such as iron and copper, significantly different from those of endosperm (Fig. 3A-C, with H2O2 produces hydroxyl radicals that can attack both light gray bars). In fact, the peroxyl radical-scavenging activ- sugar and base moieties, leading to sugar fragmentation, ity value of every sample is not significantly different from strand scission and base adducts (Park and Imlay, 2003). The each other (Fig. 3C). DNA damage prevention activity of G. gnemon methanolic The relative decreases in hydrophilic antioxidant activi- extracts was analyzed using several concentrations of the ties upon boiling are similar among young and mature leaves extract and a typical result is shown in Fig. 5. All of the ex- (Fig. 3A-C). The % decreases in DPPH radical-scavenging amined samples show a significant capacity to prevent DNA activity, hydrophilic ORAC, and peroxyl radical-scavenging damage. The calculated IC50 values show that, for the un- activity between boiled and unprocessed samples are 38.7, processed samples, the activity is highest for the young leaf, 41.2 and 24.2% for young leaf, 27.5, 17.1 and 23.5 % for followed by mature leaf, endosperm and seed skin (Table 2). mature leaf, and 29.6, 21.9 and 15.4% for endosperm, re- The boiled samples have higher IC50 values compared to the spectively. These values correlate rather nicely with the unprocessed ones, except for the seed skin. The IC50 values decrease in total phenolic, polyphenol and gnemonoside of young and mature leaves after boiling increased to 2.1 and contents, suggesting that the polyphenols, in particular those 2.5 times compared with those of the unprocessed samples. from the gnemonoside group and/or its derivatives, contrib- The endosperm exhibited the most significant increase in ute significantly to the hydrophilic antioxidant activity of G. IC50 value, to 4.2 times. For comparison, Trolox was used gnemon leaf and endosperm. as a standard. The IC50 of Trolox was determined to be 2.66 The lipophilic ORAC value of the young and mature mM. leaves and endosperm was decreased following boiling, while that of seed skin increased slightly, although not signif- Discussion icantly (Fig. 3D). The decrease is not significant in the young Here we report the distribution and activity of the anti- Antioxidant activity of Gnetum gnemon 555
antioxidant activity. The methanolic extracts were also effec- tive in inhibiting the Fenton reaction-mediated DNA damage. Boiling decreased the amount and activity of the antioxidant components for all samples except the seed skin. It is likely that the extraction of antioxidant components from seed skin was more efficient in the boiled sample than that in the un- processed sample, contributing to the increase in antioxidant activity. A similar result was also reported by Dewanto et al. (2002), in which the antioxidant activity and lycopene con- tent of tomato increased following thermal processing, most likely caused by the disruption of the cell wall matrix of to- mato skin. It has been reported that the tomato skin contains the highest amount of lycopene compared with other parts (Sharma and Le Maguer, 1996). It is likely that the same reason applies for the increase in hydrophilic antioxidant components and activities of G. gnemon seed skin, although Fig. 4. Typical results of the DNA damage prevention experiment. there is a possibility of additional heat stable antioxidant A. Agarose gel showing the inhibition of Fe-mediated DNA damage with the addition of unprocessed young leaf extract. The upper and components in the seed skin, the presence of which remains lower bands correspond to nicked (damaged) and supercoiled (un- to be determined. damaged) DNA, respectively. Lane 1: plasmid DNA, lane 2: DNA The young and mature leaves possess comparable hydro- 2+ 2+ + H2O2 + Fe , lane 3: DNA + H2O2 + Fe + 0.005 µg/mL extract, 2+ philic antioxidant activities (Fig. 3A-C). They also possess lane 4: DNA + H2O2 + Fe + 0.01 µg/mL extract, lane 5: DNA + 2+ 2+ comparable total phenolic content and total polyphenol (Fig. H2O2 + Fe + 0.0125 µg/mL extract, lane 6: DNA + H2O2 + Fe + 2+ 0.025 µg/mL extract, lane 7: DNA + H2O2 + Fe + 0.05 µg/mL ex- 1A and B). In contrast, the total ascorbic acid of the mature 2+ tract, lane 8: DNA + H2O2 + Fe + 0.1 µg/mL extract, lane 9: DNA 2+ 2+ leaf is two times higher than the young leaf (Fig. 1D). These + H2O2 + Fe + 0.125 µg/mL extract, lane 10: DNA + H2O2 + Fe 2+ data indicate that polyphenols contribute much more to the + 0.25 µg/mL extract, lane 11: DNA + H2O2 + Fe + 0.5 µg/mL ex- 2+ tract, lane 12: DNA + H2O2 + Fe + 1 µg/mL extract, lane 13: DNA antioxidant activity of the leaf as compared to ascorbic acid. 2+ + H2O2 + Fe + 5 µg/mL extract, lane 14: DNA + H2O2 + Fe2+ + 10 The lipophilic ORAC values of the samples are much µg/mL extract. B. A best-fit plot on the percentage of undamaged lower than the corresponding hydrophilic ORAC values (Fig. DNA against the concentration of unprocessed young leaf extract. The concentration of extract required to inhibit 50% of DNA dam- 3B and D), indicating that the edible parts of G. gnemon age (IC50) is 75.85 ng/mL. contain more hydrophilic antioxidants than lipophilic ones. It is also noted that the lipophilic ORAC value of the endo-
Table 2. The IC50 values of DNA damage prevention activity sperm is relatively high and comparable with that of mature of the methanolic extract of the edible parts of Gnetum gnemon leaf (Fig. 1D); however, its tocopherol content is much lower with or without boiling treatment. (Table 1). The presence of other lipophilic antioxidants in the Sample Treatment IC50 (ng/mL) edible parts of G. gnemon may be possible, especially in the
Young leaf Unprocessed 75.85 endosperm, since the high lipophilic ORAC value of the en- Boiled 158.94 dosperm does not correlate with its low tocopherol content. Unprocessed 109.75 Mature lea The type and amount of lipophilic antioxidant in the endo- Boiled 273.14 sperm is yet to be determined. Unprocessed 419.55 Seed skin Boiled 164.57 The consumption of G. gnemon seed skin as human food Unprocessed 297.55 is not very common, although it does occur. The results Endosperm Boiled 1257.83 shown here suggest that seed skin is a potential source of antioxidants, with comparable activities to those of the other oxidant components in the edible parts of G. gnemon (young edible parts of G. gnemon after boiling. We have also shown and mature leaves, seed skin and endosperm) as well as the that the methanolic extract of G. gnemon unprocessed leaves effects of boiling. We also examined the DNA damage pre- and endosperm possesses a high potential to be used as a vention activity of the methanolic extract of each part. Our source of natural antioxidants. However, the methanolic leaf results show that all the edible parts we examined possess extracts have a strong green color, which may interfere their significant amounts of antioxidant components and levels of use as food additives. The endosperm extract, on the other hand, is clear and colorless and thus possesses the greatest 556 M. Santoso et al. potential as a food additive rich in natural antioxidants. from the seeds of Melinjo (Gnethum gnemon. L.) and their bio- We have also performed a limited scission of the circular logical activity. J. Agric. Food Chem., 57, 2544-2549. plasmid DNA strands, in which a low concentration of Fe2+ Kishida, E., Nishimoto, Y. and Kojo, S. (1992). Specific determina- will produce only a limited amount of hydroxyl radical that tion of ascorbic acid with chemical derivatization and high-per- will cause a single nicking of the DNA, producing the linear formance liquid chromatography. Anal. Chem., 64, 1505-1507. or relaxed circular DNA. This type of experiment has been Luo, Y., Henle, E.S., Chatopadhyaya, R., Jin, R. and Linn, S. (1994). shown to represent DNA oxidative damage in vivo (Madhujith Detecting DNA damage caused by iron and hydrogen peroxide. et al., 2004), which not only resulted in nicked DNA but also Methods Enzymol., 234, 51-59. in base modification and single or double strand breaks that Madhujith, T., Amarowicz, R. and Shahidi, F. (2004). Phenolic can lead to cancer or other non-cancerous diseases (Cooke antioxidants in beans and their effects on inhibition of radical- et al., 2003). In the presence of the methanolic extracts, the induced DNA damage. J. Am. Oil Chem. Soc., 81, 691-696. DNA scission was inhibited in a concentration-dependent Manner, H.I. and Elevitch, C.R. (2006). Gnetum gnemon (gnemon), manner, showing that the extracts are highly beneficial for ver. 1.1. In “Species Profiles for Pacific Island Agroforestry,” human health. It is noted that the cellular bioavailability of ed. by C.R. Elevitch. Permanent Agriculture Resources (PAR), the antioxidant components of the extracts is not yet fully Hawai‘i.