Functional Foods for Disease Prevention II - American Chemical Society

Chapter 11

Antioxidative Activity of Brewed Coffee Extracts 1

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Downloaded by UNIV OF NEW SOUTH WALES on September 19, 2017 | http://pubs.acs.org Publication Date: August 21, 1998 | doi: 10.1021/bk-1998-0702.ch011

Apasrin Singhara, Carlos Macku , and Takayuki Shibamoto

Department of Environmental Toxicology, University of California, Davis, CA 95616 Dichloromethane extracts and their components isolated from brewed coffee were evaluated for antioxidative activity, measured by the oxidative converson of pentanal or hexanal to a corresponding acid. Extracts were fractionated using column chromatography and each fraction was tested for antioxidative activity. A fraction eluted from the headspace sample with apentane/ethyl acetate (95/5) solution inhibited the pentane/pentanoic acid conversion for more than 10 days. A fraction eluted from a dichloromethane extract of brewed coffee with 100% acetone inhibited the hexanal/hexanoic acid conversion by 100% for more than 14 days. Maltol and 5-hydroxy methylfurfural (5-HMH) were identified in the fraction eluded with 100% acetone as major components. Maltol inhibited the acid formation by 100% at levels higher than 250 mg/mL. Dose-related inhibitory activity was observed in the case of 5-HMF which inhibited the acid formation by 95% and 50% at the levels of 500 mg/mL and 50 mg/mL, respectively. These activities were comparable to those of known antioxidants, BHT and a-tocopherol in this testing system. Production of antioxidative compounds in processed foods has been reported many times in literature. Formation of these antioxidants is most likely related to the Maillard reaction. In the late 1950s and early 1960s, the addition of sugar and/or amino acids to baked foods, such as cookies, was found to enhance the browning reaction that subsequently increased their stability against oxidative rancidity (7, 2). It was obvious that the baking process produced some antioxidants. Many researchers reported that heat treatment improved the oxidative stability of various foods, including milk products (5), cereals (4), and meats (5). The higher molecular weight substances, such as melanoidins, produced from a sugar/amino acid model system by the Maillard reaction, have received much attention as antioxidants. For example, melanoidin and its ozone oxidation products 1

Current address: Planters LifeSavers Company, 200 DeForest Avenue, East Hanover, NJ 07936. Corresponding author.

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©1998 American Chemical Society Shibamoto et al.; Functional Foods for Disease Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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significantly inhibited linoleic acid oxidation (6). Recently, low molecular weight volatile compounds obtained from a glucose/cysteine browning model system were reported to possess certain antioxidative activities (7). Also, column chromatographic fractions prepared from a dichloromethane extract of a glucose/cysteine browning model system inhibited the oxidative transformation from hexanal to hexanoic acid. Additionally, several nitrogen- and/or sulfur-containing heterocyclic compounds, which are major flavor compounds formed by the Maillard reaction (8), exhibited antioxidative activity in two testing systems (9). In the present study, antioxidative activity of volatile compounds obtained from a dichloromethane extract of brewed coffee, which reportedly contained numerous heterocyclic flavor compounds (10), was investigated.

Experimental Materials. Pentanal, hexanal, 2,5-dimethylhexane, 5-hydroxymemylfurfural (5-HMF), maltol, caffeine, and a-tocopherol were purchased from Aldrich Chemical Co. (Milwaukee, WI). Butylated hydroxymethyl toluene (BHT) was bought from Sigma Chemical Co. (St. Louis, MO). A l l authentic samples were obtained from reliable commercial sources. Regular and decaffeinated ground roasted coffees were purchased from a local market.

Sample Preparation with Simultaneous Purging and Solvent Extraction (SPE). Ground roasted regular coffee (25 g) was brewed using a stove-top coffee brewer with 300 mL tap water. The freshly brewed coffee was mixed with 17.5 g of sodium chloride. The solution was placed in a 500 mL, two-neck, round-bottom flask interfaced to a simultaneous purging and solvent extraction apparatus (SPE) developed by Umano and Shibamoto (77). The headspace of the brewed coffee mixture was purged into 250 mL of deionized water with a purified nitrogen stream while the mixture (10 mL/min) was stirred at 60 °C; the water solution was extracted with 50 mL of dichloromethane simultaneously and continuously for 3 h. The extract was dried over anhydrous sodium sulfate for 12 h. After removal of the sodium sulfate, the extract was concentrated using fractional distillation, and subsequently further concentrated under a nitrogen stream to 1 mL. The concentrated sample was placed in a glass column (15 cm X 1 cm i.d.) packed with silica gel (Kieselgel 60, E. Merck, Darmstadt, Germany). The sample was sequentially developed into five fractions with a 30 mL solvent mixture of different ratios of pentane and ethyl acetate (100/0,100/0,95/5,5/95,0/100). Each fraction was concentrated to a final volume of 0.5 mL by fractional distillation and stored at -5 °C for subsequent experiments. The experiment was replicated four times.

Sample Preparation with Liquid-Liquid Continuous Extraction (LLE). Ground roasted regular or decaffeinated coffee (75 g) was brewed with 600 mL tap water. The freshly brewed coffee was extracted with 100 mL dichloromethane using a

Shibamoto et al.; Functional Foods for Disease Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


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liquid-liquid continuous extractor for 6 h. The extract was dried over anhydrous sodium sulfate for 12 h. After removal of the sodium sulfate, the extract was concentrated usingfractionaldistillation, and subsequently further concentrated under a nitrogen stream to 1 mL. The concentrated sample was placed in a glass column (15 cm X 1 cm i.d.) packed with 160-200 mesh silica gel (J. T. Baker Inc., NJ) and the sample was sequentially developed into sevenfractionswith a 100 mL solvent mixture of different ratios of pentane and ethyl acetate (100/0,95/5, 80/20, 50/50,20/80,0/100) and finally with 200 mL acetone. Eachfractionwas concentrated to a final volume of 1 mL by fractional distillation and stored at -5 °C for subsequent experiments. The experiment was replicated four times. These experiments were simultaneously performed with controls that contained aldehydes and a GC internal standard only. Antioxidation Test. Antioxidative activity of the samples was tested using their inhibitory effect toward conversion of aldehyde to acid (72). A testing sample (100 mL) was added to a 1 mL dichlomethane solution of pentanal (1 mg/mL) containing 0.1 mg/mL of 2,5-dimethyl hexane as a gas chromatographic internal standard or a 1 mL dichloromethane solution of hexanal (3 mg/mL) containing 0.1 mg/mL of undecane as a gas chromatographic internal standard. The oxidation of the sample solution was initiated by bubbling air at 70 °C for 1 min. The increase in acid or decrease in aldehyde was monitored at 2-day time intervals. The authentic chemicals of caffeine, 5-HMF, maltol, BHT, and a-tocopherol were also examined for their antioxidative activity using this same testing method. Quantitative Analysis of Aldehydes and Acids. The quantitative analysis was conducted according to the internal standard method. A Hewlett-Packard (HP) model 5790 gas chromatograph (GC) equipped with a 60 m X 0.25 mm i.d. DB-5 bonded-phase fused-silica capillary column (J & W Scientific, Folsom, CA) and a flame ionizatio detector (FID) was used to monitor the relative amounts of pentanal present in the samples prepared by SPE. The injector temperature was 250 °C and the detector temperature was 300 °C. The oven temperature was programmedfrom50 °C to 200 °C at 6 °C/min. An HP model 5890 GC equipped with a 30 m X 0.25 mm i.d. DB-1 bonded-phase fused-silica capillary column (J & W Scientific, Folsom, CA) and an FID was used for the samples obtained by liquid-liquid continuous extraction. The injector and detector temperatures were 250 °C and 275 °C, respectively. The oven temperature was held at 40 °C for 5 min and then was programmed to 180 °C at 3 °C/min. An HP 5890 series II gas chromatograph interfaced to an HP 5971 A mass selective detector (GC/MS) was used for mass spectral identification of the GC components at MS ionization voltage of 70 eV. Column and oven conditions were as stated above.


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Results and Discussion The aldehyde/carboxylic acid test is a fast and simple method to assess the antioxidative properties of chemicals or groups of chemicals. Fatty aldehydes are converted readily to a corresponding fatty acid in the oxygen-rich dichloromethane solution through a radical-type reaction (13). Figure 1 shows the relative peak area of pentanal for column chromatographic fractions prepared from the extract of SPE and for the control throughout a storage period of 10 days. Fraction III which was eluted with a pentane/ethyl acetate (95/5) solution inhibited the aldehyde/carboxylic acid conversion for more than 10 days. All fractions except fraction III exhibited weak antioxidative activity. 1 -Methylpyrrole and 1 -furfurylpyrrole were identified in fraction III. 1 -Methylpyrrole, which was also found in the headspace of the heated corn oil/glycine model system, inhibited conversion of pentanal to pentanoic acid for more than 80 days at a level of 500 mg/mL (12). The five-membered heterocyclic aromatic ring is reportedly able to scavenge reactive radicals (such as a hydroxy radical) (14, 15). As hypothesized in Figure 2, 1-methylpyrrole formed a hydroxy adduct with a hydroxy radical. The water elimination step may not occur under neutral pH (14). Many pyrroles have been reported in numerous heat-treated foods such as cooked meats, roasted beans and nuts, and brewed coffee (16, 17). In order to validate the testing method, activities of known antioxidants, a-tocopherol and BHT, were measured in a hexanal/hexanoic acid system. The activity was monitored by measuring amount of hexanoic acid formed. Figure 3 shows the results of the antioxidative activity test on a-tocopherol and BHT (along with maltol, 5-HMF, and caffeine). Both antioxidants exhibited dose-related activity. They inhibited hexanoic acid formation by 100% at the level of 250 mg/mL; at the level of 8 mg/mL, both antioxidants inhibited the acid formation by 50%. BHT inhibited hexanoic acid formation by 100% at the level of 50 mg/mL, whereas a-tocopherol inhibited the hexanoic acid formation by 90% at the same level. These results are consistent with previous reports (7,13). The results indicate that this method is useful for examining the antioxidative activity of chemicals. Figure 4 shows the results of the antioxidative test onfractionsobtained from a dichloromethane extract of regular brewed coffee using a liquid-liquid extraction method. All fractions except Fraction I (100% pentane eluate) inhibited the acid formation by almost 100% over 14 days. In particular, Fraction VII (100% acetone eluate) exhibited strong antioxidative activity. Hexanoic acid contained originally in the testing solution of hexanal disappeared by the action of Fraction VII after 4 days, suggesting that this fraction contained some reducing agents. Among the 26 compounds identified in Fraction VII, maltol and 5-HMF were tested for antioxidative activity. The results are shown in Figure 3. Maltol inhibited hexanoic acid formation by 100% at levels higher than 250 mg/mL. Dose-related inhibitory activity was observed in the case of 5-HMF which inhibited hexanoic acid formation by 95% and 50% at the levels of 500 mg/mL and 50 mg/mL, respectively. Hypothesized mechanisms of a hydroxy radical abstraction with maltol and 5-HMF based on the previous reports (14, 15, 18, 19) are shown in Figure 5.



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Figure 1. Relative amounts of remaining pentanal in samples containing column chromatographic fractions from headspace of brewed coffee throughout a storage period of 10 days.

7E-electron density

OH

•OH 1.106

1.106

1.035

1.035

N

I CH

I CH.

3

H0 2

13.26

Functional Foods for Disease Prevention II - American Chemical Society

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