Screening on antioxidant activity of vegetable and fruit by-products

Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 335 SCREENING ON ANTIOXIDANT ACTIVITY OF VEGETABLE AND FRUIT BY-PRODUCTS Ha Thi Hue; *Phan Thi Anh Dao HCMC University of Technology and Education Email: *daopta@hcmute.edu.vn ABSTRACT In this study, we collected and prepared ethanol extracts from 30 vegetable and fruit by-products. The extracts were screened on antioxidant activities using three methods including determination of total phenolic content (TPC), DPPH free radical scavenging assay (DPPH assay), and ferric reducing/antioxidant power (FRAP) assay. The extracts prepared ginger skin (TN-17) and coffee sediment (TN-16) showed strong activity. The total phenolic contents in ginger skin and coffee sediment extracts were 146.52 mgGAE/g and 66.14 mgGAE/g, respectively. For the DPPH assay, the IC50 showed the values of 16.73 µg/mL with ginger skin extract and 33.57 µg/mL with coffee sediment extract. For the FRAP, at the highest sample concentration (1.0 µg/mL), a significant difference about absorbance was observed between all samples (p<0.05). In addition, both ginger skin and coffee sediment showed significant higher activity than others samples (p<0.05). TN-16 was selected for further identification of constituents by HPLC-EIS-MS method. 15 compounds may present in coffee grounds extract namely, metiamide, manitol, 3- amino phenol, malicyamide, phenyl ethanolamine, 3-methoxyamphetamine, caffeine, nisoldipine, doxenitoin, dicyclohexyl phthalate, febuprol, 2.4 xylidine, glycerol 2-palmitate, 4-(benzylamino) phenol, o-toluidine. Key words: Natural antioxidant, DPPH, TPC, FRAP, by-product, coffee sediment. INTRODUCTION Although oxygen is necessary for aerobic life, it can also participate in potentially toxic reactions involving oxygen free radicals or reactive oxygen species (ROS). Free radicals are formed in the human body to prevent from virus and bacterial infections. However, they react with macromolecules including protein, lipid, DNA causing serious diseases such as heart disease, macular degeneration, cancer, diabetes, and more. Therefore, antioxidant substances are required for the protection against the oxidizing agents. Many synthetic antioxidant compounds have shown toxic and/or mutagenic effects, which have stimulated the interest of many investigators to search natural antioxidant (Nagulendran et al., 2007). Most of the waste in the processing and processing of fruits and vegetables are: nuts, leaves, stems, bark and roots contain high value natural compounds, good for human health (Chala Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 336 Gowe, 2015). The fruit and vegetable by-products should be used as a potential source of natural antioxidants compounds. Besides, the utilization of these by-products contributes significantly to reducing the amount of waste, and to enhance the environmental protection of the fruit and vegetable processing industry. The by-products can be processed into functional food rather than being discarded (Chala Gowe, 2015). Vietnam, a tropical Southeast Asian country, has extremely rich cuisine and food. Hence, there are a great amount of by-products here. Therefore, we selected 30 by-products casually to investigate their antioxidant activity by using different antioxidant tests, including DPPH free radical scavenging assay, Ferric reducing/antioxidant power (FRAP) assay and determination of total phenolics contents. We hope that the selected by-products could be a potential source of natural antioxidants that could have great importance as therapeutic agents in preventing or slowing the progress of aging and age associated and oxidative stress related degenerative diseases. METERIALS AND METHODS Samples: By-products were obtained from the shops at Long Phuoc Market, District 9 in August 2017. The voucher specimens (number sample on table 1) is preserved at the Department of Food Technology of the HCMC of Technology and Education. Chemicals 2, 2 – Diphenyl – 1 – picrylhydrazyl (DPPH) were purchased from Merck (Darmstadt, Germany). Gallic acid and Folin-Ciocalteu were purchased from Sigma Chem. Co. Ethanol solvent (EtOH), Potassium hexacyanoferrate K3[Fe(CN)6], acid trichloroacetic (TCA), ferric chloride (FeCl3), Na2CO3 were purchased from China. Preparation of samples: The by-products were cleaned with water to remove other impurities and cut into small pieces (100-200g). Then they were dried at 60 (this temperature does not affect the antioxidant compounds). After drying, the by-products were ground into fine powder and soaking extracted at room temperature with EtOH for 4 days. The mixtures were filtered and added new solvent each one days. The EtOH solution was evaporated under reduced pressure to give ethanol extract. The extracted powder were stored in a sealed box and covered with silver foil to prevent light. Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 337 Table 1: The list of 30 by-products and results of DPPH assay and TPC Sign Local name Scientific name Family Part Used IC50 (μg/mL) TPC (mgGAE/g) TN- 1 Soy Bean Glycine max Fabaceae Sediment > 50 18.56 ± 0.45h TN- 2 Carrot Daucus carota Apiaceae Sediment > 50 2.67 ± 0.2a TN- 3 Corn Zea mays Poaceae Core > 50 11.31 ± 0.44ef TN- 4 Guava Psidium guajava Myrtaceae Seed > 50 12.56 ± 0.84fg TN- 5 Pine-apple Ananas comosus Bromeliceae Peel > 50 6.45 ± 0.57bc TN- 6 Pine-apple Ananas comosus Bromeliceae Sediment > 50 12.16 ± 0.24fg TN- 7 Guava Psidium guajava Myrtaceae Peel > 50 12.72 ± 0.28fg TN- 8 Kumquat Citrus japonica Citruseae Sediment > 50 55.74 ± 1.84n TN- 9 Tomato Solanum lycopersicum Solanaceae Sediment > 50 25.67 ± 0.65j TN- 10 Wet rice Oryza sativa Poaceae Peel brain > 50 12 ± 0.21f TN- 11 Sticky pineapple leaves Pandanus amaryllifolia Roxb Pandanacese Sediment > 50 21.72 ± 1.64i TN- 12 Green Bean Vigna radiata Fabaceae Sediment > 50 10.16 ± 0.13def TN- 13 Grapes Vitis vinifera Vitidaceae Sediment > 50 18.84 ± 1.33hi TN- 14 Dragon fruit Hylocereus undatus Hylocereusease Peel > 50 37.53 ± 0.48m TN- 15 Aloe vera Aloe vera Asphodelaceae Peel > 50 20.39 ± 1.33hi TN- 16 Coffee Coffea arabica Rubiaceae Sediment <50 66.14 ± 1.15p TN- 17 Ginger Zingiber officinale Zingiberaceae Peel <50 146.52 ± 1.86q TN- 18 Logan Dimocarpus longan Sapindaceae Seed > 50 33.63 ± 0.38l TN- 19 Mandarin Citrus reticulata Blanco Rutaceae Peel > 50 30.35 ± 0.16k TN- 20 Grapes Vitis vinifera Vitidaceae Peel > 50 8.62 ± 0.13cde TN- 21 Passion fruit Passiflora incarnata Passiflora Peel > 50 61.5 ± 3.02o TN- 22 Gac fruit Momordica cochinchinensis Cucurbitaceae Peel <50 26.4 ± 0.6j Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 338 Sign Local name Scientific name Family Part Used IC50 (μg/mL) TPC (mgGAE/g) TN- 23 Grapefruit Citrus maxima Rutaceae Peel > 50 5.78 ± 0.17bc TN- 24 Vietnam's Apple Ziziphus mauritiana Rhamnaceae Seed > 50 25.17 ± 0.18i TN- 25 Watermelon Citrullus lanatus Cucurbitaceae Peel > 50 12.95 ± 0.27fg TN- 26 Sapodilla Manilkara zapota Sapotaceae Peel > 50 15.06 ± 0.57g TN- 27 Sapodilla Manilkara zapota Sapotaceae Seed > 50 25.49 ± 0.17j TN- 28 Star apple Chrysophyllum cainino Sapotaceae Peel > 50 7.75 ± 0.2cd TN- 29 Ambarella Spondias dulcis Anacardiaceae Peel > 50 11.32 ± 0.14ef TN- 30 Banana Musa basloo Musacae Peel > 50 4.33 ± 0.03ab Values are expressed as mean ± standard deviation (n = 3). Means with different letters in each column indicate statistically significant differences between treatments for the same species according to Duncan’s multiple range test (p < 0.05). DPPH free radical scavenging assay The stable DPPH free radical was used for determination of free radical scavenging activity of the extracts (P. Molyneux et al, 2004). Briefly, a 0.1 mM solution of DPPH in 90% ethanol was prepared and then 1.5 mL of this solution was mixed with 1.5 mL of each sample (crude extract) at concentrations of 100, 50, 25, 10μg/mL in 90% ethanol. After 30 min incubation in the dark, the decrease in the solution absorbance was measured at 517 nm by Hitachi UH-530 spectrophotometer (Japan). DPPH inhibitory activity was expressed as the percentage inhibition (I%) of DPPH in the above assay system, calculated as (1−B/A) x100, where A and B are the activities of the DPPH without and with test material. IC50 (inhibitory concentration, 50%) values were calculated from the mean values of data from three determinations. Vitamin C at various concentrations (1.0, 2.5, 5.0, 10.0 μM) was used as a positive control. Dertemination of the total phenolic content The total phenolic content (TPC) was determined using the Folin– Ciocalteu reagent. The experimental procedure based on the method of Velioglu et al. 1998, along with some changes to suit the experimental conditions (Velioglu et al. 1998). Firstly, 1300 μL of sample solutions mixed with 1000 μL of Folin-Ciocaltue reagent (1:5) and incubated for 5 minutes. Then, 700 μL of Na2CO3 1M solution were added and mixed thoroughly. After 30 minutes of incubation in the dark, the absorption of samples was measured at 730 nm wavelength. Results were expressed as mg gallic acid equivalents in 1 g of dried sample (mg GAE/g), which is based on the standard curves at concentrations of 1, 2, 4, 6, 8, 10, 15 and 20 μg. / mL (R2 = 0.9976). Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 339 Ferric reducing/antioxidant power (FRAP) assay The FRAP assay is a method of measuring the ability of reductants to reduce Fe3+ –Fe2+. The process of reducing Fe (III) is used to indicate the ability of giving electrons of antioxidants such as polyphenols. The experimental procedure based on the method of Benzie, 1999 (Benzie et al, 1999). Extracts (0.1, 0.5 and 1 mg) were mixed with 1.0 mL 2.0 M phosphate (pH 6.6) and 1 mL potassium ferricyanide 1%. The mixture was incubated at 50°C for 20 minutes. Then 1 mL of 10% trichloroacetic acid was added and centrifuged at 2000×g for 10 minutes. The top of the solution (1 mL) was mixed with distilled water (1 mL) and (0.2 mL) ferric chloride 0.1%. The absorption was measured at 700 nm wavelength. HPLC -EIS-MS analysis of coffee grounds extract RP-HPLC was performed to determinate compounds present in the ethanol extract prepared coffee sediment (TN-16). The separation module consisted of Agilent 1200 series HPLC (USA) equipped with ESI-MS system (micrOTOF-QII Bruker Daltonic, Germany). The samples was eluted on a column ACE3- C18 (4.6 150 mm, 3.5 µm, Merck, Germany) with a gradient system consisting of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in methanol) used as the mobile phase, with a flow rate of 0.5 mL/min. The temperature of the column was maintained at 40 and the injection volume 20 µL. For ESI-MS, full scan mass spectra were measured between m/z 150 and 2000. High purity nitrogen was used as nebulizer gas at 1.2 bar, 200°C and at a flow rate of 0.8 mL/min. RESULT AND DISCUSSION Dertemination of the total phenolic content The total polyphenol contents in the extract samples were shown in Table 1. The values of total polyphenol content of the samples were from 2.67 to 146.52 mgGAE/g. The extract from ginger skin (TN-17) showed the highest value of total polyphenol content (146.52 mgGAE/g), following by the extract from coffee sediment (TN-16) and kumquat skin (TN-8) with 66.14 mgGAE/g and 55.74mgGAE/g, respectively. The lowest values of total polyphenol content belonged to the extract from carrot sediment (TN-2) with 2.67 mgGAE/g. In general, the values of total polyphenol content in the extract samples showed significant difference (p<0.05). DPPH free radical scavenging assay Table 1 shows the results of the antioxidant activity assay, which are shown IC50 values of 30 by-product extract samples. Based on the stable free radical (DPPH), the highest antioxidants activity were ginger skins (TN-17) and coffee sediment extracts (TN-16) with IC50 at 16.73 μg/mL and 33.57 μg/mL, respectively. The following high antioxidant activity extract was the Gac fruit (TN-22) with IC50 at 44.58 μg/mL. The weak antioxidant active samples were soybeans sediment, carrots sediment, corncobs, guava seeds and pineapples sediment. These Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 340 samples inhibited only less than 10% of DPPH at concentrations of 50 μg/mL. The positive control was gallic acid with an IC50 value at 7.18 μM. The correlation between antioxidant capacity and total phenolic content in foods has been extensively studied (Shan et al., 2005; Velioglu et al., 1998). According to a study by Panusa et al., 2013, the values of antioxidant activity in the DPPH test correlate with the total polyphenol content in coffee grounds under different extraction conditions. Two sample extracts ginger skin (TN-17) and coffee sediment (TN-16) not only DPPH high inhibitory activity but also contains phenolic content. Ferric reducing/antioxidant power (FRAP) assay The reduction potential of the samples increased with increasing of sample concentration levels were shown in Table 2. At the highest sample concentration (1.0 mg), there was a significant difference between the two most active samples, ginger skin (TN-17) and coffee sediment (TN- 16) (p<0.05). By increasing the concentration, the optical absorbance values of the incremental samples increase (statistically significant differences). The higher the concentration, the greater the reduction capacity and the stronger the antioxidant activity (table 2). When comparing between the extracts with one another, in the same concentration, the ginger skin (TN-17) showed the ability to reduce Fe3+ to Fe2 + was rather high and higher than that of the other samples. Specifically, at the highest sample concentration of 1 μg/mL, TN-17 samples had the highest optical absorption (2.004) and significant differences with the other samples; TN-1 samples had the lowest optical absorption (0.331). Table 2: Absorption at 700 nm wavelengths of 30 extracts Sign Local name 0.1 g/mL 0.5 g/mL 1.0 g/mL TN- 1 Soy Bean 0.104 ± 0.003b 0.209 ± 0.005cd 0.331 ± 0.005bc TN- 2 Carrot 0.057 ± 0.001a 0.148 ± 0.007b 0.407 ± 0.008d TN- 3 Corn 0.121 ± 0.004bcd 0.228 ± 0.005cde 0.504 ± 0.009e TN- 4 Guava 0.128 ± 0.007cde 0.317 ± 0.006f 0.548 ± 0.012f TN- 5 Pine-apple 0.113 ± 0.009bc 0.249 ± 0.005de 0.417 ± 0.003d TN- 6 Pine-apple 0.107 ± 0.004bc 0.215 ± 0.002cd 0.551 ± 0.005f TN- 7 Guava 0.103 ± 0.003b 0.234 ± 0.008cde 0.402 ± 0.011d TN- 8 Kumquat 0.189 ± 0.009gh 0.472 ± 0.004h 0.779 ± 0.01i TN- 9 Tomato 0.046 ± 0.002a 0.237 ± 0.006de 0.428 ± 0.008d TN- 10 Wet rice 0.103 ± 0.003b 0.218 ± 0.005cd 0.36 ± 0.004c Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 341 Sign Local name 0.1 g/mL 0.5 g/mL 1.0 g/mL TN- 11 Sticky pineapple leaves 0.129 ± 0.004cde 0.329 ± 0.009f 0.414 ± 0.004d TN- 12 Green Bean 0.208 ± 0.009h 0.523 ± 0.005ij 0.738 ± 0.003h TN- 13 Grapes 0.147 ± 0.002ef 0.35 ± 0.003f 0.557 ± 0.004f TN- 14 Dragon fruit 0.046 ± 0.001a 0.053 ± 0.002a 0.066 ± 0.002a TN- 15 Aloe vera 0.167 ± 0.002fg 0.526 ± 0.001j 0.793 ± 0.01ij TN- 16 Coffee 0.264 ± 0.002i 0.814 ± 0.002m 1.063 ± 0.001l TN- 17 Ginger 0.51 ± 0.003kl 1.49 ± 0.004n 2.004 ± 0.0260 TN- 18 Logan 0.489 ± 0.01k 1.106 ± 0.047o 1.574 ± 0.009m TN- 19 Mandarin 0.532 ± 0.004l 1.298 ± 0.005p 1.266 ± 0.01n TN- 20 Grapes 0.141 ± 0.005de 0.263 ± 0.004e 0.538 ± 0.008ef TN- 21 Passion fruit 0.204 ± 0.003h 0.524 ± 0.029j 0.824 ± 0.04j TN- 22 Gac fruit 0.5 ± 0.014k 0.681 ± 0.023l 0.873 ± 0.006k TN- 23 Grapefruit 0.331 ± 0.005j 0.481 ± 0.006hi 0.668 ± 0.017g TN- 24 Vietnam's Apple 0.28 ± 0.006i 0.348 ± 0.006f 0.546 ± 0.006f TN- 25 Watermelon 0.1 ± 0.007b 0.194 ± 0.008c 0.305 ± 0.001b TN- 26 Sapodilla 0.262 ± 0.01i 0.493 ± 0.01hij 0.643 ± 0.006g TN- 27 Sapodilla 0.282 ± 0.008i 0.527 ± 0.004j 0.647 ± 0.005g TN- 28 Star apple 0.279 ± 0.018i 0.395 ± 0.013g 0.501 ± 0.017e TN- 29 Ambarella 0.336 ± 0.011j 0.603 ± 0.025k 0.764 ± 0.013hi TN- 30 Banana 0.179 ± 0.003g 0.337 ± 0.01f 0.558 ± 0.01f Reducing properties is related to the presence of strong reducing agents in the acid as well as in the neutral and alkaline environments (Negi, P. S.et al, 2005). The antioxidant activity of the strong reducing agents is attributed to the breakdown of free radicals by the release of a hydrogen atom (Gordon, M. H. 1990). This suggests that antioxidant properties coincide with an increase in reducing capacity. Therefore, the strong antioxidant activity of the extract from the ginger shell and coffee grounds may have a correlation with the reducing capacity of the extracts Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 342 Chemical composition of coffee sediment Fifteen compounds have been identified in TN-16 by high performance liquid chromatography with MS probes (table 3). Table 3: Identification of 15 compounds in coffee by HPLC-ESI-MS No. Compounds [M-H]- (m/z) Predicted formula 1 Metiamide 245,0871 C9H16 N4 S2 2 Manitol 205,0683 C6H14O6 3 3-amino phenol 110,0601 C6H7NO 4 Salicyamide 138,0551 C7H7NO2 5 Phenyl ethanolamine 138,0919 C8H11NO 6 3- Methoxyamphetamine 166,1225 C10H5NO 7 Caffeine 217,0696 C8H10N4O2 8 Nisoldipine 411,1502 C20H24N2O6 9 Doxenitoin 239,1179 C15H14N2O 10 Dicyclohexyl phthalate 331,1908 C20H26O4 11 Febuprol 225,1485 C13H20O3 12 2,4 Xylidine 122,0965 C8H11N 13 Glycerol 2-palmitate 330,2773 C19H38O4 14 4-(Benzylamino) phenol 200,1069 C13H13NO 15 o-Toluidine 108,0808 C7H9N Among them, 3-amino phenol, Salicyamide, Phenyl ethanolamine, Caffeine, and 4- (Benzylamino) phenol belong to phenolic group. In particular, caffeine is a strong antioxidant compound. The high levels of chlorogenic acid and caffeine in coffee grounds suggest the potential for using them as a natural source of antioxidants. (Panus et al., 2013). CONCLUSIONS In conclusion, we have carried out a systematic investigation of vegetable and fruit by-product for DPPH assay, determination phenolic content and ferric reducing/antioxidant power (FRAP) assay. The results indicate a number of by-products that may be useful for the treatment of diseases relating free radical damages such as, ginger skin, coffee sediment, Gac skin and Ambarella skin. In commerce, coffee sediment is very abundant and low-cost raw materials source thus, the potential for using the by-product as a natural source of antioxidants is going to be helpful. Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 343 REFERENCES [1] NAGULENDRAN, KR., VELAVAN, K., MAHESH, R., AND HAZEENA BEGUM, V., In vitro antioxidant activity and total polyphenolic content of Cyperus rotundus Rhizomes. E-Journal of Chemistry. 4, 440-449 (2007). [2] CHALA GOWE, 2015. Review on Potential Use of Fruit and Vegetables By-Products as A Valuable Source of Natural Food Additives. Food Science and Quality Management ISSN 2224-6088 (Paper) ISSN 2225-0557 (Online) Vol.45, 2015, pp 57-58 [3] P. MOLYNEUX, SONGKLANAKARIN J. Sci. Technol, Vol 26 pp 211-219 (2004). [4] VELIOGLU, Y. S., MAZZA, G., GAO, L., & OOMAH, B. D. (1998). Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. Journal of agricultural and food chemistry, 46(10), 4113-4117. [5] BENZIE, I. F., & STRAIN, J. J. (1999). Ferric reducing/antioxidant power assay: Direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. In Methods in enzymology (Vol. 299, pp. 15-27). Academic Press. [6] D. H. BICH: Vietnamese medicinal plants and animals, Science and Technology publisher, Ha Noi (2004). [7] D. T. LOI: Vietnamese medicinal plants, Medicinine publisher, Ha Noi (2009). [8] SHAN, B., CAI, Y. Z., SUN, M., & CORKE, H. (2005). Antioxidant capacity of 26 spice extracts and characterization of their phenolic constituents. Journal of agricultural and food chemistry, 53(20), 7749-7759. [9] PANUSA, A., ZUORRO, A., LAVECCHIA, R., MARROSU, G., & PETRUCCI, R. (2013). Recovery of natural antioxidants from spent coffee grounds. Journal of agricultural and food chemistry, 61(17), 4162-4168. [10] NEGI, P. S., CHAUHAN, A. S., SADIA, G. A., ROHINISHREE, Y. S., & RAMTEKE, R. S. (2005). Antioxidant and antibacterial activities of various seabuckthorn (Hippophae rhamnoides L.) seed extracts. Food Chemistry, 92(1), 119-124. [11] GORDON, M. H. (1990). The mechanism of antioxidant action in vitro. In Food antioxidants (pp. 1-18). Springer, Dordrecht. TÓM TẮT Trong nghiên cứu này, chúng tôi tiến hành thu thập và điều chế các mẫu cao trích ethanol từ 30 loại phụ phẩm rau củ, bã ép. Sàng lọc hoạt tính chống oxy hóa của các mẫu cao bằng ba phương pháp thử là ức chế gốc tự do DPPH (phép thử DPPH), xác định tổng hàm lượng phenol (TPC) và xác định năng lực khử sắt (FRAC), nhận thấy mẫu cao trích từ vỏ gừng (TN-17) và bã cà phê Hội nghị Khoa học An toàn dinh dưỡng và An ninh lương thực lần 2 năm 2018 344 (TN-16) là những mẫu thể hiện hoạt tính mạnh. Hàm lượng phenol tổng trong cao trích vỏ gừng, bã cà phê lần lượt là: 146,52 mgGAE/g và 66,14 mgGAE/g. Giá trị IC50 trong phép thử DPPH của hai mẫu TN-17 và TN-16 lần lượt là 16,73 µg/mL và 33,57 µg/mL. Đối với xác định năng lực khử thì tại nồng độ mẫu cao nhất (1,0 µg/mL) có sự khác biệt đáng kể giữa hai mẫu có hoạt tính mạnh nhất là vỏ gừng và bã cà phê (p< 0,05) và giá trị độ hấp thu quang của hai mẫu này cao hơn nhiều so với các mẫu còn lại (p < 0,05). Mẫu TN-16 được lựa chọn để phân tích thành phần hóa học bằng phương pháp HPLC-EIS-MS. 15 hợp chất trong mẫu cao trích từ bã cà phê đã được định danh là: Metiamide, Manitol, 3-amino phenol, Salicyamide, Phenyl ethanolamine, 3- Methoxyamphetamine, Caffeine, Nisoldipine, Doxenitoin, Dicyclohexyl phthalate, Febuprol, 2,4 Xylidine, Glycerol 2-palmitate, 4-(Benzylamino) phenol, o-Toluidine. Từ khóa: Chất chống oxy hóa tự nhiên, DPPH, TPC, FRA assay, phụ phẩm, bã cà phê.