Antioxidant Properties and Phenolic Phytochemicals from Various Cultivars of Thai Mango Seed Kernels
Maisuthisakul, P.1* and Pasuk, S.2 * School of Science, University of the Thai Chamber of Commerce, Bangkok 10400. Thailand 2 Faculty of Science and Technology, Valaya Alongkorn Rajabhat University under Royal Patronage, Prathumthani 12150. Thailand *Tel. 0-2697-6525 Fax. 0-2277-7007 E-mail address: pitchaon@yahoo.com

Abstract Thai mango (Mangifera indica Linn.) seeds were collected from marketed place and kernels were separated and dried. This study was carried out on antioxidant activities using the stable radicals 1-diphenyl-2-picrylhydrazyl (DPPH•) and phenolic compound of the ethanolic seed kernel extracts of the eleven Thai mango cultivars, which are commonly known as Kaew (KW), Nam Dokmai (NM), Khiew Sawoey (KS), Pimsaen (PS), Chok-Anan (CA), Rad (RD), Phalun (PL), Hua Chang (HC), Mun Duan Kao (MK), Okrong (OR) and Maha Chanok (MN). The results showed that all the mango seed kernels showed antioxidant activities comparable to the α-tocopherol. Cultivar KW and CA extracts showed the highest antioxidant efficiency, resulted in 110-113% amplification in antioxidant activity comparing to reference substance. The phenolic acids of mango seed kernels were in form of free phenolic acid (42-56%) more than esterified phenolic acid (10-19%) and insoluble bound phenolic acid (15-20%). The phenolic acid of KW were the highest values, 74.99 + 0.65 milligrams of gallic acid equivalents per 100 grams of fresh weight (mg of GAE/100 g fresh), whereas CA gave the highest flavonoid content (30.16 + 0.21 milligrams of rutin equivalents per 100 grams of fresh weight (mg of RUE/ 100 g fresh)). Most of the phenolic compound of KS, PS, PL and MK was phenolic acids and flavonoids, whereas the other phenolic constituents were found in KW, ND, CA, RD, HC, OR and MN extracts. The antioxidant activities in the mango seed kernel extracts did not correlate with the yields of extracts or yields of seed kernels from mango seeds, however the activities related with phenolic, flavonoid and phenolic acid contents. Mango seed kernels will be a potential material for extracting natural antioxidants. Keywords: Thai, mango seed kernel, antioxidant, phenolic, flavonoid, phenolic acid

total phenolic content.. 1990). Folin Ciocalteu reagent. Pimsaen (PS). Hence. Mango seed kernel is by product obtained during the processing and it was shown to enhance oxidative stability of fresh type cheese and ghee. and then sun dried for 24 h. by products such as peel and kernel are generated. This could be attributing to the phospholipids and phenolic compounds in mango seed extract. sodium carbonate. Louise. Hence. Moreover. During processing of mango.. phytosterols and microelements like selenium. 2007).1 Materials Eleven cultivars of ripened mangoes which are commonly known as Kaew (KW). industry has increasing cost for its waste treatment to utilize it as a good source of natural antioxidants and lipid (Arogba. total flavonoid content.2 Extraction of mango seed kernel antioxidant The freezing kernel (80 g) were blended for 1 min with ethanol at -20°C and the containers were then flushed with nitrogen and shaken for 4. stigmasterol and also contain tocopherols (Soong and Barlow. Gallic acid and Ellagic acid were purchased from Acros Organics (New Jersey. 2006). mango kernel was also shown to be a good source of phytosterols as campesterol. Falun (FL). 2004). USA). Mun Duan Kao (MK). 2003).. Khiew Sawoey (KS). insoluble bound phenolic acid and yield were determined with the aim of exploiting the high potential source from mango kernel cultivars as natural antioxidant. copper and zinc (Schiber et al.2-diphenyl-1-picrylhydrazyl (DPPH). pickles. it is discarded as a waste and becoming a source of pollution. sesquiterpenoids. Germany). Hua Chang (HC).. we reported the antioxidant activity of eleven cultivars of Thai mango seed kernels. about 20% of fruits are processed for products such as puree. DPPH activity. 2. Nam Dokmai (NM). 2000). 2004). 2. 2. The dried material was kept in freezer at -20°C no longer than two months. Ltd (St. Kernel contributed about 17-22% of the fruit (Soong et al. rutin were purchased from Sigma Chemical Co. Chok-Anan (CA).. hexamethyltetramine. Its extract extended their shelf life (Parmar and Sharmar. nectar. The seeds were washed and the kernels were obtained by manually removing shells..5 hours in the dark at 30°C (Maisuthisakul et al. β-sitosterols. The other chemicals and solvents used in this experiment were analytical grade purchased from Sigma-Aldrich Co. 2007). Materials and Methods 2. Kaew and Rad. There are many cultivars of Thai mangoes such as Num-Dokmai. This waste should be treated as a specialized residue due to their high levels of phenolic compounds and stable fat rich in saturated fatty acids. Rad (RD). Okrong (OR) and Maha Chanok (MN) were purchased from a marketed place during March to April 2007. aluminium chloride. esterified phenolic acid.) is one of the most important tropical fruits in the world and currently ranked 5th in total world production among the major fruit crops (FAO. after filtration through cheesecloth and . USA). Ltd (Steinheim. The supernatant. As mango is a seasonal fruit. The antioxidant effect of mango seed kernel was due to their high content of polyphenols. free phenolic acid. leather. Introduction Mango (Mangifera indica Linn. canned slices and chutney and these products experience worldwide popularity and have also gained increased importantce in the US and European markets (Abdalla et al.2 1.As seed kernel is not currently utilized for any commercial purposes.

2007). The absorbance at 517 nm was measured against a blank of pure methanol at 30 min of reaction used to estimate the remaining radical levels. 20 ml of acetone. . The total flavonoid content was determined in 10 ml of the extract using 1 ml of 2% AlCl3 in methanol solution (5% acetic acid in methanol). The resulting solution was filtered. Antiradical activity reported in form of 1/EC50. 2007). Results of total phenolic content were expressed as milligrams of gallic acid equivalent per 100 gram of fresh weight (mg GAE/100 g fresh). Shimadzu. Japan)..5 Determination of total phenolic content The amount of total phenolic content in 11 mango seed kernel extracts was determined using Folin-Ciocalteu reagent (Kähkonen et al. The tubes were mixed with vortex and allowed to stand for 30 min in the dark. the volume was leveled to 100 ml with acetone.4 Determination of radical DPPH scavenging activity Free radical scavenging capacity of Betel leaf extract was evaluated according to the previous reported procedure using the stable 2. 2001). 2. Shimadzu. Torrento & Lorente.mL-1). total phenolic content. Each extracted sample (200 µl) was introduced into test tubes and then one ml of Folin-Ciocalteu reagent and 0. The final concentration was 100 µM for DPPH●. Ten milliters of the extract was put into a separation funnel. Absorbance was read at 425 nm (UV/Vis 1601 spectrophotometer. and 2 ml of 0. total flavonoid content. 2.3 Whatman No 4 filter paper.6 Determination of total flavonoid content Total flavonoid content of 11 samples from various cultivars of mango seed kernels was measured by a colorimetric assay (Bonvehí. The DPPH activity was expressed in term of EC50 (µg. Yield (%.1 mol/l HCl were added to the finely ground frozen sample (5 g) and set to boil with reflux for 30 min. Absorption was measured at 765 nm (UV/Vis 1601 spectrophotometer.3 Determination of the yield of extracts The dried extracted sample was weighed for calculating the yield by the following equation..5% hexamethyl tetramine (w/v). was evaporated under vacuum. The extract was washed twice. esterified phenolic acid and insoluble bound phenolic acid.5% w/v) were added. and the residue was washed with 20 ml of acetone. 2. then centrifuged at 3300g for 5 min. 2. using 50 ml of H2O each time. along with 20 ml of H2O and 25 ml of ethyl acetate. Extraction with ethyl acetate was carried out three times. Sample was dried in a freeze dryer and stored in aluminum foil after flushing with nitrogen at -20°C until analysis by determining their yield. wet weight basis) = (W1 x100)/W2 Where W1 is the weight of extracts after freeze drying and W2 is the weight of fresh samples.2-diphenyl -1-picrylhydrazyl radical (DPPH●) (Masuda et al.8 ml of sodium carbonate (7. One milliliter of 0. and flavonoid percentage was estimated using calibration curves and expressed as rutin equivalents in milligrams per 100 gram fresh weight (mg RUE/100 g fresh). free phenolic acid. Japan). and was subsequently made up to 100 ml with ethyl acetate. radical DPPH scavenging activity.

05. and IBPA) were dissolved in methanol. EPA. 2004). 3.8 Statistical analysis Results are presented as mean value + standard deviation (at least three replicate experiments). The reidue. Solvent extraction is frequently used for isolation of antioxidants. The extract (2 g) was extracted six times with 40 ml of water-methanol-acetone (6:7:7. using Folin-Ciocalteu reagent as total phenolic ontent determination. The high moisture of fresh products investigated here required a slightly cost drying process with sun dryer. The samples were centrifuged at 5000 × g and supernatants were extracted 6 times with diethyl ether-ethyl acetate mixture (1:1. samples were centrifuge for 15 min at 5000 × g and supernatants were collected (residual kept for further analysis).7 Determination of free phenolic acid. both yields and economic parameters were primarily emphasized before detailed study of their antioxidant potential. The extracted phenolic acids contained free phenolic acids (FPA). esterified and insoluble-bound phenolic acids were expressed as ma of gallic acid equivalents per 100 gram of fresh weight (mg GAE/100 g fresh).4 2. 2. The dried phenolic acids (FPA. v/v/v) at room temperature. and contents of phenolic acids were determined colorimetrically. The contents of free. v/v) at a supernatant to solvent ratio of 1:1 (v/v). as before.Combined supernatants were evaporated at 30°C under vacuum to 40 ml and the pH of the mixture was adjusted to 2 using 6 M HCl. Results and Discussion 3.1 Determination of yields Taking into consideration the industrial requirements for extraction. The antioxidant activities of the extracts are strongly dependent on the solvent. esterified phenolic acid and insoluble bound phenolic acid The free. to yield esterified phenolic acids (EPA). separately. and extractes were evaporated to dryness under vacuum to obtain insoluble-bound phenolic acids (IBPA). esterified and insoluble-bound phenolic acids of mango seed kernel extract were isolated using the procedure of Krygier et al. due to the different antioxidant potentials of compounds with different polarity (Soong et al. then acidified with 6 M HCl to pH 2 and centrifuged at 5000 × g for 15 min.. After each extraction. kept for further analysis after extraction of free and esterified phenolic acids. The supernatants were extracted six times with diethyl ether-ethyl acetate mixtures. Statistic analysis among treatments were determined at the significance level of P <0. The ether extracts were combined and evaporated to dryness at 30°C under vacuum. as before. The resultant hydrolysate was acidified to pH 2 using 6 M HCl and extracted into diethyl ether-ethyl acetate mixtures. The ethanol . using a vortex for 2 min. (1982) with slightly modification. The aqueous layer remaining after extraction of free phenolic acids. and the residue after centrifugation were mixed together and then treated with 30 ml of a 4 M NaOH solution under nitrogen for 4 h at room temperature to release esterified phenolic acids. was treated with 20 mL of 4M NaOH under nitrogen at room temperature.

the antiradical activities (except a few) correlated with their total phenolic contents.90) (Figure 5b) of all extracts was slightly higher than that between phenolic acids and antiradical activity (R2 = 0. After ranking of both the phenolic yield and practical issues. KW. Yields of the kernels from several cultivars of mango seeds showed in Figure 1. Similar results were reported for different plants by various studies (Soong and Barlow. 3. quality (homogeneity). content of phenolic compounds correlated strongly with their antiradical activity (R2 = 0. additional pretreatment required) were assessed. pretreatment (low influence of treatment on active compounds. NM had showed the high antiradical activity comparable to the α-tocopherol (Table 1). CA seed had the largest size comparing to the other mango seed. Meanwhile. we can note that extracts from KW cultivar showed the highest total phenolic content (116. the solvent is compatible with food. possible standardization according to good agricultural practice guidelines). the following CA was selecting for further investigation. It can be observed that most cultivar of mangoes flesh which have sour taste. The results also indicated that. economic characteristics such as availability (sufficient amount. It can be seen that extracts obtained using ethanol solvent composed high polarity phenolic compounds. also.37 mg GAE/100 g fresh). Furthermore.98 + 0. hence there was no doubt that the yield of kernel from CA cultivar was the highest value. MN. showed the high phenolic content.5 was used as extraction solution in the present study as it is the most widely used solvent for hygiene and abundance reasons. flavonoid. 2004). Thus even KW seed with remarkable phenolic compounds were regarded of little amount from factories. the CA seed also gave the highest yield of the extract (Table 1). Most of the phenolic constituents of KW. and phenolic acid content varied considerably from one cultivar of mango seed kernels to another. season-independence). To evaluate the procurement of raw material.2 Determination of radical DPPH scavenging activity and phenolic compounds of mango seed kernel extracts A screening of 11 mango cultivars recently carried out had demonstrated the kernel to be a rich source of phenolic acid. we found that the amount of free phenolic acids of studied kernel samples were higher than esterified and insoluble bound phenolic acids (Figure 3). total phenolic. Moreover.3 Relationship of antiradical activity with yields and phenolic compounds The antiradical activity in all extracts did not correlated yields of kernels from mango seeds or yields of the extracts (Figure 4).92) (Figure 5a). . These can be confirmed that the phenolics are likely to contribute to the radical-scavenging activity of kernel extracts. On the basis of fresh weight basis. These results suggested that the antioxidant capacity of extracts is due to the contribution of flavonoids (90%) and phenolic acids (86%). 3. The antiradical activity. transport. CA. PS. According to the resulted presented in Figure 2. The correlation between flavonoids and antiradical activity (R2 = 0. PA and MK cultivars were phenolic acids and flavonoids (Figure 2). market opportunities and cost (price. The main phenolic compound of mango seed kernel of 11 cultivars was phenolic acid. for a given raw materials.86) (Figure 5c). When we considered only phenolic acid contents. A multiple mixture of different phenolic classes in a wide range of polarities can be assumed for mango kernel extract yields. extracts obtained from MK cultivar was less value of total phenolic content.

Antioxidant activity and phenolic content of selected fruit seeds. 88: 411-417. Pihlaja. L. Oyama. S. 47.. A. Journal of Agricultural and Food Chemistry. Chok-Anan cultivar may be considered suitable for further investigation of their potential antioxidant agent. Tanaka. (2007). S... H. M. Quantification of gallic acid and ellagic acid fron Longan (Dimocaepus longan Lour) seed and mango (Mangifera indica L. Ayad... S. Berardini. R. P. Food Chemistry. M. Evaluation of polyphenolic and flavonoid compounds in honey-bee collected pollen produced in Spain. and Barlow. Soong. Y.S.. Y. E. total phenolic. Y. S. Andoh. P. Identification of flavonol and xanthol glycosides from mango peels by HPLC.. Rauha.6 4. Egyptian mango by product 2: Antioxidant and antimicrobial activities of extract and oil from mango seed kernel. P. S. 334–336. M. Acknowledgement This study was supported by a grant from University of the Thai Chamber of Commerce (UTCC). Food Chemistry. E. 3954–3962. S. 51: 5006-5011. and Hogge. Food Chemistry. (1982). Maisuthisakul. Arogba. 97: 524-530. However. A. J. Conclusion The cultivars of mango seed kernels significantly affected extraction yields. J. (2007). C. (2003). Food Chemistry. Bonvehí. P. 13: 149-156. Y. Free. 1749–1754... and Carle. I. Vuorela. and stability study of the associated polyphenol oxidase activity.. T. The authors would like to thank Dr. S. N.. F. Kähkonen. R. (2004). W. S.. Effect of mango (Mangifera indica L. cultivar Kaew and Chok-Anan were more efficient as antioxidant agents than did the corresponding ones. (2006).. since we considered economic characteristics. 30. Leonard Fweje for any suggestions. K. (2000). Masuda.. Darwish. Torrent. Food Chemistry. (2001). Hopia. Assessment of phenolic content and free-radical scavenging capacity of some Thai indigenous plants. Journal of Agricultural and Food Chemistry.. H. Pongsawatmanit. and Sharmar. K. J..) kernel and their effects on antioxidant activity. Parmar. M. Soong. J. Krygier. (1999). Yonemori. Journal of Agricultural and Food Chemistry. T. H. Kujala. and Barlow... R. esterified and insoluble bound phenolic acids 2. 35: 99-107. R. T. (1990). Mango (Mangifera indica) kernal: Chromatographic analysis of the Tannin.. It was notable that extracts from Mangifera indica Linn. and El-Hamahmy. T. phenolic acid content. 1848-1853. Journal if Food Composition and analysis. Schiber. and Gordon.. Journal of Agricultural and Food Chemistry. Takeda. M. E. Composition of phenolic acids in rapeseed flour and hulls. 1409-1418. Evaluation of the antioxidant activity of environmental plants: activity of leaf extracts from seashore plants. A. flavonoid. and Lorente. S.. References Abdalla. 47.. 103: 1141-1152. P. 100.. Sosuiski. . E.) seed kernel pre-extract on the oxidative stability of buffalo ghee. Journal of Agricultural and Food Chemistry. (1999). M. 49. Antioxidant activity of plant extract containing phenolic compounds..

03 ab 3.49 ± 0.01 f 1.02 c 2.94 ± 0.14 g 16.33 ± 0.01 ± 0.48 ± 0.07 ± 0.14 e 14.03 b 3.04 f 2.73 ± 0.69 ± 0.30 cd 19.04 ab 3.20 h 2.09 ± 0.04 ± 0.65 ± 0.01 a 2.05 b 3.93 ± 0.90 ± 0.07 cd 3.25 ± 0.07 b 20.08 bc 3.11 ± 0.06 ± 0.22 b α.02 b 2.11 ± 0.44 ab 15.25 ± 0.54 ± 0. .53 ± 0.06 d 3.80 ± 0.05.04 ef Data followed by different letters within each column are significantly different according to Duncan's multiple range test at P < 0.27 ± 0.01 ± 0.75 ± 0.41 ± 0.21 f 13.43 de 18.03 a EC50 (µg/ml) 14.11 ± 0.04 c 3.67 ± 0.04 bc 3.06 c 2.09 ± 0.tocopherol Kaew Numdokmai Keawsawuar Pimsaen Chokeanan Rad Phalun Huachang Munduankao Okrong Mahachanok Note : § Antiradical activity (1/EC50) 2.05 d 2.86 a 14.41 ± 0.02 b 2.09 g 2.23 c 13.22 bc 16.68 + 0. Data obtained from at least three replicates.03 ab 3.04 e 3.16 fg 14.17 ± 0.95 + 0.72 ± 0.40 ± 0.33 ± 0.7 Table 1 Yield and Antioxidant activity of mango seed kernel extracts and reference compounds determined by the DPPH method § Mango cultivars Yield (%) of extracts 3.

8 Figure caption Figure 1 The yields (%) of mango seed kernel from various mango cultivars. flavonoid and phenolic acid contents of mango seed kernel from various mango cultivars. Figure 4 The relationship between yield of kernel from mango seed. The total phenolic acid calculated in term of mg/ 100 g of fresh sample. Figure 2 The total phenolic. yield of extracts and antiradical activity of mango seed kernel extracts. . (b) flavonoid and (c) phenolic acid content of mango seed kernel extracts. Figure 5 The relationship between antiradical activity and (a) total phenolic. Figure 3 The fraction of free phenolic acid. esterified phenolic acid and insoluble bound phenolic acid in mango seed kernel.

34 77.08 86.87 70.11 75.76 Varieties of mango seed Cultivar of mango .88 72.Figure 1 Yield (%) of kernel from mango seed 100 90 80 70 60 50 40 30 20 10 0 78.67 Nu Kaew m Ke d ok m aw saw ai u Pim ar sa e Ch n ok e an an 9 80.60 Ra d Ph Hu alu n Mu ach a n nd u an g kao Ma Ok ro n ha ch a g no k 74.55 73.69 78.75 68.

36 30.68 23.32 24.63 66.05 102.98 30 60 90 120 Total phenolic content (mg of GAE/ 100 g fresh) Total flavonoid content (mg of RUE/ 100 g fresh) Total phenolic acid content (mg of GAE/ 100 g fresh) Figure 2 .95 115.65 75.83 116.19 24.38 72.20 29.42 98.39 79.36 74.63 100.42 71.67 65.33 101.10 Mahachanok Okrong Munduankao Huachang Phalun Rad Chokeanan Pimsaen Keawsawuar Numdokmai Kaew 0 25.43 45.27 19.32 67.16 22.72 50.15 56.29 65.45 21.22 25.59 26.58 59.99 93.67 109.45 63.

11 Mahachanok Okrong Munduankao Huachang Phalun Rad Chokeanan Pimsaen Keawsawuar Numdokmai Kaew 0 0.4 0.2 0.6 0.8 1 Total phenolic acid Free phenolic acid Esterified phenolic acid Insoluble bound phenolic acid Figure 3 .

0 3.2 2. db) of kernel from mango seed .0 Antiradical activity Figure 4 Yield (%.5 40 3.8 2.0 60 3.5 4. wb) of kernel Yield (%.8 3. db) of extracts Yield (%.6 2.0 80 4.12 100 5. wb) of extract 0 1.4 2.0 2.5 2.2 2.0 20 yield (%.

0 2.0 60.90 30.86 70. db) (mg GAE/g g fresh) fresh) (mg RUE/gg fresh) fresh) (mg GAE/g fresh) (mg GAE/100 (mg RUE/100 (mg GAE/100 g fresh) Figure 5 .5 Antiradical activity 3.5 1.5 0.0 2 R = 0.0 35.0 1.0 50.0 40. db) (mg RUE/g.0 80.0 20.13 3.5 2.0 80.0 2 R = 0. db) (mg GAE/g.0 50.0 R = 0.92 110.0 140.0 25.0 15.0 2 Total phenolic content Total flavonoid content Total phenolic acid Total phenolic acid Total phenolic Total phenolic content Total flavonoid content Total phenolic acid (mg GAE/g.0 0.

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