Polyphenols, carotenoids, and ascorbic acid in underutilized medicinal vegetables

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Polyphenols, carotenoids, and ascorbic acid in underutilized medicinal vegetables Nuri Andarwulana,b,*, Dewi Kurniasihb, Riza Aris Apriadyb, Hardianzah Rahmatb, Anna V. Rotoc, Bradley W. Bollingc a

Southeast Asian Food and Agricultural Science and Technology (SEAFAST) Center, Bogor Agricultural University, Bogor, Indonesia Department of Food Science and Technology, Bogor Agricultural University, Bogor, Indonesia c Department of Nutritional Sciences, University of Connecticut, Storrs, CT, USA b

A R T I C L E I N F O

A B S T R A C T

Article history:

The polyphenols, carotenoids, ascorbic acid, and protein were determined in 24 underuti-

Received 9 September 2011

lized medicinal vegetables from Indonesia. Anacardium occidentale, Sauropus androgynus (L.)

Received in revised form

Merr., and Moringa pterygosperma Gaertn. leaves were rich sources of flavonoids, with

27 December 2011

118–144 mg/100 g fresh weight. Quercetin, kaempferol, and chlorogenic acid were the

Accepted 5 January 2012

predominant polyphenols among those measured in vegetables. Polyscias pinnata leaves

Available online 30 January 2012

and Solanum torvum Swartz fruits had the most phenolic acids, with 53 and 36 mg/100 g, respectively. Moringa pterygosperma had the most carotenoids among vegetables, with

Keywords:

14 mg b-carotene equivalents (bCE)/100 g. Ascorbic acid content of fresh vegetables was

Medicinal vegetable

12.03–494.43 mg/100 g. A. occidentale, S. androgynus, Ocimum americanum L., Cosmos caudatus

Phenolic acid

H.B.K., and Carica papaya L. (papaya) leaves had more than 100 mg ascorbic acid/100 g.

Flavonoid

Thus, a number of underutilized vegetables from Indonesia may be rich sources of func-

b-Carotene

tional components including polyphenols and ascorbic acid.

Ascorbic acid

1.

Introduction

Vegetables may confer a variety of health benefits. Increased vegetable consumption is correlated with reduced risks of cardiovascular disease, stroke, arthritis, inflammatory bowel diseases, and some cancers (Amre et al., 2007; Dauchet et al., 2010; Griep, Verschuren, Kromhout, Ocke, & Geleijnse, 2011; Jain, Hislop, Howe, & Ghadirian, 1999). Many underutilized vegetables are also used medicinally (Andarwulan, Batari, Sandrasari, Bolling, & Wijaya, 2010). Vitamins, minerals, dietary fiber, and phytochemicals contribute to the functionality of vegetables. Characterizing the distribution of these functional components can inform dietary and horticultural efforts to increase intake of these potentially beneficial components from underutilized vegetables.

 2012 Elsevier Ltd. All rights reserved.

Among functional components, polyphenols are particularly promising for their role in health-promotion. Polyphenols or their metabolites modulate gene expression, epigenetic regulation, cell signaling, inflammation, antioxidant function, detoxification, and immune function (Bolling, Ji, Lee, & Parkin, 2011; Kang, Shin, Lee, & Lee, 2011; Yun, Jialal, & Devaraj, 2010). Vegetables contain a number of polyphenol classes, including hydroxycinnamic and hydroxybenzoic acids, flavonols, flavan-3-ols, flavones, flavanones, anthocyanins, phenolic aldehydes, stilbenes, hydrolyzable tannins, and proanthocyanidins, among others (Naczk & Shahidi, 2006; Shahidi, Chandrasekara, & Zhong, 2011). Ascorbic acid and carotenoids are also important functional vegetable components. Ascorbic acid is an antioxidant vitamin that acts synergistically with tocopherol to preserve

* Corresponding author at: Southeast Asian Food and Agricultural Science and Technology (SEAFAST) Center, Bogor Agricultural University, Jl Puspa No. 1, Kampus, IPB Darmaga, Bogor, Indonesia. Tel./fax: +62 251 8629903. E-mail address: [email protected] (N. Andarwulan). 1756-4646/$ - see front matter  2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jff.2012.01.003

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antioxidant function in chronic disease states (Bruno et al., 2006; Traber & Stevens, 2011). Vegetable carotenoids have antioxidant functions and can prevent vitamin A deficiencies when consumed with lipids to increase their bioavailability (Ribaya-Mercado et al., 2007; Shahidi & Zhong, 2010). This is particularly important in developing countries at risk for vitamin A deficiency (Tan et al., 2002). Polyphenols, ascorbic acid, and carotenoids have not been adequately characterized in a number of underutilized vegetables, particularly in Indonesia. We previously reported that Indonesian medicinal vegetables contain the flavonols quercetin, kaempferol, and myricetin and the flavones luteolin and apigenin, and possessed antioxidant activity (Andarwulan et al., 2010). Particularly, Sauropus androgynus (L.) Merr. (katuk), Cosmos caudatus H.B.K. (kenikir), and Polyscias pinnata (kedondong cina) were identified as rich sources of dietary flavonoids (Andarwulan et al., 2010). The aim of this study was to provide a more comprehensive characterization of polyphenols, ascorbic acid, carotenoids, and protein in underutilized Indonesian vegetables. Therefore, we quantified flavonols, flavones, phenolic acids, anthocyanins, total phenols, b-carotene, ascorbic acid, and protein in 24 Indonesian vegetables. This study is significant in that it is apparently the first report of polyphenols in a number of these vegetables.

2.

Materials and methods

Butylated hydroxyanisole (BHA), tert-butylhydroquinone (TBHQ), gallic acid, caffeic acid, ferulic acid, chlorogenic acid, and b-carotene were from Sigma–Aldrich (St. Louis, MO, USA). Methanol, acetic acid, chloroform, acetonitrile, ethanol, HPLC grade water, HPLC grade methanol, Folin–Ciocalteu reagent, Na2CO3, KH2PO4, HCl, soluble starch, KI, and iodine were from Merck (Darmstadt, Germany). Hexane and acetone were from Brataco Chemica (Bandung, Indonesia), KOH was from BDH (Leicestershire, UK). The vegetables sampled in the present study represent a new sampling and analysis from our previous report (Andarwulan et al., 2010). Samples of 0.5–1.0 kg of fresh vegetables free from obvious defects were obtained in Bogor, Indonesia (Table 1). C. caudatus H.B.K. leaves, Etlingera elatior (Jack) R.M.Sm. flowers, Ocimum americanum L. leaves, S. androgynus leaves, Pilea melastomoides (Poir.) Bl. leaves, Talinum triangulare (Jacq.) Willd. leaves, Allium schoenoprasum L., Solanum torvum Swartz fruits, Vigna unguiculata (L.) Walp. leaves, Saccharum edule Hassk flowers, Sechium edule (Jacq.) Swartz leaves, Carica papaya L. flowers, Anacardium occidentale L. leaves, and Arcypteris irregularis (C.Presl) Ching leaves were obtained from the Bogor traditional market, while the rest were harvested from uncultivated fields near Bogor Agricultural University. The vegetables were identified and classified by Dr. Eko Baroto Waluyo, APU, Indonesian Institute of Science, Research Center for Biology. Vegetables were washed, drained, cut into pieces, and frozen at 20 C. Within 12 h of freezing, samples were lyophilized for at least 48 h, powdered, passed through 30 mesh sieves, and stored at 20 C until further analysis. The total phenols content of extracts was determined using a modified method of Singleton, Orthofer, and

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Lamuela-Ravent (1999) as previously described (Andarwulan et al., 2010). Total phenols contents were expressed as gallic acid equivalents. For anthocyanin analysis, lyophilized vegetable powder, was extracted 1:10 (w/v) with 10 mL of 5% HCl in water at 4 C overnight (Raharja & Dianawati, 1995). The mixture was filtered and analyzed by the pH differential method (Lees & Francis, 1982). Flavonoids were extracted and quantified by a method by Hertog et al. (1995), with modifications (Andarwulan et al., 2010). Briefly, lyophilized vegetable powder was incubated with acidified aqueous methanol and TBHQ to hydrolyze and release bound flavonoids. The resulting extract was analyzed with a Shimadzu HPLC with UV detection (Kyoto, Japan) and a Develosil ODS column (Nomura Chemical, Seto, Japan). Flavonoids were separated with an acetonitrile gradient in phosphate buffer, tentatively identified based on UV absorbance and retention time, and quantified using dilutions of authentic standards. A representative chromatogram for flavonoid analysis is in Fig. 1. On column limits of detection were 0.78 pg myricetin, 1.12 pg luteolin, 0.56 pg quercetin, 4.4 pg apigenin, 0.94 kaempferol, determined experimentally as three times the standard deviation of the area under the curve for each compound (Rounds & Nielsen, 2000). Limits of quantification were >10 times the standard deviation of the area under the curve for each compound. Extraction and quantification of phenolic acids was performed by acid hydrolysis and release of bound phenolics according to the method described by Mattila and Kumpulainen (2002). Lyophilized vegetable powder (0.5 g) was reconstituted in 7 mL of 62.5% methanol in 15% acetic acid in water with 2 g/L BHA, sonicated for 30 min (Branson ultrasonic 3510, Danbury, CT, USA), and made to 10 mL with distilled water. Extract was filtered through a 0.45 lm membrane, and 20 lL filtrate was injected to the HPLC and column used for flavonoid analysis, with the following modifications. Isocratic methanol:0.4% acetic acid (80:20, v/v) at 1 mL/min was used to elute phenolic acids with detection at 290 nm (Singh et al., 2008). Phenolic acids were tentatively identified based on UV absorbance and retention time, and quantified using standard curves of authentic compounds. A representative chromatogram for phenolic acid analysis is in Fig. 1. Limits of detection and quantification were determined as described above. Limits of detection were 16 pg ferulic acid, 19.4 pg chlorogenic acid, and 16.6 pg caffeic acid on column. Additional acid and base hydrolysis of extraction residues did not yield additional phenolic acids. Total carotenoids and b-carotene were determined after saponification and extraction (Zakaria-Rungkat, Djaelani, Setiana, & Nurrochmah, 2000). Hexane and acetone (1:1, v/v) was used to extract 0.25 g lyophilized vegetable powder (20:1, v/w) thrice and passed through Whatman 42 filter paper. Solvent was removed by rotary evaporation at 45 C. Extract was mixed with 4 mL of 5% KOH in methanol, sonicated, and incubated at 70 C for 30 min. The extracts were cooled and mixed with 4 mL deionized water and 8 mL hexane. Following centrifugation, the organic phase was withdrawn, and the aqueous phase reextracted with 6 mL hexane and 3 mL of 5% acetic acid. Organic phases were combined, dried by rotary evaporation at 45 C, and reconstituted in 4 mL hexane. Absorbance

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Table 1 – Underutilized vegetables of Indonesian origin and their uses analyzed in the present study. Common names

Reported medicinal or traditional usesa

Allium schoenoprasum L.; all parts Anacardium occidentale L. leaf Arcypteris irregularis (C.Presl) Ching; leaf Carica papaya L.; flower Centelia asiatica (L.) Urb.; all parts

kucai, wild chives jambu mete, cashew pakis, fern pepaya, papaya antanan, Indian pennywort

Cosmos caudatus H.B.K.; leaf

kenikir, wild cosmos

Etlingera elatior (Jack) R.M.Sm.; flower Hydrocotyle sibthorpioides Lmk.; all parts

kecombrang, torch ginger antanan beurit, lawn marshpennywort

Morinda citrifolia L.; leaf Moringa pterygosperma Gaertn.; leaf

mengkudu, Indian mulberry kelor, horseradish tree

Nothopanax scutellarius (Burm.f.) Merr.; leaf Ocimum americanum L.; leaf

mangkokan kemangi, basil

Antihypertensive, intestinal antibiotic, blood thinner Anti-rheumatic Anthyhypertensive, tonsils, anti-rheumatic Antipyretic, antimalarial Diuretic, antihypertensive; memory enhancer, ergogenic aid for elderly, blood detoxicant; for hemorrhoids, liver disease, antitusive, sore throat, asthma, colitis, kidney stones, and Celiac disease Appetite stimulant, bone strengthener, insect repellant, for weak stomachs Food antibacterial, deodorant Memory enhancer, antipyretic, anti-inflammatory, diuretic, antibacterial, insecticide, antihistamine; for blood detoxification, circulation, bleeding Anthelmintic Antihypertensive, anti-diarrheic for diabetes mellitus and heart disease Lactation aid; for breast edema, hair loss, urination For cough, skin diseases, and rheumatism; treat headache, conjunctivitis, malaria, and fragrance

Pilea melastomoides (Poir.) Bl.; leaf Pluchea indica (L.) Less.; leaf

pohpohan beluntas, Indian camphorweed

Polyscias pinnata; leaf Polyscias scutellaria (Burm.f.) Fosb.; leaf

kedondong cina, balfour aralia mangkokan putih, shield aralia

Portulaca oleracea L.; leaf and stem

krokot, little hogweed, purslane

Saccharum edule Hassk.; flower Sauropus androgynus (L.) Merr.; leaf Sechium edule (Jacq.) Swartz; leaf

terubuk, vegetable cane katuk, chekkurmanis labu siam, chayote

Sesbania grandiflora (L.) Pers.; flower Solanum torvum Swartz; fruit

turi, vegetable hummingbird takokak, turkey berry

Talinum triangulare (Jacq.) Willd.; leaf

daun ginseng, Ceylon spinach

Vigna unguiculata (L.) Walp.; leaf

lembayung, blackeyed pea

Species; edible part

a

Appetite stimulant, anti-diaphoretic, antipyretic, digestive aid, deodorant, antibacterial, anti-diarrheal, antitusive, emollient Deodorant, appetite suppressant, eyewash Diuretic, anti-diaphoretic, deodorant, anti-alopecia, hairloss Anti-diarrheal, anti-inflammatory, anthelmintic, laxative; for appendix, breast inflammation, hemorrhoids Lactation aid, antipyretic, colorant Antihypertensive, anti-coagulant; for arteriosclerosis, kidney stones, respiratory and digestive systems, circulation For headache, as eyedrop Anti-edemic, analgesic, circulation improvement, alleviate pain, antitusive, anti-inflammatory; for stomach pain, toothache, cataracts, menstruation disorders, hemorrhoids, sore breasts, influenza, swelling, ulcers, soreness, sore waist, high uric acid, bone loss, heart palpitations, and detoxification For circulation, mucous membranes, stimulate mucous membranes, anti-edemic, antibacterial, anti-viral, appetite stimulant Dermatitis and swellings

References for these functions are provided in Supplemental Table 1.

was measured at 450 nm, with hexane as a blank. Total carotenoids (lg/g) were determined as: Absorbance at 450 nm · [10 lg/lL/E1%] · [4000 lL/mass powdered vegetable (g)] · dilution factor, where E1% is was the extinction value of a 1% b-carotene solution at 450 nm (=2600) and DF was the dilution factor. For b-carotene quantification, total carotenoid extracts were dried by rotary evaporation, dissolved in 5% chloroform in methanol, and kept at 20 C overnight. Solutions were then filtered with 0.22 lm membrane, dried, and dissolved in 2 mL mobile phase. Extract (20 lL) was analyzed by HPLC-UV with a Eclipse XDB-C18 column (Agilent, Santa Clara, CA, USA) and isocratic mobile phase of

methanol:acetonitrile:chloroform (48.5:48.5:3.0, v/v/v) at 0.8 mL/min and detection of b-carotene at 450 nm. Ascorbic acid was determined in fresh vegetables homogenized in water (2:1, w/v) by titration (Jacobs, 1951). Water and protein content were determined by AOAC (1984, 1995) methods 930.04 and 9260.5, respectively. Data are reflective of two independent experiments with duplicate analysis. Analysis of variance, Duncan’s test, and Tukey’s test (P < 0.05) were used to determine statistical significance, using SPSS 13.0 (IBM Corp., New York, NY, USA). Relationships between phenolic and non-phenolic compounds were determined by Principal Component

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3.

Fig. 1 – Representative chromatograms of HPLC analysis of vegetable extracts for (A) H. sibthorpioides flavonoids; and (B) P. scutellaria phenolic acids.

Analysis (PCA) using Minitab 16 (Minitab Inc., State College, PA, USA).

Results and discussion

Vegetables contain a wide variety of both essential and nonessential nutrients. The relative abundance of these nutrients determines the contribution of specific vegetables to prevent deficiencies, optimize health or performance, or prevent diseases. Many nutrients from Western foods have been characterized and quantified. However similar analysis of vegetables from Indonesia is lacking. Many vegetables have traditional medicinal uses in Indonesia, particularly among the Javanese population. We hypothesized these vegetables would be sources of carotenoids, ascorbic acid, polyphenols, and proteins. Therefore we quantified these components in 24 underutilized vegetables of Indonesian origin (Table 1). The contents of flavonoids quercetin, kaempferol, myricetin, luteolin, and apigenin of vegetables ranged from to 0.30 mg/100 g fresh weight (fw) for Portulaca oleracea L. to 143.58 mg/100 g fw for A. occidentale L. leaves (Table 2). Together, quercetin and kaempferol constituted more than 60% of the sum of vegetable flavonoids. Quercetin was the only flavonoid detected in E. elatior, P. oleracea, and Sa. edule. No flavones were detected in leaves or flowers of T. triangulare, S. androgynus, P. pinnata, C. caudatus, Nothopanax scutellarius (Burm.f.) Merr., Morinda citrifolia L., A. irregularis, and Sesbania grandiflora (LOD 5.6 lg luteolin, 22 lg apigenin per gram dry vegetable matter). A. occidentale, S. androgynus, and M. pterygosperma Gaertn. leaves had more than double the flavonoid content of other vegetables. A. occidentale and M. pterygosperma flavonoid content were 87% and 81% quercetin, respectively. In contrast, S. androgynus flavonoids were 97% kaempferol. Se. edule (Jacq.) Swartz leaves and A. occidentale

Table 2 – Flavonoid content of underutilized vegetables of Indonesian origin. Vegetable

Flavonoids (mg/100 g fw) Myricetin

A. occidentale S. androgynus M. pterygosperma P. pinnata C. caudatus H. sibthorpioides V. unguiculata C. papaya Se. edule M. citrifolia P. scutellaria S. grandiflora C. asiatica A. schoenoprasum A. irregularis O. americanum P. indica N. scutellarius T. triangulare S. torvum fruits P. melastomoides E. elatior Sa. edule P. oleracea

8.28 ± 0.17 nd nd nd nd 1.57 ± 0.01 nd nd 12.49 ± 0.14 nd nd nd 0.13 ± 0.00 2.69 ± 0.02 nd nd 0.90 ± 0.03 nd nd 2.30 ± 0.06 nd nd nd nd

Luteolin

Quercetin

Apigenin

Kaempferol

Total

nd nd 1.32 ± 0.04 nd nd nd nd nd nd nd nd nd nd nd nd 2.12 ± 0.05 nd nd nd nd 0.33 ± 0.02 nd nd nd

125.39 ± 1.3 4.50 ± 0.22 95.84 ± 1.98 28.48 ± 1.88 51.28 ± 4.06 37.51 ± 0.66 27.35 ± 0.75 18.85 ± 0.11 13.81 ± 0.17 23.67 ± 1.62 12.67 ± 0.12 2.76 ± 0.05 12.31 ± 0.41 4.46 ± 0.08 7.42 ± 0.06 1.89 ± 0.10 5.21 ± 0.26 3.69 ± 0.09 0.41 ± 0.03 0.66 ± 0.01 1.76 ± 0.20 1.18 ± 0.06 0.44 ± 0.01 0.30 ± 0.02

nd nd nd nd nd nd 12.97 ± 0.3 11.95 ± 0.28 nd nd 6.87 ± 0.33 nd nd nd nd 0.74 ± 0.04 nd nd nd nd nd nd nd nd

9.91 ± 0.07 138.14 ± 5.81 20.79 ± 0.35 23.71 ± 1.38 0.90 ± 0.05 10.85 ± 0.16 3.33 ± 0.11 5.47 ± 0.22 9.72 ± 0 .15 9.75 ± 0.69 12.95 ± 0.26 18.47 ± 0.31 8.57 ± 0.38 7.65 ± 0.15 2.10 ± 0.01 2.47 ± 0.18 0.28 ± 0.02 1.74 ± 0.07 3.52 ± 0.16 nd 0.25 ± 0.03 nd nd nd

143.58 142.64 117.95 52.19 52.19 49.93 43.65 36.27 36.03 33.42 32.49 21.23 21.00 14.79 9.52 7.22 6.39 5.43 3.93 2.96 2.34 1.18 0.44 0.3

nd, not detected above the limit of detection (3.9 lg myricetin, 5.6 lg luteolin, 22 lg apigenin, 4.7 lg kaempferol per g dry weight basis).

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leaves had significant myricetin content with 12.49 and 8.28 mg/100 g fw, respectively. Although flavones were not as prevalent as flavonols in vegetables, V. unguiculata (L.) Walp leaves and C. papaya L. flowers had 13 and 12 mg apigenin/ 100 g, respectively. To the best of our knowledge, this is the first report of flavonoid content in the edible parts of A. irregularis (C.Presl) Ching (9.52 mg/100 g), Polyscias scutellaria (Burm.f.) Fosb. (32.49 mg/100 g), Sa. edule Hassk (0.44 mg/100 g), and S. grandiflora (L.) Pers. (21.23 mg/100 g). The flavonoid content of vegetables in the present study was similar to those measured previously (Andarwulan et al., 2010). However, Centelia asiatica (L.) Urb. and Pluchea indica (L.) Less had more than 60-fold higher myricetin content compared to our previous study. While the reasons for these differences are unclear, environmental, seasonal, and soil variation may impact leafy green vegetable polyphenol content (Bano et al., 2004; Reimberg, Renata, & Yariwake, 2009). We quantified common vegetable flavonoids as aglycone equivalents following their hydrolysis. This approach may underestimate flavonoid content, due to the diversity of flavonoids in plants. For example, S. torvum fruits also contain isoflavones (Arthan et al., 2002) and A. occidentale leaves also contain robustaflavone, agathisflavone, and amentoflavone (Arya, Babu, Ilyas, & Nasim, 1989). In addition, it should be noted that plant flavonoids are mainly conjugated to sugars. For example, glycosylated or rhamnosyl flavonoids were previously identified in M. citrifolia leaves (Deng, West, & Jensen, 2008) and C- and O-glycosyl flavones were identified in Se. edule (350 mg/100 g dw) (Siciliano, De Tommasi, Morelli, &

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Braca, 2004). Nonetheless, this work is an important first step in characterizing the flavonoids in a number of underutilized medicinal vegetables. Chlorogenic acid, caffeic acid, and ferulic acid were present at 0.04–52.53 mg/100 g fw in vegetables (Table 3). P. pinnata leaves had the most phenolic acids among the vegetables studied, with a distribution of 90% chlorogenic acid and 10% ferulic acid. All vegetables contained phenolic acids, although T. triangulare leaves, A. schoenoprasum, and S. grandiflora flowers had less than 1 mg/100 g. M. citrifolia, A. occidentale, P. indica, and P. pinnata leaves did not have caffeic acid above the LOD. A. irregularis leaves and Hydrocotyle sibthorpioides Lmk. consisted only of chlorogenic and caffeic acid, while S. grandiflora flowers had solely ferulic acid. To the best of our knowledge, this is the first report of phenolic acid content in P. pinnata, M. pterygosperma Gaertn., A. irregularis (C.Presl) Ching, N. scutellarius (Burm.f.) Merr., Sa. edule Hassk, Se. edule (Jacq.) Swartz, and S. grandiflora (L.) Pers. Chlorogenic acid was the most abundant phenolic acid in 18 of the 24 vegetables. Caffeic acid was the second most abundant phenolic acid among the vegetables studied. Caffeic acid was most enriched in P. indica and C. caudatus leaves with 8.65 and 3.64 mg/100 g fw, respectively. P. pinnata, M. pterygosperma, and C. caudatus leaves had the greatest ferulic acid content among vegetables, with 3–5 mg/100 g fw. It should be noted that phenolic acids besides those quantified in the present study could significantly contribute to vegetable phenolic acid content. For example, gallic, protocatechuic, p-hydroxybenzoic, cinnamic, and p-coumaric acids are also found in A. occidentale leaves (Ko¨gel & Zech, 1985).

Table 3 – Specific phenolic acids and sum phenolic acids in 24 medicinally used Indonesian vegetables. Vegetable

Phenolic acids (mg/100 g fw) Chlorogenic acid

P. pinnata S. torvum P. indica H. sibthorpioides P. melastomoides A. occidentale P. scutellaria E. elatior M. pterygosperma C. asiatica C. caudatus leaves V. unguiculata P. oleracea Se. edule Sa. edule S. androgynus M. citrifolia A. irregularis C. papaya O. americanum N. scutellarius T. triangulare A. schoenoprasum S. grandiflora

47.02 ± 0.81 33.14 ± 1.63 20.00 ± 0.24 24.27 ± 0.51 17.47 ± 0.39 13.53 ± 0.38 14.13 ± 0.41 14.06 ± 1.11 6.65 ± 0.16 9.22 ± 0.34 4.54 ± 0.18 4.26 ± 0.12 5.79 ± 0.07 5.80 ± 0.12 4.17 ± 0.13 3.38 ± 0.32 2.31 ± 0.05 2.58 ± 0.16 0.77 ± 0.00 0.32 ± 0.01 0.86 ± 0.02 0.38 ± 0.01 0.08 ± 0.01 nd

Caffeic acid

Ferulic acid

Total

nd 2.56 ± 0.08 8.65 ± 0.46 1.35 ± 0.03 1.11 ± 0.01 nd 1.69 ± 0.03 0.96 ± 0.03 2.93 ± 0.08 1.19 ± 0.02 3.64 ± 0.14 2.02 ± 0.04 0.55 ± 0.00 0.55 ± 0.00 1.05 ± 0.02 1.13 ± 0.02 nd 0.47 ± 0.01 1.03 ± 0.00 2.03 ± 0.06 1.15 ± 0.01 0.41 ± 0.00 0.36 ± 0.01 nd

5.02 ± 0.16 0.32 ± 0.01 nd nd 0.17 ± 0.00 2.88 ± 0.12 0.80 ± 0.04 0.13 ± 0.00 4.41 ± 0.16 1.81 ± 0.09 3.14 ± 0.28 1.38 ± 0.09 0.22 ± 0.01 0.13 ± 0.00 0.16 ± 0.00 1.10 ± 0.04 0.76 ± 0.04 nd 0.75 ± 0.02 0.16 ± 0.00 0.24 ± 0.00 0.09 ± 0.00 0.10 ± 0.00 0.04 ± 0.00

52.53 ± 0.97 35.76 ± 1.71 28.48 ± 0.67 25.51 ± 0.54 18.57 ± 0.40 16.70 ± 0.32 16.30 ± 0.47 15.01 ± 1.15 13.50 ± 0.40 11.86 ± 0.44 10.92 ± 0.44 7.34 ± 0.24 6.32 ± 0.06 6.20 ± 0.12 5.13 ± 0.15 5.12 ± 0.29 3.24 ± 0.09 2.88 ± 0.16 2.30 ± 0.02 2.23 ± 0.06 1.82 ± 0.03 0.68 ± 0.01 0.35 ± 0.01 0.04 ± 0.00

nd, not detected above limit of detection (1.6 lg ferulic acid/g dry weight, 1.9 lg chlorogenic acid/dry weight, and 1.6 lg caffeic acid/g dry weight).

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S. torvum fruits also have methyl caffeate (Takahashi et al., 2010). Chlorogenic acid is present in a number of vegetables including lettuce (3.78 mg/100 g fw), broccoli (1.78 mg/100 g fw), and endive (101 mg/100 g fw) (Neveu et al., 2010). Caffeic acid is present in fresh herbs such as rosemary, sage, thyme, and oregeno from 2.08 to 11.7 mg/100 g (Neveu et al., 2010). Ferulic acid has previously been identified in vegetables from 0.21 to 3.01 mg/100 g fw, and is also found in beans and dried herbs (Neveu et al., 2010). Thus, consumption of P. pinnata, M.pterygosperma, and C. caudatus may increase dietary chlorogenic acid and ferulic acid from vegetables. Animal studies suggest that chlorogenic acid and ferulic acid improve antioxidant status and increase phase 2 detoxification enzymes. Dietary chlorogenic acid improved hepatic glutathione redox status in rats subjected to azoxymethane-induced colon cancer (Park, Davis, Liang, Rosenberg, & Bruno, 2010). Ferulic acid supplementation increased intestinal glutathione expression of glutathione-S-transferase and quinone reductase in rats (Bolling et al., 2011). Total content of phenols of vegetables ranged from 21.01 to 847.41 mg GAE/100 g (Table 4). A. occidentale and P. indica leaves had more than twofold higher total phenols content than other vegetables. A. occidentale leaves also had the greatest flavonoid content and the 6th highest phenolic acid content. The comparatively higher total phenols content suggests that flavones or other polyphenols are abundant in A. occidentale leaves (Siciliano et al., 2004). Similarly, P. indica flavonoid, phenolic acid, and anthocyanin contents do not appear to account for its high total phenols content. Other non-phenolic reducing agents present in the ethanol extracts of vegetables,

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such as ascorbic acid, are known to contribute to total content of phenols. However, ascorbic acid was not correlated with total phenols of vegetables (Fig. 2). More work is needed to define the constituents in A. occidentale and P. indica leaves that contribute to their increased total content of phenols. The vegetables in the present study did not have particularly abundant anthocyanin content, ranging from 0.07 to 4.44 mg/100 g fw (Table 4). M. pterygosperma leaves, S. torvum Swartz fruits, and E. elatior flowers had 3–4 mg/100 g, which is considerably less than the 26–589 mg/100 g fw found in berries (Neveu et al., 2010). Nevertheless, the anthocyanin content illustrates the diversity of polyphenolic compounds, and may contribute synergistically or additively to the medicinal properties of these vegetables. Leafy green carotenoids are composed of b-carotene, lutein, neoxanthin, zeaxanthin, and violaxanthin, with varying bioaccessibility (Chandrika, Basnayake, Athukorala, Colombagama, & Goonetilleke, 2010). It is estimated that 85% of total vitamin A activity from leafy green carotenoids is from b-carotene (Ball, 2000). The b-carotene content of vegetables in the present study ranged from 0.13 to 2.25 mg/100 g. For comparison, iceberg lettuce (0.30 mg/100 g), green leaf lettuce (4.4 mg/100 g), and spinach (5.6 mg/100 g) have equivalent or more b-carotene than the vegetables in the present study (USDA-ARS., 2011). The total carotenoid content of vegetables analyzed was 0.36– 13.96 mg b-carotene equivalents (bCE)/100 g fw (Table 4). M. pterygosperma leaves had the most carotenoids with 13.96 mg b-carotene equivalents (bCE)/100 g and 2.25 mg b-carotene/ 100 g. C. caudatus and P. indica leaves also had relatively higher carotenoid content compared to other vegetables, with 9.55 and 8.74 mg bCE/100 g FW. The ratio of b-carotene to total

Table 4 – Total phenols, anthocyanin, and carotenoids in 24 medicinally used Indonesian vegetables. Vegetable A. occidentale P. indica C. caudatus E. elatior C. asiatica P. pinnata P. scutellaria S. torvum H. sibthorpioides S. androgynus P. melastomoides V. unguiculata M. pterygosperma Sa. edule O. americanum P. oleracea M. citrifolia C. papaya Se. edule T. triangulare A. irregularis N. scutellarius S. grandiflora A. schoenoprasum

Total phenols (mg GAE/100 g fw)

Total anthocyanin (mg/100 g fw)

b-Carotene (mg/100 g fw)

Total carotenoids (mg bCE/100 g fw)

847.41 ± 15.05 742.54 ± 24.78 342.06 ± 0.37 256.99 ± 0.98 200.52 ± 2.05 189.08 ± 0.32 179.88 ± 2.00 158.92 ± 0.94 144.81 ± 1.14 138.01 ± 0.47 121.51 ± 1.64 112.55 ± 2.20 107.00 ± 2.87 87.65 ± 3.43 86.89 ± 0.70 82.66 ± 1.12 72.72 ± 0.38 66.75 ± 0.20 66.46 ± 0.71 64.64 ± 0.50 61.56 ± 1.25 40.36 ± 0.09 38.43 ± 1.53 21.01 + 0.13

0.37 ± 0.02 0.27 ± 0.01 0.78 ± 0.05 4.42 ± 0.11 1.08 ± 0.05 0.41 ± 0.01 1.64 ± 0.08 4.44 ± 0.14 0.77 ± 0.03 1.53 ± 0.11 0.75 ± 0.01 1.23 ± 0.08 3.25 ± 0.01 2.38 ± 0.13 0.11 ± 0.01 0.24 ± 0.01 1.12 ± 0.04 1.33 ± 0.08 0.78 ± 0.04 0.23 ± 0.01 0.07 ± 0.00 1.42 ± 0.07 0.22 ± 0.01 0.46 ± 0.01

0.73 ± 0.06 1.70 ± 0.05 1.35 ± 0.03 nd 1.16 ± 0.12 0.51 ± 0.10 0.85 ± 0.11 0.13 ± 0.01 0.24 ± 0.01 1.63 ± 0.02 1.48 ± 0.16 0.38 ± 0.07 2.25 ± 0.05 0.02 ± 0.00 1.56 ± 0.20 0.94 ± 0.08 0.33 ± 0.00 0.13 ± 0.01 1.77 ± 0.23 0.97 ± 0.01 0.89 ± 0.07 0.21 ± 0.02 0.01 ± 0.00 0.08 ± 0.00

5.42 ± 0.36 8.74 ± 0.34 9.55 ± 0.27 0.83 ± 0.05 5.95 ± 0.38 3.29 ± 0.27 7.64 ± 0.57 0.87 ± 0.03 2.75 ± 0.32 5.15 ± 0.07 5.12 ± 0.20 3.31 ± 0.09 13.96 ± 0.19 1.29 ± 0.08 7.35 ± 0.45 5.48 ± 0.22 3.28 ± 0.21 0.84 ± 0.06 2.83 ± 0.03 4.22 ± 0.14 6.15 ± 0.08 2.69 ± 0.01 0.36 ± 0.03 0.65 ± 0.06

nd, not detected above limit of detection; GAE, gallic acid equivalents; bCE, b-carotene equivalents.

Ascorbic acid (mg/100 g fw) 494.43 ± 0.94 15.44 ± 0.00 108.83 ± 0.50 16.90 ± 0.00 24.04 ± 0.01 12.03 ± 0.19 15.61 ± 0.00 54.78 ± 0.04 21.85 ± 0.00 190.83 ± 0.82 45.13 ± 0.00 58.87 ± 0.84 90.28 ± 0.85 29.16 ± 0.00 146.37 ± 0.17 21.48 ± 0.43 30.71 ± 0.00 117.15 ± 0.01 31.74 ± 0.42 25.52 ± 0.01 69.85 ± 0.99 34.30 ± 0.13 33.52 ± 0.03 24.59 ± 0.02

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Fig. 2 – (A) Principal component scores and (B) loading plot of Principal Component Analysis of functional components of vegetables of Indonesian origin.

carotenoids was greatest in Se. edule (63%), S. androgynus (32%), and P. melastomoides leaves (29%). Minimal to no b-carotene was detected in S. grandiflora (2%), Se. edule (3%), and E. elatior flowers (none detected). Thus, a substantial amount of other carotenes or xanthophylls may account for the total carotenoid values for these vegetables. Many of the vegetables sampled contained significant quantities of ascorbic acid. Ascorbic acid content ranged from 12.03 to 494.43 mg/100 g fw (Table 4). A. occidentale leaves had the most ascorbic acid with 494.43 mg/100 g, while S. androgynus leaves. A 30 g serving (1 c) of A. occidentale leaves would provide twice the ascorbic acid of an orange (USDA-ARS, 2011). O. americanum, C. caudatus, and C. papaya leaves had more than 100 mg/100 g FW. The majority of Indonesian vegetables had ascorbic acid content greater than typical western vegetables such as spinach (28 mg/100 g), green leaf lettuce (9.2 mg/ 100 g), and iceberg lettuce (2.8 mg/100 g) (USDA-ARS, 2011). Carotenoid and anthocyanin pigmentation is dependent upon protein denaturation following heat processing of certain vegetables, such as sweet potatoes. While carotene and protein content were not correlated in tubers, this relationship in other vegetables is unclear (Novita, Hartana, & Soeharsono, 1996). The vegetables analyzed had 0.4–8 g protein/100 g fw (Table 5). The protein content among vegetables analyzed varied more than 10-fold, and are comparable to spinach and lettuce contents (USDA-ARS, 2011). Protein content in vegetables was not correlated with polyphenol, carotenoid, or ascorbic acid content. The moisture content of vegetables was 75–92%. Thus, on a dry weight basis, some vegetables may comparatively be better sources of functional constituents than others. This is a consideration to utilizing these vegetables for ingredients or supplements. On a dry weight basis, A. occidentale leaves and P. indica leaves had the highest total phenols with 4.4 and 3.9 g GAE/100 g. S. androgynus leaves was the best source of flavonoids with 831 mg/100 g. S. torvum fruits and P. pinnata leaves were the best sources of phenolic acids, with 179 and 357 mg/100 g. PCA was used to examine the relationships among the functional components of vegetables. Principal component scores were able to distinguish vegetables, with the first two

components explaining 33% and 24% of the data variation (Fig. 2). The loading plot illustrates the relationships among variables, where two vectors with an angle less than 90 are positively correlated and two vectors with an obtuse angle (>90) are negatively correlated (Sartono, Affendi, Syahfitri, Sumertajaya, & Anggraeni, 2003). Total phenols was positively correlated with the flavonoid and phenolic acid content. Ascorbic acid was more strongly correlated with flavonoid and total phenol content than total carotenoids or phenolic acids. Carotenoid and b-carotene content was not well-correlated with polyphenol content. A. occidentale L. (jambu mete) was distinguished by its high ascorbic acid, flavonoids, and

Table 5 – Moisture and protein of underutilized medicinal vegetables of Indonesian origin. Vegetable A. schoenoprasum T. triangulare S. grandiflora E. elatior A. irregularis C. papaya Sa. edule P. oleracea P. melastomoides O. americanum Se. edule M. citrifolia P. pinnata V. unguiculata H. sibthorpioides P. scutellaria N. scutellarius C. asiatica A. occidentale P. indica C. caudatus S. torvum S. androgynus M. pterygosperma

Moisture (g/100 g fw)

Protein (g/100 g fw)

92.30 ± 0.32 91.83 ± 0.00 90.23 ± 0.09 89.77 ± 0.21 89.27 ± 0.89 88.91 ± 0.45 88.39 ± 0.10 88.07 ± 0.35 87.68 ± 0.01 87.42 ± 0.47 86.68 ± 0.48 85.46 ± 0.06 85.43 ± 1.57 84.36 ± 0.38 84.30 ± 0.11 82.31 ± 0.18 82.28 ± 0.13 81.72 ± 0.30 80.82 ± 0.09 80.81 ± 1.50 80.3 ± 2.53 79.89 ± 1.22 78.19 ± 0.49 75.27 ± 0.14

0.43 ± 0.03 1.79 ± 0.04 2.41 ± 0,01 0.96 ± 0.05 3.61 ± 0.09 3.50 ± 0.02 2.69 ± 0.03 2.36 ± 0.01 3.33 ± 0.21 4.56 ± 0.07 3.33 ± 0.03 2.50 ± 0.02 3.08 ± 0.03 5.95 ± 0.03 3.01 ± 0.05 2.44 ± 0.06 4.32 ± 0.04 0.79 ± 0.03 6.96 ± 0.16 4.49 ± 0.10 4.22 ± 0.12 3.11 ± 0.06 8.31 ± 0.11 7.76 ± 0.11

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total phenols values. E. elatior (Jack) R.M.Sm. (kecombrang) was distinguished from other vegetables by its relatively higher anthocyanin and phenolic acid content. O. americanum L. (kemangi) were distinguished by the combination of increased ascorbic acid and carotenoids. The unique distributions of functional components in these plants make them intriguing candidates for further research into their traditional medicinal uses.

4.

Conclusions

In conclusion, underutilized vegetables of Indonesian origin were rich sources of polyphenols, ascorbic acid, and carotenoids to varying degrees. This work provides a basis for further characterization of polyphenols, carotenoids and other constituents of these vegetables. A. occidentale, S. androgynus, M. pterygosperma, and P. indica were rich in polyphenol content and are promising candidates to further investigate for functional uses. More work is needed to determine the safety of increased consumption of these plants, particularly outside the context of their traditional uses and intakes. Further studies are necessary to establish the efficacy and plausible mechanisms for the medicinal uses of these vegetables.

Acknowledgement This work was supported by the Southeast Asian Food and Agricultural Science and Technology (SEAFAST) Center.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jff.2012.01.003.

R E F E R E N C E S

Amre, D. K., D’Souza, S., Morgan, K., Seidman, G., Lambrette, P., Grimard, G., Israel, D., Mack, D., Ghadirian, P., Deslandres, C., Chotard, V., Budai, B., Law, L., Levy, E., & Seidman, E. G. (2007). Imbalances in dietary consumption of fatty acids, vegetables, and fruits are associated with risk for Crohn’s disease in children. American Journal of Gastroenterology, 102, 2016–2025. Andarwulan, N., Batari, R., Sandrasari, D. A., Bolling, B., & Wijaya, C. H. (2010). Flavonoid content and antioxidant activity of vegetables from Indonesia. Food Chemistry, 121, 1231–1235. AOAC. (1984). Official method 930.04. In Official method of analysis of AOAC international. Washington, DC: AOAC International. AOAC. (1995). Official method 9260.5. In Official Methods of Analysis (16th ed.). Gaithersburg, MD: AOAC International. Arthan, D., Svasti, J., Kittakoop, P., Pittayakhachonwut, D., Tanticharoen, M., & Thebtaranonth, Y. (2002). Antiviral isoflavonoid sulfate and steroidal glycosides from the fruits of Solanum torvum. Phytochemistry, 59, 459–463. Arya, R., Babu, V., Ilyas, M., & Nasim, K. T. (1989). Phytochemical examination of the leaves of Anacardium occidentale. Journal of the Indian Chemical Society, 66, 67–68. Ball, G. F. M. (2000). The fat soluble vitamins. In L. M. L. Nollet (Ed.), Food analysis by HPLC 2nd ed.: Revised and expanded (pp. 321–402). New York: Marcel Dekker, Inc..

4 ( 2 0 1 2 ) 3 3 9 –3 4 7

Bano, M. J., Lorente, J., Castillo, J., Benavente-Garcia, O., Marin, M. P., Del Rio, J. A., Ortuno, A., & Ibarra, I. (2004). Flavonoid distribution during the development of leaves, flowers, stems, and roots of Rosmarinus officinalis. Postulation of a biosynthetic pathway. Journal of Agricultural and Food Chemistry, 52, 4987–4992. Bolling, B. W., Ji, L. L., Lee, C.-H., & Parkin, K. L. (2011). Dietary supplementation of ferulic acid and ferulic acid ethyl ester induces quinone reductase and glutathione-S-transferase in rats. Food Chemistry, 124, 1–6. Bruno, R. S., Leonard, S. W., Atkinson, J., Montine, T. J., Ramakrishnan, R., Bray, T. M., et al. (2006). Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation. Free Radical Biology and Medicine, 40, 689–697. Chandrika, U. G., Basnayake, B. M. L. B., Athukorala, I., Colombagama, P. W. N. M., & Goonetilleke, A. (2010). Carotenoid content and in vitro bioaccessibility of lutein in some leafy vegetables popular in Sri Lanka. Journal of Nutritional Science and Vitaminology, 56, 203–207. Dauchet, L., Montaye, M., Ruidavets, J. B., Arveiler, D., Kee, F., Bingham, A., et al. (2010). Association between the frequency of fruit and vegetable consumption and cardiovascular disease in male smokers and non-smokers. European Journal of Clinical Nutrition, 64, 578–586. Deng, S., West, B. J., & Jensen, C. J. (2008). Simultaneous characterisation and quantitation of flavonol glycosides and aglycones in noni leaves using a validated HPLC-UV/MS method. Food Chemistry, 111, 526–529. Griep, L. O., Verschuren, W. M., Kromhout, D., Ocke, M. C., & Geleijnse, J. M. (2011). Raw and processed fruit and vegetable consumption and 10-year stroke incidence in a populationbased cohort study in The Netherlands. European Journal of Clinical Nutrition, 65, 791–799. Hertog, M. G., Kromhout, D., Aravanis, C., Blackburn, H., Buzina, R., Fidanza, F., Giampaoli, S., Jansen, A., Menotti, A., Nedeljkovic, S., et al. (1995). Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study. Archives of Internal Medicine, 155, 381–386. Jacobs, M. B. (1951). The chemical analysis of foods and food products (2nd ed). New York: D. Van Nostrand Company, Inc.. Jain, M. G., Hislop, G. T., Howe, G. R., & Ghadirian, P. (1999). Plant foods, antioxidants, and prostate cancer risk: Findings from case–control studies in Canada. Nutrition and Cancer, 34, 173–184. Kang, N. J., Shin, S. H., Lee, H. J., & Lee, K. W. (2011). Polyphenols as small molecular inhibitors of signaling cascades in carcinogenesis. Pharmacology & Therapeutics, 130, 310–324. Ko¨gel, I., & Zech, W. (1985). The phenolic acid content of cashew leaves (Anacardium occidentale L.) and of the associated humus layer, Senegal. Geoderma, 35, 119–125. Lees, D. H., & Francis, F. J. (1982). Analysis of anthocyanins. In P. Markakis (Ed.), Anthocyanins as food colors. New York: Academic Press. Mattila, P., & Kumpulainen, J. (2002). Determination of free and total phenolic acids in plant-derived foods by HPLC with diode-array detection. Journal of Agricultural and Food Chemistry, 50, 3660–3667. Naczk, M., & Shahidi, F. (2006). Phenolics in cereals, fruits and vegetables: Occurrence, extraction and analysis. Journal of Pharmaceutical and Biomedical Analysis, 41, 1523–1542. Neveu, V., Perez-Jime´nez, J., Vos, F., Crespy, V., du Chaffaut, L., Mennen, L., Knox, C., Eisner, R., Cruz, J., Wishart, D., & Scalbert, A. (2010). Phenol-Explorer: An online comprehensive database on polyphenol contents in foods. Database, 2010, 1–9. Novita, L., Hartana, A., & Soeharsono, D. (1996). Cross compatibilities among orange-flesh sweet potato clones. Hayati, 3, 37–42.

JOURNAL OF FUNCTIONAL FOODS

Park, H. J., Davis, S. R., Liang, H. Y., Rosenberg, D. W., & Bruno, R. S. (2010). Chlorogenic acid differentially alters hepatic and small intestinal thiol redox status without protecting against azoxymethane-induced colon carcinogenesis in mice. Nutrition and Cancer, 62, 362–370. Raharja, S., & Dianawati, E. (1995). Study in extraction of anthocyanins from Erpa leaf (Aerva sp.) with acidic solvent. Journal Teknologi Industri Pertanian, 11, 49–52. Reimberg, M. C., Renata, C., & Yariwake, J. H. (2009). Multivariate analysis of the effects of soil parameters and environmental factors on the flavonoid content of leaves of Passiflora incarnata L., Passifloraceae. Revista Brasileira de Farmacognosia, 19, 853–859. Ribaya-Mercado, J. D., Maramag, C. C., Tengco, L. W., Dolnikowski, G. G., Blumberg, J. B., & Solon, F. S. (2007). Carotene-rich plant foods ingested with minimal dietary fat enhance the totalbody vitamin A pool size in Filipino schoolchildren as assessed by stable-isotope-dilution methodology. American Journal of Clinical Nutrition, 85, 1041–1049. Rounds, M. A., & Nielsen, S. S. (2000). Basic principles of chromatography. In L. M. L. Nollet (Ed.), Food analysis by HPLC (2nd ed.). New York: Marcell Dekker. Sartono, B., Affendi, F. M., Syahfitri, U. D., Sumertajaya, I. M., & Anggraeni, Y. (2003). Analisis Variabel Ganda. Bogor, Indonesia: Statistics Dept. Bogor Agricultural University. Shahidi, F., & Zhong, Y. (2010). Lipid oxidation and improving oxidative stability. Chemical Society Reviews, 39, 4067–4079. Shahidi, F., Chandrasekara, A., & Zhong, Y. (2011). Bioactive phytochemicals in vegetables. In N. K. Sinha (Ed.), Handbook of vegetables and vegetable processing (pp. 125–158). Oxford, UK: Wiley–Blackwell. Siciliano, T., De Tommasi, N., Morelli, I., & Braca, A. (2004). Study of flavonoids of Sechium edule (Jacq) Swartz (Cucurbitaceae) different edible organs by liquid chromatography photodiode

4 ( 20 1 2) 3 3 9–34 7

347

array mass spectrometry. Journal of Agricultural and Food Chemistry, 52, 6510–6515. Singh, U. P., Suman, A., Sharma, M., Singh, J. N., Singh, A., & Maurya, S. (2008). HPLC Analysis of the Phenolic Profiles in Different Parts of Chilli (Capsicum annum) and Okra (Abelmoschus esculentus L.) Moench.. The Internet Journal of Alternative Medicine, 5, 2. Singleton, V. L., Orthofer, R., & Lamuela-Ravent, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods in Enzymology, 299, 152–178. Takahashi, K., Yoshioka, Y., Kato, E., Katsuki, S., Iida, O., Hosokawa, K., & Kawabata, J. (2010). Methyl caffeate as an alpha-glucosidase inhibitor from Solanum torvum fruits and the activity of related compounds. Bioscience, Biotechnology, and Biochemistry, 74, 741–745. Tan, Z., Ma, G., Lin, L., Liu, C., Liu, Y., Jiang, J., Ren, G., Wang, Y., Hao, Y., He, L., & Yao., J. (2002). Prevalence of subclinical vitamin A deficiency and its affecting factors in 8669 children of China. Zhonghua Yu Fang Yi Xue Za Zhi, 36, 161–163. Traber, M. G., & Stevens, J. F. (2011). Vitamins C and E: Beneficial effects from a mechanistic perspective. Free Radical Biology and Medicine, 51, 1000–1013. USDA-ARS. (2011). National nutrient database for standard reference, release 24. Yun, J. M., Jialal, I., & Devaraj, S. (2010). Effects of epigallocatechin gallate on regulatory T cell number and function in obese v. lean volunteers. British Journal of Nutrition, 103, 1771–1777. Zakaria-Rungkat, F., Djaelani, M., Setiana, Rumondang E., & Nurrochmah (2000). Carotenoid bioavailability of vegetables and carbohydrate-containing foods measured by retinol accumulation in rat livers. Journal of Food Composition and Analysis, 13, 297–310.