Article pubs.acs.org/JAFC
Chemical Compositions and Antioxidative and Antidiabetic Properties of Underutilized Vegetable Palm Hearts from Plectocomiopsis geminiflora and Eugeissona insignis Zabidah Ahmad Aufa,† Fouad Abdulrahman Hassan,†,§ Amin Ismail,*,†,⊥ Barakatun Nisak Mohd Yusof,†,⊥ and Muhajir Hamid‡ †
Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia § Department of Food Science, Faculty of Agriculture, Ibb University, Ibb, Yemen ‡ Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia ⊥ Research Center of Excellence for Nutrition and Non-Communicable Diseases, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia ABSTRACT: Underutilized vegetables are currently studied not only for their nutrient values but also for their healthpromoting components for protection against chronic diseases. The present study was performed to evaluate chemical compositions and antioxidant properties of underutilized vegetable palm hearts, namely, lalis (Plectocomiopsis geminiflora) and pantu (Eugeissona insignis). Additionally, the vegetable extracts were evaluated for their activities in the inhibition of digestive enzymes and effects on insulin secretion using BRIN BD11 pancreatic cell lines. Both vegetables contain valuable sources of dietary fiber, potassium, and zinc. For the first time, the phenolic compounds of the vegetables were identified and quantified using HPLC-DAD and LC-ESI-MS. Appreciable amounts of chlorogenic acid were found in the studied vegetables. The sample extracts exhibited potential antioxidant capacities through chemical and biological in vitro assays. High inhibition of α-amylase activity (>50%) was found from the extracts. Thus, it was suggested the vegetable consumption could fulfill the nutrient requirements among local communities. KEYWORDS: underutilized vegetable, proximate composition, phenolic, antidiabetic, Plectocomiopsis geminiflora, Eugeissona insignis
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INTRODUCTION Pathogenesis of human illnesses such as diabetes and cancer is generated by the oxidative damage of reactive oxygen and nitrogen species.1 Numerous antioxidant compounds such as vitamins, tocopherols, and polyphenols, which naturally present in plant species, are able to protect against oxidative stress. Diabetes mellitus is a major health problem, and its incidence and prevalence are growing rapidly, especially in developing countries. One effective therapeutic approach for type 2 diabetes management is the inhibition of pancreatic α-amylase and intestinal α-glucosidase.2 The presence of inhibitors of these enzymes can decelerate carbohydrate digestion and thus finally prevent the postprandial plasma glucose rise.3 Natural resources of underutilized plant species have potential for supplying food security, especially to the poor, income generation, and environmental services.4 The underutilized species grow wildly and are considered to be adaptive and resilient and to tolerate poor climatic conditions better than exotic plant species.5 In Malaysia, various type of indigenous vegetables grow wildly in the tropical rain forest. Most of these plant species are recognized as “underutilized” due to unknown features and are familiar only among the local community.6 Nowadays, several palm heart species have been cultivated and commercialized widely in many countries such as Euterpe oleracea, Calamus tenuis, and Euterpe edulis Mart.7 In this study, © 2014 American Chemical Society
lalis (Plectocomiopsis geminiflora) and pantu (Eugeissona insignis) are tropical underutilized palm hearts in the Borneo islands and commonly consumed among local people in East Malaysia. Lalis is light yellow in color and has a bitter and slightly sweet taste; its tree can grow up to 30 m high. It is known as wi lalis in the Iban culture; rotan, baluak, or tambarluak for Kadazandusun and Murut peoples; and mudmua for the Dayak population in Sarawak, Malaysia. Pantu, known as pantu ketajau, has a slightly bitter taste and a texture similar to that of bamboo shoot. Local communities usually cook it with other vegetables or meat in soups. Many studies have revealed the compositions and antioxidant properties of Malaysian underutilized fruits.6,8 Nevertheless, limited studies have been performed on the nutritional values and health benefit properties of underutilized vegetables. Investigations of nutritional compositions and antioxidant and antidiabetic properties of these vegetable palm hearts are important to provide new information for future researchers and policy-makers as well as local communities where these plants are grown and consumed. To promote consumption of Received: Revised: Accepted: Published: 2077
August 21, 2013 February 4, 2014 February 6, 2014 February 6, 2014 dx.doi.org/10.1021/jf403481p | J. Agric. Food Chem. 2014, 62, 2077−2084
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implemented using an eclipse XDB-C18 column (250 mm × 4.6 mm i.d.) (Hewlett-Packard, Palo Alto, CA, USA). The mobile phases consisted of 0.5% acetic acid (v/v) (A) and methanol (B). The applied gradient elution composition was a linear increase to 90% B in 20 min, then isocratic for 5 min, and returned to the initial condition in 5 min. Run time and column temperature were set at 30 min and 30 °C, respectively. Flow rate was 0.6 mL/min, and injection volume was 20 μL. The wavelength was set at 280 nm. The compounds in extracts were identified by comparison of their retention times and UV−vis absorption spectra with authentic standards and further confirmed by spiking of extract with authentic standards. External standards with different concentrations (20−100 μg/mL) diluted in ethanol were performed to determine the quantity of identified compounds. This linearity of detector response was assessed using five different concentrations, with three injections for all concentrations. A linear relationship was found between peak area and concentration (20−100 μg/mL). The correlation coefficient ranges from r = 0.985 to r = 0.998. After completion of HPLC analysis, the liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) analysis was conducted using an Applied TSQ Quantum Ultra-LCMS (Thermo Fisher, San Diego, CA, USA) to check the molecular mass of detected compounds in the HPLC chromatogram. A reversed-phase Lichrospher C18 column (250 mm × 4 mm, i.d., 5 μm; Merck, Damstadt, Germany) was used. Both positive and negative ion modes were used, and optimum resolution used was 3000 Da. The spray voltage was maintained at 3.5 kV. The sheath/auxiliary/sweep gas was 99% pure. The sheath and auxiliary gas pressures were set at 30 and 5 psi, respectively. The capillary temperature was 270 °C. The flush speed was 100 μL/s, and the injection volume was 10 μL. Samples were run using a gradient elution system as described by Hassan et al.18 Continuous full mass spectral data were achieved by scanning from m/z 100 to 800. Determination of phenolic compounds was carried out by comparing the retention times, UV−vis absorption spectra, and reference standards available. In Vitro Antioxidant Capacities. Ferric Reducing Antioxidant Power (FRAP) Assay. The ability of extract to convert Fe3+/ ferricyanide complex to Fe2+ ions was assessed on the basis of the method of Benzie and Strain,19 with some modifications. Concentration of samples was at 0.2 mg/mL. A preliminary test had been carried out to determine the concentration of samples that gave readings within the standard curve range. Oxygen Radical Absorbance Capacity (ORAC) Assay. The ORAC assay20 was based on the inhibition of peroxyl radical-induced oxidation initiated by thermal decomposition of 2,2′-azobis(2methylpropionamidine) dihydrochloride (AAPH). Different dilutions of Trolox (12.5−200 μM) and sample extracts were prepared in phosphate buffer (10 mM, pH 7.4). A concentration of samples was at 0.5 mg/mL. A preliminary test was carried out to determine the concentration of samples that gave readings within the range of Trolox standard curve. Briefly, 10 nM fluorescein (150 μL) was added into a black 96-well plate (Nunc, Roskilde, Denmark) and followed by 25 μL of Trolox, sample, or blank (phosphate buffer). The plate was covered and incubated for 10 min at 37 °C. After incubation, fluorescence measurements (excitation wavelength, 485 nm; emission wavelength, 520 nm) were taken every 90 s to determine the background signal. The fluorescence measurement was performed using microplate reader FLUOstar Omega (BMG LABTECH, Offenburg, Germany). After four cycles, 25 μL of 240 mM AAPH was injected via onboard injector. The test was continued, and fluorescence intensity measurements were taken up to 120 min. ORAC values were calculated on the basis of the difference in area under the fluorescence decay curve between the blank and sample. The antioxidant capacity (ORAC) was calculated according to the following equation:
underutilized palm heart species and support the claims of locals with regard to their health potential in treating diabetes, these two vegetables were investigated using related in vitro assays.
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MATERIALS AND METHODS
Preparation of Samples. Lalis (P. geminif lora) and pantu (E. insignis) were purchased at Satok market in Kuching, Sarawak, Malaysia. About 15 kg of each sample was collected and stored in a freezer (−20 °C) and then brought back to the laboratory by flight within 3 days. Voucher specimens of the samples, lalis (SK2276/13) and pantu (SK2277/13), were deposited at the Biodiversity Unit, Bioscience Institute, Universiti Putra Malaysia, Malaysia. The peels of sample were removed, and edible parts were cleaned and chopped into small pieces. Samples were subjected to lyophilization for 72 h using a benchtop freeze-dryer (Virtis, Gardiner, NY, USA). Subsequently, the dried vegetables were ground into fine particles and stored at −40 °C. Proximate Analyses. The moisture content of the studied vegetables was evaluated through a direct drying method (AOAC method 964.22) using a convection oven (Memmert Universal, Schwabach, Germany). Ash content was measured using a dry ashing method (AOAC method 923.03) whereby the samples were incinerated at 550 °C in a furnace (Furnace 62700, Barnstead/ Thermolyne, Dubuque, IA, USA). Lipid content was determined using the Soxhlet method (AOAC method 920.39C), and protein content (N × 6.25) was measured according to the Kjeldahl method (AOAC method 955.04) by Kjeltec instrument (Kjeltec 2200, Foss, Höganäs, Sweden). The above analyses followed AOAC methods.9 Total carbohydrate was determined using a Clegg anthrone method.10 Total (TDF) and insoluble dietary fiber (IDF) contents were measured using a gravimetric method.11 Dietary fiber analyses were carried out using a Fibertec system (Fibertec System E 1023 filtration module, Sweden). Result was obtained after subtracting the protein and ash contents of residue. Soluble dietary fiber (SDF) was calculated as TDF − IDF value. Mineral Analysis. The resulting ash sample obtained from the dryashing method was dissolved in 100 mL of 1 N hydrochloric acid solution. Subsequently, the ash solution was used in determination of the mineral contents. Minerals (sodium, potassium, calcium, magnesium, iron, zinc, phosphorus, manganese, and copper) were determined using an inductively coupled plasma optical emission spectrometry (ICP-OES) (ICP-OES, PerkinElmer DV2000, Boston, MA, USA) method.12 Sample Extraction. The extraction method was according to Liu et al.13 with slight modification. Four grams of powder extract was mixed with 100 mL of 70% ethanol containing 1.2 M HCl (ratio of 1:25, g/v) and gently shaken at room temperature using an incubator shaker (Heidolph Instruments Incubator1000, Schwabach, Germany) set at 200 rpm for 5 h. After that, sample extracts were filtered through Whatman paper no. 1, and the filtrate was used for total phenolic and flavonoid content determination. The remaining extract was evaporated using a rotary evaporator (Buchi Rotavapor R-200, Essen, Germany) to remove the solvent and then lyophilized. The yield of extracts was subjected to antioxidant and antidiabetic in vitro assays. Total Phenolic and Flavonoid Contents. Total phenolic content (TPC) of studied samples was determined according to method of Velioglu et al.14 Results were generated from the standard calibration curve of gallic acid (20−100 μg/mL). Total flavonoid content (TFC) of studied samples was evaluated by an aluminum chloride colorimetric assay15 with a slight modification. A standard calibration curve was generated using rutin (0.01−0.08 μg/mL). Phenolic Compounds Analysis. Sample extract was prepared according to a method described by Liu et al.16 with modifications. Quantification of phenolics in the studied vegetable extracts was performed using a HPLC system consisting of an Agilent 1100 series liquid chromatographic system equipped with an Agilent 1100 series diode array detector (DAD) (Agilent Technologies, Santa Clara, CA, USA) as described by He and Xia.17 Separation of compounds was
ORAC value = [(AUCsample − AUC blank )/(AUCTrolox − AUC blank ) × [Trolox] dilution factor
Inhibition of Lipid Membrane Erythrocytes Oxidation Assay. This assay was done according to the method described by Rodriguez et 2078
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al.21 with some modifications. The blood sample (10 mL) was obtained from a volunteer and collected into EDTA tubes. Then, the tube was immediately centrifuged at 3000 rpm and 4 °C for 10 min. After removal of plasma, the packed red blood cells (RBC) were directly washed three times with 0.9% NaCl (2 mL). After the third wash, packed RBC were gently resuspended in phosphate buffer saline (PBS) (pH 7.4) to obtain a 5% hematocrit in the presence of 1 mM NaN3 and preincubated at 37 °C for 10 min. Then, the mixture was divided into aliquots (1.6 mL) for each experimental treatment. Concentrations of samples and standard were 0.05 and 0.2 mg/mL, respectively. All treatment groups and BHT (0.2 mL) were challenged with 0.01 M H2O2 (0.2 mL). Meanwhile, negative and positive controls were made with solvent and H2O2 alone, respectively. After 60 min of incubation at 37 °C, the tube was kept in an ice bath for 1 min. Malondialdehyde (MDA) produced from membrane lipid oxidation of erythrocyte oxidation was measured using the TBARS method of Buege and Aust,22 with some modifications. One milliliter of erythrocyte solution was vortexed with 1 mL of TCA/TBA/HCl reagent (15% TCA/0.375% TBA/0.25 mol of HCl) (1:1:1, v/v/v) for 10 s. Then, the mixture was heated in a boiling water for 15 min. Subsequently, the mixture was cooled at room temperature for 10 min and centrifuged at 3000 rpm for 10 min. The absorbance of supernatant was read at 535 nm against a blank of 15% TCA/ 0.375% TBA/0.25 mol of HCl reagent. The results were expressed as micromolar MDA. The MDA level was calculated using the equation
Cell Viability Assay. The viability of the BRIN BD11 cell line in the presence of sample extracts was determined using an MTT (3-(4,5dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) assay.26 BRIN BD11 cell line was obtained from the Malaysian Institute Nuclear Technology (MINT). Cells were cultivated in T-25 cm2 culture flasks using Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco BRL Products, Rockville, MD, USA) with 2.0 g/L sodium bicarbonate, antibiotics (100 units of penicillin/mL and 100 μg of streptomycin/ mL), and 10% fetal bovine serum (FBS). Cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C and were harvested at 80− 90% confluence to maintain exponential growth. The extracts were dissolved in water and filtered through a sterile 0.22 μm syringe filter. Insulin Secretion Assay. Insulin secretion activity was evaluated according to the method of Hamid et al.27 The BRIN BD11 cell line was seeded at a density of 1.0 × 105 cells per well in a 24-well plate cultured in 1 mL of RPMI-1640 culture medium containing 11.1 mM glucose supplemented with 10% (v/v) of fetal bovine serum (FBS) and antibiotics (100 IU mL−1 penicillin and 0.1 g L−1 streptomycin) to allow attachment overnight prior to the test. Cells were washed three times with Krebs−Ringer bicarbonate buffer (KRBB, pH 7.4) supplemented with 0.5% (w/v) bovine serum albumin (BSA) and 1.1 mM glucose and preincubated for 40 min at 37 °C. The buffer was removed, and cells were then incubated for 30 min with 1 mL of KRBB test buffer supplemented with 0.5% (w/v) BSA and 1.1 mM glucose in the presence of sample extract (0.625−5 mg/mL). Insulin concentration was measured using rat insulin ELISA kit (Mercodia AB, Uppsala, Sweden). Statistical Analysis. All results were expressed as the mean ± standard deviation (SD) for three replicates unless specified. The data were analyzed by statistical software, SPSS version 20.0 for windows (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) and Tukey’s test were performed to determine differences of means among groups. Statistical significance was set at p < 0.05.
concentration (M) = absorbance/(molar extinction × length of cuvette used)
where molar extinction was 1.56 × 105 M−1 cm−1. Inhibition of Erythrocyte Hemolysis. Erythrocyte hemolysis was performed on the basis of method of Huang et al.23 The packed RBC or erythrocytes obtained from previous analysis were diluted in PBS. The suspension of 2% erythrocytes in PBS (1 mL) was mixed with 0.5 mL of the extract. Concentrations of samples and standard were 0.05 and 0.2 mg/mL, respectively. Subsequently, 50 μL of H2O2 (100 mmol/L) was added to the mixture. The reaction mixture was incubated at 37 °C for 1 h and then diluted with 8 mL of PBS. The diluted reaction mixture was centrifuged at 1000g for 10 min. The absorbance of supernatant was measured at 415 nm. Erythrocytes were treated with 100 mmol/L H2O2 and without extract to obtain a complete hemolysis (control). The percentage of inhibition was calculated by using the following equation:
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RESULTS AND DISCUSSION Nutritional Values of Underutilized Vegetables. The nutritional compositions of the studied vegetables are presented in Table 1. These vegetables were high in moisture content (>87%). The moisture content in the studied vegetables is Table 1. Nutrient Compositions of Underutilized Vegetablesa nutrient composition
inhibition of hemolysis (%)
lalis
Macronutrients (g/100 g fw) moisture 87.97 ± 0.13b ash 1.09 ± 0.27a protein 3.86 ± 0.04a lipid 0.56 ± 0.04a total carbohydrate 1.56 ± 0.01b total dietary fiber (TDF) 4.54 ± 0.01a soluble dietary fiber (SDF) 0.48 ± 0.01a insoluble dietary fiber (IDF) 4.06 ± 0.00a Micronutrients (mg/100 g fw) calcium 47.07 ± 2.78a magnesium 30.62 ± 0.47a sodium 1.74 ± 0.10a potassium 343.03 ± 2.19a iron 0.32 ± 0.01a copper 0.18 ± 0.00a zinc 1.76 ± 0.05a phosphorus 41.18 ± 0.29a manganese 13.16 ± 0.20a
= (absorbance of H 2O2 ‐induced hemolysis − absorbance of sample /absorbance of H 2O2 ‐induced hemolysis) × 100
In Vitro Antidiabetic Properties. α-Amylase Inhibition Assay. This assay was performed according to the method of Apostolidis et al.24 Acarbose (62.5−1000 μg/mL) was used as positive control. The percentage of inhibition was calculated by using the equation inhibition (%) = (Ac − Ae)/Ac × 100
where Ac is absorbance of control and Ae is absorbance of sample extracts or standard. Control was using phosphate buffer to replace extracts. α-Glucosidase Inhibition Assay. The α-glucosidase inhibition assay was carried out according to the method of Kim et al.25 The αglucosidase inhibitory activity was expressed as percent inhibition and was calculated as inhibition (%) = (ΔAc − ΔAe)/ΔAc × 100
where ΔAc and ΔAe are the difference of absorbance before and after incubation of control and sample extracts, respectively. Acarbose (0.125−2 mg/mL) dissolved in phosphate buffer was used as a positive control.
pantu 90.70 0.36 1.67 0.33 2.43 3.32 0.22 3.10
± ± ± ± ± ± ± ±
0.07a 0.03b 0.03b 0.02b 0.00a 0.26b 0.25a 0.01b
27.75 33.57 1.62 236.94 0.22 0.22 1.08 34.96 1.63
± ± ± ± ± ± ± ± ±
4.52b 4.98a 0.83a 32.09a 0.03a 0.01a 0.15b 5.53a 0.24b
Values are the mean ± SD (n = 3), except for dietary fiber (n = 2). Values with different letters are significantly different at p < 0.05 within the same row. a
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Table 2. Antioxidant Contents and Capacities of Underutilized Vegetablesa
a
sample
TPC (mg GAE/100 g dw)
TFC (mg RE/100 g dw)
FRAP (μmol Fe2+/100 g dw)
ORAC (μM TE/100 g dw)
lalis pantu
2504.88 ± 61.15a 1959.70 ± 122.32b
6.85 ± 0.27b 10.63 ± 0.35a
226.83 ± 14.43b 396.83 ± 38.19a
159.21 ± 19.88b 203.58 ± 8.63a
Values are the mean ± SD (n = 3). Values with different letters are significantly different at p < 0.05 within the same column.
similar to those of some other Malaysian vegetables such as Etlingera elatior (Jack), Kaempferia galangal L., and Apium graveolons L.28 Additionally, comparable findings also have been demonstrated in three Caulerpa species (82−92%).29 Lalis contained significantly higher ash, protein, lipid, and dietary fiber compared to pantu, except for total carbohydrate. However, a lower carbohydrate content was found for the studied vegetables compared with the nutrient databank value for raw palm hearts (25.6 g/100 fw).30 It was observed that dietary fiber (DF) content was an abundant nutrient in both vegetables, followed by protein value in lalis and total carbohydrate in pantu. A predominant portion of dietary fiber in the vegetables was insoluble DF compared to soluble DF. It was observed that total dietary fiber (TDF) in the samples was higher than their total available carbohydrate content. This may due to a high content of total indigestible carbohydrates in these samples.18 It was noted that TDF contents of the studied vegetables exceeded the content in common Malaysian vegetables such as bamboo shoot, celery, and kantan.31 The TDF of studied vegetables was considerably higher compared to other nutrients examined in this study, with the ratio of soluble to insoluble within 8.5−14.1. The ratio was higher compared to other vegetables such as seaweed due to more fibrous tissue.32 A higher level of potassium, zinc, calcium, phosphorus, and magnesium was observed in the studied vegetables as shown in Table 2. A similar pattern was observed for calcium, magnesium, and phosphorus levels with the same vegetables studied by Voon and Kueh.33 Interestingly, higher amounts of potassium, magnesium, and phosphorus were found compared to common tomato.34 It was found that the potassium level was higher than the sodium level, as is ordinary for plant foods. The high potassium content together with low sodium had shown a protective effect toward several diseases.35 The studied vegetables could be a potential zinc source for daily intake as it contributes about 25% of zinc in the Recommended Nutrient Intake (RNI) of Malaysia.36 Antioxidant Properties of Underutilized Vegetables. The total phenolic (TPC) and flavonoid (TFC) contents of studied vegetables are depicted in Table 3. Lalis had significantly higher phenolic content (2505 mg GAE/100 g dw). In contrast, pantu exhibited a significantly higher TFC value (11 mg RE/100 g dw). The TPC values were much higher than those reported in vegetables from Indonesia (440− 1143 mg GAE/100 g dw).37 The studied vegetables showed a comparable TPC content and a lower flavonoid content compared to an ethanolic extract of Barringtonia racemosa stem.38 As total phenolic and flavonoid levels were determined, further identification and quantification of the polyphenol profile were performed using HPLC-DAD (Figure 1). To confirm the result of HPLC analysis, nine phenolic compounds were determined using LC-ESI-MS. MS data for the compounds are shown in Table 3. From the results, major compounds were detected in ESI(−) mode compared to ESI(+) mode. Seven compounds (protocatechuic acid,
Table 3. Phenolic Compounds Detected in Lalis and Pantu by HPLC-DAD and LC-ESI-MS compound protocatechuic acid catechin epigallocatechin gallate chlorogenic acid methyl gallate 4-hydroxybenzoic acid myricetin morin genistein a
tR (min)
MW
[M + H]+ (m/z)
[M − H]− (m/z)
15.4 15.9 16.1
154 290 458
nda 291.2 nd
153.1 nd 457.2
16.4 16.7 17.8 21.8 22.6 24.2
354 184 138 318 302 270
nd nd nd nd nd nd
353.2 183.2 137.0 317.2 301.0 269.1
nd, not detected above the limit of detection.
catechin, epigallocatechin gallate, chlorogenic acid, methyl gallate, 4-hydroxybenzoic acid, and myricetin) were present in pantu. Meanwhile, a total of five compounds (protocatechuic acid, chlorogenic acid, myricetin, morin, and genistein) were detected in lalis. Due to a lack of availability of standard phenolic compounds, certain peaks cannot be identified. Chlorogenic acid was the most abundant phenolic found in lalis (4.8 mg/g dw) and pantu (16.8 mg/g dw) (Table 4). This content was higher compared to chlorogenic content of commercial vegetables such as carrot, sprout, cauliflower, courgettes, and broccoli (0.01−0.44 mg/g dw).39,40 The phenolic acid compounds identified in our vegetable extracts are quite similar to those of bamboo shoots.41 The myricetin content in both of the studied vegetables was higher than the content in vegetables from Indonesia.37 Comparable protocatechuic acid was found in wild mushroom, Ramaria botrytis (0.34 mg/g dw).42 There is considerable interest as 4hydroxybenzoic acid functions as a polyester precursor in plants and protocatechuic acid as an antioxidant.43 Antioxidant activity of extracts was evaluated by measuring the ability to reduce ferric ions in ferric reducing antioxidant power (FRAP) assay. Significant FRAP values of both studied vegetables were observed, as shown in Table 3. The ORAC assay determined the scavenging capacity of vegetable extracts against a peroxyl radical. The ORAC values for lalis and pantu were 159.2 and 203.6 μM TE/100 g dw, respectively (Table 3). In inhibition of lipid membrane erythrocyte oxidation assay (Table 5), the result demonstrated that both vegetables had good protective effects on membrane erythrocyte oxidation. The malondialdehyde (MDA) formation exhibited by lalis and pantu extract is comparable with that of BHT. In addition, pantu exhibited a significantly higher inhibition on erythrocyte hemolysis as compared to standard (Table 5). The inhibition activities of both samples were higher than that of gallic acid. The FRAP value (226.8−396.8 μmol Fe2+/100 g) was comparable with values reported for pumpkin leaves and butternut (104−744 mmol/L Fe[II]/g extract).43 However, the studied vegetables showed a lower FRAP valued compared to commercial carrot (680 μmol Fe2+/100 g).44 Similar findings were found in ORAC values of some commercial vegetables 2080
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Figure 1. HPLC-DAD chromatograms of phenolic compounds in (A) lalis and (B) pantu. Detection was done at 280 nm. Peaks: (1) protocatechuic acid; (2) catechin; (3) epigallocatechin gallate; (4) chlorogenic acid; (5) methyl gallate; (6) 4-hydroxybenzoic acid; (7) myricetin; (8) morin; (9) genistein.
greater the ability the extract has to neutralize lipid peroxidation.46 Inhibitory Activities of Extracts against Digestive Enzymes. Consumption of foods that contain potential αamylase and α-glucosidase inhibitors can be dietary solutions for management of type II diabetes. α-Amylase enzyme is able to catalyze the hydrolysis of glycosidic linkages in starch. The αamylase inhibitors are considered starch blockers as they bind with the reactive sites of amylase enzyme and consequently control the blood glucose level. The studied vegetables showed high inhibition of α-amylase activity (>50%) (Figure 2a). This result was higher than that of wild legume grains (10.6− 18.5%)47 and comparable with those of finger and Proso millets (77−78%).48 α-Glucosidase is an enzyme situated in the epithelium of the small intestine and catalyzes the breakdown of disaccharides into glucose for subsequent absorption. An inhibitor of αglucosidase could delay the glucose absorption rate through the intestine. The impediment of glucose absorption may give a positive effect in controlling the postprandial blood sugar level. In contrast with α-amylase inhibition, the studied vegetables exhibited low levels of α-glucosidase inihibition activity (0.99. nd, not detected above the limit of detection of phenolic compounds.
a
Table 5. Antioxidant Capacities of Underutilized Vegetables Using Inhibition Assays on RBC Cell Damagea sample
inhibition of lipid membrane of erythrocyte oxidation (MDA, μM)
inhibition of erythrocyte hemolysis (%)
lalis pantu standard
0.67 ± 0.02a 0.77 ± 0.01a 0.65 ± 0.19α,a
48.70 ± 1.47ab 53.51 ± 1.11a 44.96 ± 0.28β,b
Values are the mean ± SD (n = 3). Values with different letters are significantly different at p < 0.05 within the same column. α, BHT; β, gallic acid. a
such as Brussels sprout, eggplant, and green beans.45 Furthermore, no significant difference of ORAC values was found among the vegetables and standard. Due to the high hydrophilic phenolic compound content in lalis (Table 4), this may contribute to hinder MDA formation. It was noted that the more polar hydrophilic compounds present in the extract, the 2081
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In conclusion, this research signifies the initial effort to evaluate the antioxidant and antidiabetic properties of these underutilized vegetables. It is revealed that they are providing valuable sources of dietary fiber, potassium, and zinc contents and exhibited potential antioxidant and antidiabetic properties. Different phenolic acids and flavonoid compounds were found in the studied vegetables such as chlorogenic acid and morin. High inhibition activity (>50%) was exhibited by all samples in α-amylase inihibition assay. Nevertheless, low activity (
