EFFECT OF SELECTED INSECTICIDE ON WHITEFLY (Bemisia tabaci) INFESTING BRINJAL PLANTS 35 BIOACTIVE COMPOUNDS AND BIOCHEMICAL AND ANTIOXIDANT PROPERTIES OF SELECTED MINOR INDIGENOUS FRUITS IN BANGLADESH Molla, M.M.a Sabuz, A.A.b Chowdhury, M.G.F.c Khan, M.H.Hd Alam, M.e Nasrin,T.A.A.f Khatun, A.g Islam, M.M.h a,b,c,d,ePostharvest Technology Division, Bangladesh Agricultural Research Institute (BARI), Bangladesh. fPostharvest Technology Section, HRC, BARI, Gazipur, Bangladesh. gInstitute of Food Science and Technology, BCSIR, Dhaka, Bangladesh. hNutrition Unit, Bangladesh Agricultural Research Council (BARC), Dhaka, Bangladesh.  mainuddinmolla@yahoo.com (Corresponding author) Article History ABSTRACT Received: 10 November 2020 Revised: 18 December 2020 Accepted: 4 January 2021 Published: 25 January 2021 Keywords Minor fruit Nutritional composition Minerals Phytochemicals compound Antioxidants properties. Minor fruits are a potential source of antinutrients, but there is no complete primary data source in the Bangladeshi context. Therefore, the present study was undertaken to acquire documentation for a database of the composition of selected minor fruits. The total phenolic (TPH), vitamin C, total carotene, and ß-carotene contents and antioxidant activity of selected minor fruits were determined by 1,1- diphenyl-2picryl hydrazyl (DPPH) scavenging and reducing power assays (RPA). Phenolic compounds were assessed using high- performance liquid chromatography coupled with a photodiode array detector and autosampler. Results revealed that minor fruits contain different phytochemicals, particularly TPH, ascorbic acid, total flavonoid (TF), ß-carotene, total carotenoid (TC), and total anthocyanin content (TAC); values ranged, respectively, 0.23-176.50 mg GAE/g, 16.67-664.92 mg/100 g, 2.26-150.02 mg QE/100 g, 1.41- 6897.57 µg/100 g, 1.26-98.24 mg/100 g and 1.15-47.46 mg/100 g. In the parameters antioxidant activity, total antioxidant capacity, DPPH, reducing power capacity (RPC), ferric reducing antioxidant power (FRAP), metal chelating capacity (MCC), nitric oxide (NO), and free radical scavenging activity, IC50 ranged 0.01-278.24 µg of ascorbic acid/mg of extract, 39.70-250.00%, 3.21-634.00%, 0.02-1817.88 µM Fe2SO4/100g, 22.29-210.43%, 0.02-70.50%, and 4.98-856.70 µg/g, respectively. Among the identified and quantified phenolic acids, leading examples were gallic acid (279.06 mg/100 g), vanilic acid (43.77 mg/100 g), Þ-courmaric acid (178.96 mg/100 g), ferulic acid (20.44 mg/100 g), and lutein (91.13 µg/100 g) in aonla, day fruit, elephant apple, and bilimbi. Moreover, all selected minor fruits are rich sources of bioactive, biochemical, and antioxidant compounds with potential for use in therapeutic applications. Contribution/Originality: The study contributes the first logical analysis of selected minor indigenous fruits grown in Bangladesh in terms of phytochemical and antioxidant activities. The nutritional, phytochemical, and antioxidant profiles of these fruits may encourage their consumption rather than that of major and exotic fruits. Pharmaceutical researchers may apply this phytochemical and antioxidant profile for pharmaceutical research purposes. Asian Journal of Agriculture and Rural Development Volume 11, Issue 1 (2021): 35-46 http://www.aessweb.com/journals/5005 https://orcid.org/0000-0002-3851-7094 https://orcid.org/0000-0002-9266-7093 https://orcid.org/0000-0003-1105-0049 https://orcid.org/0000-0001-8507-3054 https://orcid.org/0000-0003-2173-7143 https://orcid.org/0000-0003-0473-6632 https://orcid.org/0000-0002-4156-3016 https://orcid.org/0000-0003-1089-766X mailto:mainuddinmolla@yahoo.com http://www.aessweb.com/journals/5005 Asian Journal of Agriculture and Rural Development, 11(1)2021: 35-46 36 DOI: 10.18488/journal.ajard.2021.111.35.46 ISSN(P): 2304-1455/ ISSN(E): 2224-4433 How to cite: Molla, M.M. -- Sabuz, A.A. --- Chowdhury, M.G.F. --- Khan, M.H.H --- Alam, M. --- Nasrin, T.A.A. --- Khatun, A. --- Islam, M.M. (2021). Bioactive Compounds and Biochemical and Antioxidant Properties of Selected Minor Indigenous Fruits in Bangladesh. Asian Journal of Agriculture and Rural Development, 11(1), 35-46. 10.18488/journal.ajard.2021.111.35.46 © 2021 Asian Economic and Social Society. All rights reserved. 1. INTRODUCTION Bangladesh is blessed with a great diversity of fruits and, in 2017, was placed sixth in world rankings for tropical fresh fruit production (FAOSTAT, 2019). A significant quantity of tropical fruits are underexploited – generally recognized as indigenous or minor fruits. These fruits are not commercially cultivated and are not frequently found in national or international markets. Usually these fruits are found in home yards, unused high lands, hill tracts, and roadsides. No extra care and farming management are needed to grow these fruits, and that is why people describe these as underutilized minor indigenous fruits in Bangladesh; year on year, these fruit trees are being destroyed. However, these minor fruits can be a good source of micronutrients and phytochemicals. Micronutrients are essential elements in the development of physical growth and help prevent various acute and chronic diseases. Low dietary intake of micronutrient-rich foods, as well as low absorption and lower bioavailability, are the leading causes of micronutrient deficienciy. Phytochemicals with antioxidant capacity naturally present in fruits are of great interest due to their beneficial effects on human health, while regular consumption is associated with reduced risk of developing chronic diseases because they offer protection against oxidative deterioration (Paul et al., 2007). Nowadays, antioxidants are also considered as important as vitamins in the promotion of health and prevention of various diseases linked to reactive oxygen species (ROS), which have been linked to over 100 disorders (Halliwell & Gutteridge, 2000). Excess generation of ROS causes oxidative stress that damages cellular DNA, lipids, and proteins, leading to the pathogenesis of various diseases including cerebrovascular diseases. Among various antioxidants and phytochemicals, flavonoids, anthocyanin, phenolic acids, and other compounds are linked with natural protective agents, astringents, antibiotics, positive health effects, and antimicrobial properties (Osorio-Esquivel, Álvarez, Dorantes-Álvarez, & Giusti, 2011). In recent years, interest in antioxidant-rich fruits and their products has been growing in both domestic and international markets because of increasing appreciation of their role in the protection of human health. This has occurred because of their nutritional and antioxidant properties, and also because of the prospects offered to the agricultural and pharmaceutical industries and a promising future source of income for local inhabitants. A nutrition database is of great importance in addressing the nutritional health benefits of both rural and urban communities. It is essential for the formulation of a national policy on food to have a database on nutritional composition. However, little information in nutrition databases is available for minor fruits. Several studies have been performed on a few fruits only, but these are indiscriminate and have not been scientifically documented or aimed at the consumer. Therefore, documentation, conservation, and revalorization of indigenous knowledge on minor fruits is urgently needed to promote nutritional health for both rural and urban inhabitants. With that in mind, the present research waas undertaken to analyze, document, and popularize the role of phytochemical content in highlighting the phenols, flavonoids, and carotenoids present in selected minor fruits in Bangladesh. 2. MATERIALS AND METHODS 2.1. Materials Nine minor indigenous fruits were selected based on peoples’ choice and production catchments in Bangladesh, and are shown in Table 1. Analytical grade chemicals and reagents used in this study were procured from Sigma- Aldrich (Steinheim, Germany). Table 1. Selected minor indigenous fruits grown in Bangladesh. Sl. no. Bengali name English name Scientific name Family 1 Aamlaki Aonla Phyllunththus embelica L. Phyllanthaceae 2 Amra Hog plum Spondias mombin L. Anacardiaceae 3 Bilimbi Cucumber tree Averrohoa bilimbi L. Oxalidaceae 4 Chulta Elephant apple Dillenia india L. Dilleniaceae 5 Day fall Day fruit – – 6 Jara lebu Citron Citrus medica Rutaceae 7 Satkara Melanesian papeda Citrus macroptera Rutaceae 8 Sour kul Ber Ziziphus mauritania Rhammaceace 9 Toikar Toikar Garcinia pedunculata Clusiaceae Source: Project Completion Report, Competitive Research Grant (ID: 328), Bangladesh Agricultural Research Council (BARC), Farmgate, Dhaka-1215, Bangladesh. http://crossmark.crossref.org/dialog/?doi=10.18488/journal.ajard.2021.111.35.46 Asian Journal of Agriculture and Rural Development, 11(1)2021: 35-46 37 2.2. Methods 2.2.1. Sample Collection and Fruit Extraction Fruits were collected from the Regional Agricultural Research Station (RARS), Bangladesh Agricultural Research Institute (BARI), Akbarpur, Moulvibazar, Bangladesh. After collection, the fruits were washed with potable water and surface dried. The fruits were then freeze-dried and milled to powder using a laboratory grinder. Fruit powder of known quantity was extracted in a thermostatic water bath (Vision Scientific Co. Ltd.) at 60oC for 1 h using methanol (80%, v/v) maintaining a fruit:solvent ratio of 1:20 (w/v). The fruit extract was filtered under vacuum, centrifuged at 4000×g for 10 min and the supernatant was collected and kept at -18oC until used for analysis. 2.2.2. Determination of Physicochemical Parameters Physicochemical properties – moisture, protein, ash, total soluble solids (TSS), pH, and titrable acidity – were determined following the method of AOAC (2005). Ascorbic acid, starch, and total sugar content were determined based on the procedure of Ranganna (1995). Edible and non-edible portions of the fruits were measured using the gravimetric method. 2.2.3. Analysis of Minerals Minerals analyzed included sodium, potassium, calcium, magnesium, phosphorus, sulphur, boron, copper, manganese, iron, and zinc. Before quantification, fruits were first wet ashed and then digested in nitric and perchloric acid solution at 320oC, cooled, diluted to an appropriate concentration, and filtered. This filtrate was then used as the stock solution for further analysis. Atomic absorption spectrophotometry (Model-AA-7000S, Shimadzu, Tokyo, Japan) was used to assess levels of sodium, iron, copper, zinc, boron, manganese, calcium, and magnesium. Potassium content was measured using flame photometry, while phosphorous and sulphur were assessed by spectrophotometry. Individual minerals were quantified by comparison to the corresponding standard mineral procured from Sigma Chemical Co., USA. 2.2.4. Determination of Phytochemicals 2.2.4.1. Total Phenolic Content Twenty milligrams (0.02 g) of powder was dissolved in 1 ml of methanol to prepare a stock solution for experiments. A volume of 0.5 ml of the each extract (100 µg/ml) was mixed with 2 ml of Folin–Ciocalteu reagent (diluted 1:10 with deionized water) and neutralized with 4 ml of sodium carbonate solution (7.5%, w/v). The reaction mixture was incubated at room temperature for 30 min with intermittent shaking for color development. Absorbance of the colored solution was measured at 765 nm using double-beam UV-VIS spectrophotometry. Total phenolic content was determined from the linear equation of a standard curve prepared with gallic acid. Determination of total phenolic content in the extracts was determined according to the Folin–Ciocalteu method (Ough & Amerine, 1988), with gallic acid (GAE) as the standard and expressed (mg) as gallic acid equivalents (GAE)/g of extract (Aoshima, Hirata, & Ayabe, 2007). 2.2.4.2. Determination of Total Flavonoid Content Total flavonoid content (TFC) of the fruit powder was determined by the aluminium chloride method (Chang, Yang, Wen, & Chern, 2002) with slight modifications: 0.5 ml of sample solution was mixed with 1.5 ml of methanol. To this mixture 0.1 ml of 10% aluminium chloride and 0.1 ml of 1 M potassium acetate were added. The final volume was made up to 5 ml by adding 2.8 ml of distilled water, and the reaction was left for 30 min at room temperature. Absorbance of the solution was measured at 415 nm and expressed as mg QE/g extract. TFC was calculated from the calibration curve of quercetin plotted (Figure 1). The curve obtained was found to be linear with equation y = 0.0035 + 0.0056x, and the correlation coefficient was found to be R2 = 0.9993. TFC was expressed as mg quercetin equivalents per gram of extract (mg QE/g extract). Figure-1. Calibration curve of quercetin plotted. Asian Journal of Agriculture and Rural Development, 11(1)2021: 35-46 38 2.2.4.3. Determination of Total Anthocyanin (TA) The method was adapted from Burgos et al. (2013), the concentration of TA being calculated using the molar extinction coefficient and molecular weight of malvidin-3-p-coumaroyl-glucoside for blue-violet pigments (545 nm, 3.02×104 l/mol/cm, 718.5 g/mol), pelargonidin-3-glucoside for red pigments (515 nm, 2.73×104 l/mol/cm, 486.5 g/mol), and cyanidin-3-glucoside for purple pigments (535 nm, 3.43×104 l/mol/cm, 449.2 g/mol). Results were expressed as mg/100 g DW. 2.2.4.4. Determination of Total Carotenoid Content Total carotenoid was determined according to the method described by Thaipong, Boonprakob, Crosby, Cisneros- Zevallos, and Byrne (2006). Absorbance was measured at λ 470 nm. Each extract was dissolved in n-hexane pro- analysis. ß-carotene solution in various concentrations was used as standard carotenoid compound and the standard curve. A linear regression equation of the standard curve was used for calculating total carotenoid content, and expressed as beta-carotene equivalents per 100 g of extract (mg/100 g). 2.2.4.5. Determination of ß-Carotene Content β-Carotene content was determined using the procedure of Holden et al. (1999), with values expressed as µg/100g of fruit extract. 2.2.5. Determination of Antioxidant Activity 2.2.5.1. Total Atioxidant Activity Total antioxidant activity was assessed using the phosphomolybdenum system according to the technique described by Prieto, Pineda, and Aguilar (1999), and results expressed as micrograms ascorbic acid (AA) per gram (µg/ml) of the sample. 2.2.5.2 Reducing Power Assay The reducing power of the fruit extract was assessed using the approach of Guo, Saravanakumar, and Wang (2018). Ascorbic acid was used as the standard for the preparation of the calibration curve. 2.2.5.3. FRAP FRAP activity was measured following the scheme outlined by Benzie and Strain (1996). A standard curve was made using ferrous sulphate aqueous solution (1–10 mM) and FRAP values were expressed as μM Fe (II)/100g of the sample. 2.2.5.4. DPPH Radical Scavenging Activity (DPPH-RSA) and IC50 DPPH-RSA was evaluated by measuring the inhibition rate following the procedure described by Brand-Williams, Cuvelier, and Berset (1995), with modification. Exactly 0.1 ml of extract was placed in a Falcon tube and 1.4 ml of methanolic solution of DPPH added. The mix was left to rest for 30 min in the dark and absorbance at 517 nm was measured against a blank. The results are expressed as percentage radical scavenging activity: DPPH radical scavenging activity (%) = AO − As Ao × 100 where Ao is absorbance of blank and As is absorbance of sample extract. The inhibition curves were then prepared and IC50 values calculated (Brand-Williams et al., 1995). BHT was the positive control. 2.2.5.5. MCC MCC was determined based on Bahadori, Zengin, Bahadori, Dinparast, and Movahhedin (2018), with minor modification. Briefly, 2 ml of extract was placed in a glass tube to which 0.05 ml of ferrous chloride (2 mM), 3.7 ml of distilled water, and 0.2 ml of ferrozine (5 mM) were added. After 20 min of incubation under atmospheric conditions, absorbance was read at 562 nm against a blank. The following formula was applied to calculate MCC: Metal chelating capacity (%) = Absorbance (control) − Absorbance (sample) Absorbance (control) × 100 2.2.5.6. Nitric Oxide (NO) Radical Scavenging Activity Nitric oxide radical quenching activity was determined according to the procedure of Bogucka-Kocka, Zidorn, Kasprzycka, Szymczak, and Szewczyk (2018), with some modification. In a glass tube, 0.5 ml of extract was mixed with 2 ml of sodium nitroprusside (10 mM). This was followed by incubation under atmospheric conditions for 1 h. Thereafter, 0.5 ml of the incubated mixture was transferred to another centrifuge tube, Griess reagent (0.5 ml) was added, and the mixture rested for 30 min. Absorbance of the solution was read at 540 nm against a blank. The following formula was employed to calculate the result, expressed as percentage inhibition: Inhibition (%) = Absorbance (control) − Absorbance(sample) Absorbance (control) × 100 2.2.6. Assessment of Phenolic Acids by HPLC Phenolic compounds were assessed according to the method of Pandey and Negi (2018), with some adjustment, using high-performance liquid chromatography (HPLC; Shimadzu SPD-M10A) coupled with a photodiode array Asian Journal of Agriculture and Rural Development, 11(1)2021: 35-46 39 detector and autosampler at 280 and 320 nm. Separation was achieved using a C18 column (250 × 4.6 mm2) of 5 µm particle size at room temperature. The mobile phase was 1% acetic acid (A) and 80% acetonitrile in A (B). The following gradient was used: 0.01–35 min, 0% of B; 35–40 min, 50% of B; 40–45 min, 100% of B; and 45–60 min, 0% of B. The flow rate was 1 ml/min and the injection capacity was 20 µl. Six phenolic standards (Þ-courmaric acid, gallic acid, vanilic acid, caffeic acid, ferulic acid, and lutein) were used for identification of respective phenolics, and quantification was accomplished using a standard curve. 3. STATISTICAL ANALYSIS Data obtained for each analysis were expressed in duplicate as means ± standard deviation. Data were analyzed by one-way ANOVA with post hoc using Tukey’s multiple comparisons test. Significance was defined at the 95% confidence level. Statistical analysis and data processing were performed using the software SPSS 17.0 (IBM Inc., New York). 4. RESULTS AND DISCUSSION 4.1. Biochemical Properties 4.1.1. Crude Protein Analysis of crude protein content of selected minor fruits is shown in Table 2. It will be seen that in all fruits this is statistically significant, with levels of crude protein ranging from 2.39 to 4.10% (Table 2). The highest levels of protein were found in citron (4.10%) and satkara (4.06%). Total protein obtained from the Bangladeshi commercial mango fruits amrapali, chausa, fazlee, gopalbhog, himsagor, and langra was found to be 0.14, 0.26, 0.21, 0.07, 1.11, and 1.18 g/100 g, respectively (Ara, Motalab, Uddin, Fakhruddin, & Saha, 2014). The crude protein content of Indian minor fruits Aegle marmalose Correa, Cordia myxa L., Zizipus mauritiana Lam, Averrhoa bilimbi L., and Grewia asiatica Mast weas recorded as 2.2, 1.9, 1.04, 1.3, and 1.09 g/100 g, respectively (Mitra, Pathak, & Chakraborty, 2008). The crude protein content found in our minor indigenous fruits was higher than that found by both Mitra et al. (2008) and Ara et al. (2014). 4.1.2. Ash The ash content of the nine selected minor fruits was statistically significant, ranging from 1.40 to 3.78%, the maximum (3.78%) being recorded in citron (Table 2). The highest ash content found in the present study is comparable to the findings of Shukla, Dubey, Jain, and Kumar (2001), who reported that Adansonia digitata contain 3.31% ash. 4.1.3. Total Sugar Total sugar content of the nine selected minor fruit ranged from 2.83 to 5.64%. All values were statistically highly significant, with the maximum content recorded for hog plum (5.64 %) and the minimum for aonla (2.83%) (Table 2). Values for blilimbi, elephant apple, day fruit, citron, satkara, ber and toikar were 4.07, 4.13, 4.20, 4.27, 3.91, 4.35, and 4.02%, respectively. The results obtained from our study are supported by the findings of Ara et al. (2014), who reported that the total sugar content of commercial mango varieties ranged from 4.27 to 4.83%. 4.2. Starch Starch is a major carbohydrate source of immense economic and nutritional value. It is essential that the food industry search for new starch sources to meet the requirements of both the food processing industry and consumers. Table 2 shows selected minor fruits containing higher starch levels and all statistically highly significant differences. The highest starch content (8.70%) was recorded for day fruit and the lowest for satkara (3.44%). The high total sugar content in day fruit may be due to enzymatic hydrolysis of starch to sugar (Nelson, 1944). The lower yield of starch in satkara is probably due to its increased respiration directly by genetical induced. 4.3. Moisture The moisture content of the selected minor fruits varied. The data show that the moisture content of the selected minor fruits was statistically significant (P < 0.05), ranging from 76.62 to 80.73% – maximum in citron (80.73%) and minimum in aonla (76.62%) (Table 2). The results obtained from the study are comparable to those of Parveen and Khatkar (2015able 3), who reported aonla (variety Desi) with 81.26% moisture content. 4.4. Acidity Acidity varied highly significantly among the selected minor fruits, ranging from 0.79 to 3.58% – the maximum was 3.58% in day fruit and the minimum in citron (Table 2). The results obtained from this study confirm that the acidity of the selected minor fruits is much higher than that found by Akhter et al. (2010) for langra (0.68%) and chausa (0.63%) commercial mango cultivars in Bangladesh. The reason for this discrepancy may be that most minor fruits are citrus fruits with variation in maturity stage, harvesting time, soil management, and cultural practices. Asian Journal of Agriculture and Rural Development, 11(1)2021: 35-46 40 Table 2. Biochemical and mineral composition of selected minor fruits. Parameter Minor fruits Aonla Hog plum Bilimbi Elephant apple Day fruit Citron Satkara Ber (sour) Toikar Crude protein (%) 3.71± 0.03ab 3.80± 0.10ab 3.70± 0.20ab 3.26± 0.15bc 3.04± 0.01 4.10± 0.10a 4.06± 0.02a 3.18± 0.09bc 2.39± 0.57d Ash (%) 1.92± 0.01f 2.23± 0.02e 3.33± 0.01bcd 3.45± 0.02bc 1.40± 0.10g 3.78± 0.02a 3.31± 0.10cd 3.21± 0.10d 3.49± 0.02b Total sugars (%) 2.83± 0.01d 5.64± 0.03a 4.07± 0.06bc 4.13± 0.04bc 4.20± 0.10bc 4.27± 0.03bc 3.91± 0.06c 4.35± 0.40b 4.02± 0.02bc Starch (%) 5.51± 0.27b 4.45± 0.08cd 4.13± 0.02de 4.71± 0.03cd 8.70± 0.20a 3.69± 0.58ef 3.44± 0.03f 4.15± 0.02de 4.85± 0.07c Moisture (%) 76.62± 0.10g 78.44± 0.11ef 77.77± 0.10f 79.02± 0.13def 79.49± 0.54bcd 80.73± 0.25a 80.33± 0.09ab 78.61± 0.48def 79.82± 0.57abc Acidity (%) 1.82± 0.02de 2.29± 0.02b 1.84± 0.02cde 1.92± 0.03cd 3.58± 0.03a 0.79± 0.02f 1.78± 0.10e 1.95± 0.05c 2.40± 0.02b pH 2.65± 0.02e 2.94± 0.00cd 2.14± 0.02f 3.29± 0.02b 2.63± 0.10e 3.53± 0.03a 3.58± 0.09a 2.88± 0.04d 3.09± 0.02c TSS (oB) 11.40± 0.02a 10.91± 0.01b 10.18± 0.02c 9.17± 0.00d 7.01± 0.01g 7.54± 0.25f 8.50± 0.10e 10.44± 0.05c 9.20± 0.02d Edible (%) 84.62± 0.02d 47.59± 0.3f 98.00± 1.00a 79.03± 0.02e 90.95± 0.03b 79.77± 1.59e 44.70± 0.20g 88.37± 0.31c 84.01± 0.01d Non-edible (%) 15.38± 0.02 50.50± 0.16a 2.00± 1.00f 18.02± 1.01c 8.01± 0.01e 20.05± 1.20b 50.01± 0.09a 9.30± 0.09e 15.33± 0.03d Minerals Ca 0.25± 0.00e 0.27± 0.00e 0.58± 0.00cd 0.54± 0.00d 0.67± 0.02c 0.82± 0.05b 0.55± 0.00d 1.02± 0.05a 0.87± 0.06b Mg 0.15± 0.01e 0.14± 0.00e 0.31± 0.00d 0.28± 0.00d 0.29± 0.02d 0.37± 0.02c 0.29± 0.00d 0.57± 0.03a 0.49± 0.03b K 0.93± 0.00cd 0.93± 0.00cd 1.07± 0.01ab 1.08± 0.00a 0.40± 0.02f 0.64± 0.04e 0.99± 0.00bc 0.87± 0.03d 0.37± 0.04f Na 0.32± 0.01a 0.16± 0.00de 0.18± 0.01cd 0.19± 0.00c 0.12± 0.00f 0.08± 0.01g 0.15± 0.00e 0.22± 0.00b 0.18± 0.00cd P 0.42± 0.00a 0.42± 0.00a 0.11± 0.00ef 0.31± 0.01c 0.07± 0.01g 0.10± 0.00f 0.34± 0.00b 0.12± 0.00e 0.21± 0.00d S 0.07± 0.00b 0.07± 0.00b 0.06± 0.00b 0.05± 0.01b 0.02± 0.00c 0.02± 0.00c 0.06± 0.00b 0.36± 0.00a 0.36± 0.00a B 68.04± 2.16a 4.82± 0.02de 14.31± 0.02c 26.30± 0.14b 13.40± 3.51c 8.03± 0.30d 1.36± 1.01e 8.60± 0.26d 6.38± 0.40d Cu 4.82± 0.02h 128.30± 0.10c 107.53± 0.97e 157.77± 0.24b 11.67± 0.30g 114.22± 5.02d 62.61± 0.02f 155.78± 1.31b 395.52± 1.77a Asian Journal of Agriculture and Rural Development, 11(1)2021: 35-46 41 Fe 90.12± 0.02d 68.32± 0.06e 57.48± 0.21f 83.91± 0.05d 180.99± 5.86b 73.47± 2.98e 35.30± 0.03g 155.78± 1.31c 238.26± 1.07a Mn 47.92± 0.01e 27.52± 0.01f 81.78± 0.03d 151.19± 0.99a 116.68± 6.38b 16.06± 0.60gh 11.22± 0.03h 93.84± 0.78c 19.99± 0.99g Zn 18.84± 0.02ab 13.16± 0.15cd 20.00± 2.00ab 17.84± 0.28bc 23.34± 0.60a 11.20± 4.42d 10.14± 0.01d 10.73± 0.50d 22.33± 0.57ab Note: Values are mean ± standard deviation (n = 3); Ca, Mg, K, Na, P, and S expressed as mg%; B, Cu, Fe, Mn, and Zn expressed as ppm. All values are means of triplicate determinations ± SD. Means within columns with different letters a, b, c, d, e, f, g, indicate significant result (p˂0.05). Table 3. Phytochemical and antioxidant properties of selected minor indigenous fruits. Parameter Minor fruits Aonla Hog plum Bilimbi Elephant apple Day fruit Citron Satkara Ber (sour) Toikar Phytochemical propertrties TPH (mg GAE/g) 2.10± 0.09 0.23± 0.02e 105.92± 0.14b 62.85± 0.05c 176.50± 2.42a 50.00± 0.08d 1.58± 0.08e 2.04± 0.06e 1.81± 0.07e TF(mg QE/g) 45.04± 2.47b 2.84± 0.11d 150.02± 1.17a 26.16± 1.96c 27.00± 3.04c 27.85± 1.05c 23.38± 1.25c 2.26± 0.09d 18.33± 1.12 Total ß-carotene(µg/100 g) 6897.57 ±0.09a 1737.11 ±3.27d 39.33± 1.52f 41.95± 0.05f 1.41± 0.04g 9.58± 0.09g 2318.44± 5.60b 549.12± 6.42e 1815.88± 3.62c TC (mg/100 g) 83.73± 0.39c 68.45± 0.12e 4.70± 0.30f 1.58± 0.09g 1.26± 0.23g 2.07± 0.11g 93.91± 0.80b 74.89± 1.12d 98.24± 0.09a TA (mg/100 g) 15.53± 0.03b 5.11± 0.01d 47.46± 0.22a 1.74± 0.23e 1.15± 0.14f 1.19± 0.03f 6.25± 0.04c 1.66± 0.01e 6.24± 0.01c AA (mg/100 g) 664.92± 1.00a 67.90± 0.05e 28.41± 0.09 40.29± 0.11f 16.67± 0.53i 24.28± 0.61h 210.43± 0.02b 160.12± 0.11c 142.81± 0.99d Antioxidant activity TAC (µg AA/g) 0.02± 0.00f 0.01± 0.00f 108.06± 0.45c 85.90± 0.03d 122.32± 0.01b 278.24± 0.03a 0.01± 0.00f 0.02± 0.00f 1.14± 0.02e DPPH (%) 250.00± 10.00b 314.00± 5.00a 73.27± 0.25d 68.40± 4.60d 87.70± 0.49c 70.20± 0.19d 39.70± 0.20e 41.66± 0.76e 45.00± 5.00e RPA (µg AA/g) 490.50± 0.50b 634.00± 1.00a 18.71± 0.01e 5.89± 0.02f 3.21± 0.01f 3.85± 0.02f 93.00± 2.00d 105.66± 12.09d 130.00± 5.00c FRAP (µM Fe2SO4/100 g) 38.01± 9.99e 4.87± 0.10f 1817.88± 3.62b 2325± 6.09a 1213.46± 0.47d 1247.50± 3.25c 0.35± 0.05f 1.86± 0.20f 0.02± 0.00f MCC (%) 33.89± 0.02h 182.39± 0.06c 192.97± 1.01b 22.29± 0.09i 97.56± 0.10d 210.43± 0.02a 40.76± 0.09g 72.00± 1.00e 59.51± 0.01f NO (%) 7.97± 0.10e 0.01± 0.00g 70.50± 1.00b 40.18± 0.03d 41.65± 0.02c 178.87± 0.02a 1.09± 0.06f 0.02± 0.00g 1.27± 0.06f IC50 (µg/g) 255.20± 0.20d 856.70± 0.03a 30.36± 1.32g 4.98± 0.24i 49.57± 0.50f 11.26± 0.26h 362.00± 0.50c 242.78± 0.03e 504.00± 1.00b Note: All values are means of triplicate determinations ± SD. Means within columns with different letters a, b, c, d, e, f, g,h, i indicate significant result (p˂0.05). Asian Journal of Agriculture and Rural Development, 11(1)2021: 35-46 42 4.5. pH pH levels of the selected minor fruits are shown in Table 2; these ranged from 2.14 to 3.58. pH levels were highly significant, with maxima of 3.53 and 3.58 for citron and satkara, respectively, and a minimum of 2.14 for bilimbi (Averrhoa bilimbi L.). pH levels showed an inverse relationship to acidity, with a minimum of 0.79% for citron but a maximum of 3.53. A similar observation was also made by Ara et al. (2014) for major mango fruit commercial cultivars. Variation in pH among selected minor fruits may be due to variation in their internal metabolism and ripening behavior. 4.6. TSS TSS include carbohydrates, organic acids, proteins, fats, and minerals. TSS denotes a fresh weight of 10–20%, and increases with fruit maturity in regard to the production of a less acidic and sweeter fruit. In our study, TSS of the selected fruits varied significantly among species with a maximum of 11.40°B for aonla and a minimum for day fruit (7.01°B). The results obtained from our study confirmed that there was an inverse relationship between TSS and acidity. The acidity of 1.82% in aonla was influenced by TSS (11.40°B) (Table 2). Likewise, another relationship was found between TSS and fruit maturity, with increase in TSS following fruit maturity (no maturity data are shown here). For example, in the initial stage, the taste of aonla appears to be astringent but, when fully matured, it becomes sweeter with increasing TSS. 4.7. Edible Percentage Edible percentage differed significantly among the selected fruit cultivars, as shown in Table 2. The highest edible percentage was calculated at 98.0 in bilimbi (Avverhoa bilimbi L.); all of the fruit, except the pericarp, is used for consumption. The lowest edible percentage was recorded at 44.7 for satakara, due to removal of peel during preprocessing for consumption. However, the edible percentage of the selected minor fruits ranged from 44.7 to 98.0, being much higher than in major fruits (Ara et al., 2014). 4.8. Non-Edible Percentage The non-edible percentage of selected minor fruits ranged from 2.0 to 50.5. The lowest (2.00%) non-edible percentage was found in bilimbi while the highest recorded were 50.5 and 50.0, in hog plum and satkara, resoectively, due to their natural fruit structure and peel, because most of the peel goes to waste during preprocessing. 4.9. Mineral Profiling Minerals are the inorganic components present in foodstuff as ash when food is burned. Generally macro- and microminerals are present in foodstuffs and play important metabolic roles in body functions (Reilly, 2002), and contribute to our daily diet. In our study, 11 minerals were assessed from the selected minor fruits (Table 2). It will be seen that these contained a significant range of macrominerals including sodium (0.08-0.32 mg%), potassium (0.37– 1.08 mg%), calcium (0.25–1.02 mg%), magnesium (0.14–0.57 mg%), and phosphorus (0.07–0.42 mg%); and considerable amounts of microminerals including sulphur (0.02–0.36 mg%), boron (1.36–68.04 ppm), copper (4.82–395.52 ppm), iron (35.30–238.26 ppm), manganese (11.22–151.19 ppm), and zinc (10.14–23.34 ppm). Our study indicates that sour ber is a rich source of Ca, Mg, and S; aonla is a rich source of Na and P, hog plum of P, and elephant apple of K and Mn. Toikar is a rich source of Cu and Fe, and day fruit of Zn. In comparison with Indian minor fruits, our selected minor fruits possessed lower amounts of various minerals. It is claimed in different studies that soil fertility, genetic factors, fertilizers, and geographical conditions play significant roles in the mineral content of fruits (Tiburski, Rosenthal, Deliza, de Oliveira Godoy, & Pacheco, 2011). 4.10. Phytochemical properties Phytochemical components reputedly have the ability to lower the prevalence of various degenerative diseases such as cancer, heart attack, and cardiovascular disease, by blocking the activity of free radicals. The results recorded for phytochemical analyses of total phenolics, flavonoids, carotenoids, β-carotene, ascorbic acid, and anthocyanin are shown in Table 3. 4.10.1. Total Phenolic Content Total phenolic content of the selected minor fruits ranged from 0.23 to 176.50 mg GAE/g (Table 3). The maximum was recorded for day fruit (176.50 mg GAE/g) and the minimum in hog plum (0.23 mg GAE/g). Guava, pineapple, soursop, passion, acerola camarinha, and mango recorded a polyphenol content of 83.1, 21.7, 84.3, 20.2, 580.1, 492.8, and 260.21 mg GAE/100g, respectively (Kuskoski, García Asuero, Morales Millán, & Fett, 2006; Tiburski et al., 2011). 4.10.2. TFC Flavonoids are regarded as a low-molecular-weight substances in foodstuffs and boost antioxidant activity. Their content depends on the levels of polyphenols and geographical location. The value of total flavonoids found in the minor fruits studied ranged from 2.26 to 150.02 mg QE/g (Table 3). In our study, bilimbi ranked highest for flavonoid content (150.0259 mg QE/g) while the minimum was recorded as 2.26 mg QE/g in ber. This variation is somewhat similar to previous reports for exotic fruits: Osorio-Esquivel et al. (2011) found a TFC of 13.45–68.79 mg/100g in Opuntia joconostle. Since these fruits possessed a fair amount of flavonoids like other exotic fruits, its consumption would help to contribute to add antioxidants to our daily diet. Asian Journal of Agriculture and Rural Development, 11(1)2021: 35-46 43 4.10.3. Total Carotenoid Content It is evidenced from different studies that carotenoid plays a crucial role in human nutrition and health, and can lessen the risks of cancer and heart disease because of the activity of provitamin A (Tiburski et al., 2011). In this study, total carotenoid content of the selected fruits ranged from 1.26 to 98.24 mg/100g (Table 3). The total carotenoids contained in the selected fruit are comparable to reports published elsewhere: Shajib et al. (2013) reported carotenoid levels of 149 µg/100 g for wood apple, 161 µg/100 g for orboroi, and 218 µg/100 g for Burmese grape. 4.10.4. Total ß-Carotene Content Total ß-carotene content of the selected minor fruits ranged from 1.41 to 6897.57 µg/100 g. The lowest ß-carotene content was calculated as 1.41 µg/100 g in day fruit and the maximum in aonla (6897.57 µg/100 g) (Table 3). Comparatively higher values for β-carotene were reported by Tiburski et al. (2011) for yellow mombin pulp (314 µg/100 g). It is evidenced that carotenoid content in fruits depends on various factors including soil conditions, maturity stage, enzymes, phenolic content, and genetics (Leong & Oey, 2012). 4.10.5. Ascorbic Acid Content Ascorbic acid is an important bioactive compound and is considered the most powerful antioxidant in foodstuffs; regular intake lowers the risk of cancer in the human body (Almeida et al., 2011). In the selected fruits it ranged from 16.67 to 664.90 mg/100 g, with a maximum in aonla (664.90 mg/100 g) and a minimum in day fruit (16.67 mg/100 g) on a fresh weight basis (Table 3). These levels are much higher than in the report of Shajib et al. (2013) for monkey jack (14 mg/100 g), Burmese grape (12.1 mg/100 g), orboroi (20.8 mg/100 g), karanda (9.5 mg/100 g), mangosteem (14.4 mg/100 g), blackberry (25.7 mg/100 g), flacourtia (25.6 mg/100 g), rochelle (3.7 mg/100 g), and takituki (27.8 mg/100 g). According to Jukes (1974), the recommended daily intake of vitamin C (ascorbic acid) required to prevent scurvy in adults is about 10 mg, which indicates that the current study found a level of ascorbic acid in selected fruits exceeding that necessary to prevent scurvy, with a daily intake level of 100 g. 4.10.6. Anthocyanin Content One of the key bioactive compounds in foodstuffs is anthocyanin, which shows potent antioxidant capacity. The anthocyanin content of our selected minor fruits ranged from 1.15 to 47.46 mg/100 g (Table 3). The maximum content noted was 47.46 mg/100 g in bilimbi (Averrhoa bilimbi L.) and the minimum in day fruit (1.15 mg/100 g). Moreover, comparatively similar results were found for some tropical fruits (red grapes, 27 mg/100 g; cherries, 112 mg/100g; strawberries, 21 mg/100g; and red raspberries, 92 mg/100g) (Wu et al., 2006). 4.11. Evaluation of Antioxidant Activity Foodstuffs with high antioxidant properties play a crucial role in the inhibition of ROS-mediated diseases (Dutta & Ray, 2020). 4.11.1. Total Antioxidant Capacity In this investigation, the antioxidant capacity of the selected minor fruits was assessed by an assortment of different tests. It can be seen that all fruits exhibited potent antioxidant capacity, ranging from 0.01 to 278.24 µg AA/g (Table 3). It is interesting that citron possessed a higher amount of antioxidant capacity (278.24 µg AA/g) than other fruits. 4.11.2. DPPH Radical Scavenging Activity DPPH is a relatively stable free radical scavenger that donates hydrogen protons to unpaired electrons to convert them into paired ones. It is noted that DPPH radical scavenging activity of the selected minor fruits ranged from 39.7 to 314.0% (Table 3). The maximal DPPH was calculated as 314.0% in hog plum and the minimum in satkara (39.7%). However, all selected fruits in our study showed a strong capacity to scavenge free radicals. 4.11.3. RPA The RPA of the selected minor fruits ranged from 3.21 to 634.00 µg AA/mg. The maximum value of the reducing power assay was calculated as 634.00 µg AA/mg in hog plum while the lowest was 3.21 µg AA/mg in day fruit (Table 3). 4.11.4. IC50 The IC50 is a widely accepted method used to assess the antioxidant activity of foodstuffs, and its value is expected to be lower for higher free radical quenching ability (Sathyanarayanan, Chandran, Thankarajan, Abrahamse, & Thangaraj, 2018). In this study, IC50 values ranged from 4.98 to 504.0 µg/g while the maximum value was found in hog plum (856.7 µg/g) and the minimum in elephant apple (4.98 µg/g) (Table 3). Our results indicate that hog plum has potential antioxidant capacity due to its lower value of IC50 (4.98 ± 0.24 µg/g), which may be due to the presence of significant amounts of phenolics and flavonoids. This finding is also corroborated by the research of Sathyanarayanan et al. (2018). 4.11.5. MCC MCC of the selected minor fruits ranged from 22.29 to 210.43% (Table 3), which indicates the ability of minor fruits to reduce different metallic ions and create a stable chemical bond to counteract free radicals. Recent studies have Asian Journal of Agriculture and Rural Development, 11(1)2021: 35-46 44 reported that the redox properties of phenolic species enable them to work as reducing agents, by donating hydrogen and quenching singlet oxygen showing antioxidant activity and chelating metal ions (Elfalleh et al., 2011). 4.11.6. FRAP The FRAP values found ranged from 0.35 to 2325.0 µM Fe2SO4/100 g (Table 3). Our results show a high FRAP value for elephant apple (2325.0 µM Fe2SO4/100 g), demonstrating that phenolic composites are among the leading contributors to the high antioxidant properties of this fruit. Previous reports evidenced that fruits with high phenolics can react with free radicals to form a stable product that blocks the radical chain reaction (Sathyanarayanan et al., 2018). 4.11.7. NO Radical Scavenging Activity NO radical scavenging activity of the selected minor fruits ranged from 0.02 to 178.87%. The results revealed that citron is a rich source of NO radical scavenging activity, recording the highest value of 178.87%, the lowest value being 0.02% in sour ber (Table 3). Thus, our study reported high NO radical foraging activity (178.87 ± 0.02%), which may be due to the presence of different polyphenolic substances in citron. 4.12. Phenolic Compounds HPLC was used for analysis of six major phenolic acids and matching to the respective standards. Phenolic acid levels in the selected minor fruits ranged 0.94–279.0 mg/100 g for gallic acid, 0.01–43.77 mg/100 g for vanillic acid, 0.16–178.96 mg/100 g for Þ–courmaric acid, 0.01–3.04 mg/100 g for caffeic acid, 0.01–20.44 mg/100 g for ferulic acid, and 12.03–97.35 µg/100 g for lutein. These results indicate that the leading phenolic compounds were gallic and ferulic acids for aonla; gallic acid for hog plum; gallic, Þ-courmaric, ferulic, and vanilic acids for bilimbi; gallic, Þ-courmaric, ferulic, and caffeic acids for elephant apple; gallic, caffeic, and Þ-courmaric acids for day fruit; Þ-courmaric and gallic acids for citron; gallic acid for satkara; Þ-courmaric and gallic acids for ber; and ferulic and caffeic acidc for toikar (Table 4). Table 4. Phenolic acid content of selected minor indigenous fruits. Parameter Minor fruits Aonla Hog plum Bilimbi Elephant apple Day fruit Citron Satkara Ber (sour) Toikar Gallic acid (mg/100 g) 279.06 ±2.00a 58.33 ±0.33d 82.01 ±3.01b 38.14± 0.15e 3.62± 0.01g 0.94± 0.02g 66.00± 0.52c 26.50± 1.17f 62.74± 0.74c Vanilic acid (mg/100 g) 43.77± 0.15b 42.02± 0.02b 70.50± 2.43a 0.01 ±0.00d 0.04± 0.01d 0.02± 0.01d 4.04± 0.04c 2.46± 0.05c 0.03± 0.01d p-courmaric (mg/100 g) 1.06± 0.03e 0.96± 0.02e 13.16± 0.17d 11.51± 0.07d 178.96± 4.13a 19.26± 1.54c 3.00± 0.50e 38.14± 0.74b 0.16± 0.01e Caffeic acid (mg/100 g) 0.09± 0.08c 0.01± 0.00c 0.05± 0.01c 3.04± 0.05a 0.45± 0.01b 0.01± 0.00c 0.10± 0.05c 0.02± 0.01c 0.02± 0.01c Ferulic acid (mg/100 g) 5.54± 0.03c 0.21± 0.01d 9.59± 0.06b 20.44± 0.05a 0.01± 0.00e 0.01± 0.00e 0.01± 0.00e 0.20± 0.15d 0.21± 0.20d Lutein acid (µg/100 g) 12.03± 0.04f 29.74± 0.11d 91.13± 3.80b 97.35± 0.35a 71.33± 0.33c 71.33± 0.33c 12.62± 0.12ef 14.85± 0.03ef 16.20± 0.10e Note: All values are means of triplicate determinations ± SD. Means within columns with different letters a, b, c, d, e, f, g indicate significant result (p˂0.05). 5. CONCLUSION This study is the first to provide details regarding the biochemical properties, minerals, bioactive compounds, antioxidant activities, and phenolic compounds of nine key minor indigenous fruits grown in Bangladesh. The results indicate that aonla is a rich source of ascorbic acid (664.92 mg/100 g), total ß-carotene (6897.57 µg/100 g), and RPA (634.00 µAA/g); bilimbi is a rich source of total flavonoids (150.02 mg QE/g), total anthocyanin (47.46 mg/100 g), and FRAP (1817.88 µM Fe2SO4/100 g); citron is a rich source of TAC (278.24 µAA/g), MCC (210.43%), and NO (178.87%); hog plum is a rich source of DPPH (314.0%); and day fruit is a rich source of TPH (176.50 mg GAE/g). The findings suggest that all minor fruits studied possess levels of phytochemicals that may be applied as raw material for nutritional and pharmaceutical usage. Funding: This work was supported by the National Agricultural Technology Program (NATP), Phase- II, Competitive Research Grant sub-project (Grant Number: CRG-328), Bangladesh Agricultural Research Council (BARC), Dhaka, Bangladesh. Competing Interests: The authors declare that they have no competing interests. Contributors/Acknowledgement: Authors thankfully acknowledged Dr. Md. Latiful Bari, Chief Scientist and Head of Food Analysis and Research Laboratory, Center for Advance Research in Sciences, University of Dhaka, Bangladesh for his scientific advice during the study. Views and opinions expressed in this study are those of the authors views; the Asian Journal of Agriculture and Rural Development shall not be responsible or answerable for any loss, damage, or liability, etc. caused in relation to/arising out of the use of the content. Asian Journal of Agriculture and Rural Development, 11(1)2021: 35-46 45 REFERENCES Akhter, S., Naz, S., Sultan, T. 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