Nutritional value,phytochemicals and antioxidant properties of two wild edible fruits ( Eugenia operculata Roxb. and Antidesma bunius L.) from Assam,North-East India

Abstract

The purpose of present study was to investigate the nutritional composition, phytochemical contents and antioxidant capacities of two wild edible fruits viz. Eugenia operculata Roxb. and Antidesma bunius L. from Assam of North-East India. The fruits showed variable amounts of proximate and mineral compositions which are reported herein and discussed. The phytochemical screening of different solvent extracts exhibited the presence of many phytochemicals which are biologically important. The antioxidant activities were examined using DPPH (1, 1-diphenyl-2-picrylhydrazyl), ABTS (2, 2′-Azinobis (3-ethylbenothiazoline-6-sulfonic acid) diammonium salt), H₂O₂ (Hydrogen peroxide) and FRAP (Ferric reducing antioxidant power) assays. The fruits showed antioxidant properties with DPPH IC₅₀ value of 92.330±4.163 μg/mL (E. operculata) and 395.002±3.605 μg/mL (A. bunius), ABTS IC₅₀ value of 52.660±1.154 μg/mL (E. operculata) and 105.331±3.055 μg/mL (A. bunius), H₂O₂ IC₅₀ value of 20.566±0.208 μg/mL (E. operculata) and 24.366±0.057 μg/mL (A. bunius), and FRAP value of 281.583±8.799 μM TE/g DE (dry extract) in E. operculata and 61.583±3.818 μM TE/g DE in A. bunius. The total phenolic content (TPC) in E. operculata and A. bunius fruits were found to be 226.741±2.099 mg GAE/g DE and 119.356±1.395 mg GAE/g DE, respectively, while the total flavonoid content (TFC) were 108.761±7.015 mg QE/g DE and 64.323±8.828 mg QE/g DE, respectively. The fresh fruits of A. bunius and E. operculata were found to contain vitamin C of 7.30±1.452 mg/100 g and 6.60±1.123 mg/100 g, respectively. The studies revealed that E. operculata fruit had stronger antioxidant activity than A. bunius fruit showing better DPPH, ABTS and H₂O₂ scavenging activities, and higher FRAP value, TPC and TFC. TPC and TFC showed a strong positive correlation with antioxidant activity assayed by FRAP. A strong positive correlation of antioxidant activity (DPPH, ABTS and H₂O₂) was also found with vitamin C.

Keywords

Wild edible fruits proximate minerals phytochemicals antioxidant

1 Introduction

Wild edible fruits are excellent sources of nutrients and used as an important food items in the diets of indigenous people and consequently, these fruits are making an important contribution to the health of local communities. Wild fruits contain numerous natural antioxidant compounds such as carotenoids, vitamins, phenolic compounds, and many more which have the capability to scavenge reactive oxygen species such as free radicals, inhibit the oxidation process and slow down the growth of various human diseases like cancer, heart disease, diabetes, stroke, rheumatoid arthritis, obesity, cataracts, and Alzheimer’s disease [1 –3]. Antioxidants include both natural and synthetic antioxidants. The free radicals generated in the body due to various reasons are responsible for the damage of nucleic acids, proteins, and lipids in the cells that lead to various human diseases and the plant based natural antioxidants can prevent this to happening by scavenging this active radicals [4]. It was reported that plant products are also potential sources of skin-whitening compounds in cosmetic and medicinal fields [5]. Synthetic antioxidants have widely been used in the food industry as food additives to protect food items against oxidative degradation and to prolong the shelf-life, but some of the synthetic antioxidants are harmful as they possess potential toxicity and carcinogenicity [6, 7]. Fruits, spices, vegetables, medicinal plants and even microalgae have been reported to contain good sources of natural antioxidants [7 –10] and the natural antioxidants are considered as potential alternatives to synthetic ones because of their numerous health profits [11]. Hence, the researchers throughout the world are being attracted to explore natural antioxidants from fruits, vegetables and medicinal plants and the search is still increasing.

Eugenia operculata Roxb. is a perennial tree, extensively distributed and propagated in China, Vietnam and some other tropical countries [12]. The plant is found growing in Bihar, Orissa, and Northeast region of India [13]. The plant produces fruits which are eaten by local communities in Assam. Its leaves, bark and buds were studied and showed medicinal and food properties [12 , 15]. Devi et al. [13] also studied aqueous and ethanol leave extracts of E. operculata and reported that the leave contains different phytochemicals and possess antioxidant property. However, still there is no information available on nutrient and antioxidant properties from the fruits of E. operculata. Antidesma bunius (L.) Spreng. is a wild plant belonging to the family Euphorbiaceae and distributed in Thailand, Philippines, and Southeast Asia [16]. The plant has antioxidant, anticancer and antidiabetic properties [17 –19]. Its fruit has a sweet-sour taste and is popular among rural populaces. A. bunius fruits found in Northeast Thailand are good sources of nutritional and phytochemical constituents [20]. Its fruit extract also showed antibacterial activities [21]. This plant is also found in Assam of Northeast of India and no available information on food properties of its fruit is reported from this region. The aim of this study was to evaluate nutritional, mineral, phenolic and flavonoid contents along with the investigation of antioxidant properties of two wild edible fruits viz. E. operculata and A. bunius.

2 Materials and methods

2.1 Chemicals

DPPH (1, 1-diphenyl-2-picrylhydrazyl), ABTS (2, 2′-Azinobis (3-ethylbenothiazoline-6-sulfonic acid) diammonium salt) and quercetin were obtained from Himedia Laboratories Pvt. Ltd., Mumbai (India), trolox from Sigma Aldrich, Bangalore (India), H₂O₂ (Hydrogen peroxide), ascorbic acid and Folin-Ciocalteu’s reagent from Merck, Mumbai (India) and gallic acid from Central Drug House Pvt. Ltd., New Delhi (India).

2.2 Collection and identification of plants

Ripened fruits of E. operculata and A. bunius were obtained in the month of April, 2015 from the Chirang district of Assam and the plants were identified with the help of Botanical Survey of India, Shillong (Meghalaya).

2.3 Sample preparation

The fresh fruits were washed thoroughly under tap water followed by distilled water and then moisture and vitamin C contents of the fresh fruits were determined on the same day. The remaining fruits were then freeze dried (FD) for 72 h. The dried fruits were pulverized and the powdered materials were kept in a container. For screening of phytochemical constituents, the powdered materials were extracted separately with methanol, hexane, chloroform, acetone and water in 1:10 ratio (w/v), shivered, stored for 72 h, filtered (Whatman No. 1), filtrate evaporated to dryness using Buchi Rotavapor R-215 (Switzerland) and the dry extracts were kept in air-tight containers at 4C till further analyses.

2.4 Analyses of proximate composition

Proximate compositions were determined following the methods of the Association of Official Analytical Chemists [22]. Total carbohydrate and dry matter were calculated using the methods of James [23]. Calorific value was calculated following the method of Food and Agriculture Organization (FAO) [24].

2.5 Determination of mineral contents

Mineral contents were investigated at Sophisticated Analytical Instrumentation Centre (SAIC), Tezpur University using Atomic Absorption Spectrometer (AAS-ICE 3500, Thermo Scientific, UK). The powder of freeze dried (FD) fruits were digested with concentrated HNO₃. The mineral contents were presented in mg/100 g of FD sample.

2.6 Qualitative phytochemical study

The qualitative phytochemical screening of different solvent extracts of FD fruits was performed using the standard procedures [25, 26].

2.7 Evaluation of antioxidant properties

Using methanol extract of FD fruit, antioxidant activities were determined with an UV-VIS spectrophotometer (Lambda 35, Perkin Elmer, USA) following the previously reported DPPH and ABTS assays [26]. Hydrogen peroxide scavenging activity was investigated with an UV-VIS spectrophotometer (Lambda 35, Perkin Elmer, USA) at 230 nm following the method of Ruch et al. [27]. Ferric reducing antioxidant power (FRAP) value was obtained using the method of Benzie and Strain [28]. The values were presented in μM trolox equivalent (TE)/g of dry extract (DE).

2.8 Investigation of total phenolic content (TPC) and total flavonoid content (TFC)

TPC and TFC were determined from methanol extract of FD fruits using an UV-VIS spectrophotometer (Lambda 35, Perkin Elmer, USA) following the previously reported procedures [26].

2.9 Estimation of vitamin C content

Vitamin C content of the fresh fruits was estimated following the procedure of Suntornsuk et al. [29].

2.10 Statistical analysis

The experimental results were presented as mean of triplicate results±standard deviation. Microsoft Excel was used for calculation of standard deviations. Statistical analyses of results were performed by the one-way ANOVA t-test at p < 0.05 using OriginPro 8.5 software (OriginLab Corporation, MA 01060 USA). SPSS 13.0 software was used for the study of Pearson’s correlation.

3 Results and discussion

3.1 Proximate composition

The results of proximate composition of two wild edible fruits are shown in Table 1. A. bunius had the higher moisture content of 4.53±0.351 g/100 g of FD and 64.466±0.251 g/100 g fresh fruit while E. operculata showed moisture content of 3.343±0.004 g/100 g of FD and 52.53±0.404 g/100 g of fresh sample. The moisture content in A. bunius is similar to that of Arbutus pavarii (68.06±1.65 g/100 g) and Ficus palmata (67.82±2.07 g/100 g) [30] and the moisture content of E. operculata is also comparable to that of Melastoma malabathricum (56.6±0.71 g/100 g) [31]. The ash content which is an index of mineral contents was found to be 0.516±0.003 g in A. bunius and 0.343±0.004 g in E. operculata. The crude fat content of the fruits investigated were found to be 0.97±0.026 g in A. bunius and 1.86±0.02 g in E. operculata which is almost similar to the values of Rosa dumalis, Rosa pulverulenta and Rosa canina reported by Ercisli [32]. The crude protein content in A. bunius was 1.231±0.050 g and that in E. operculata was 1.323±0.035 g per 100 g of FD which are comparable to the values of Elaeagnus conferta fruit reported by Rai et al. [33] and Ziziphus spinachristi fruit reported by Feyssa et al. [34]. Higher value of crude fibre was found in E. operculata fruit (17.566±0.351 g/100 g) than A. bunius fruit (9.433±0.305 g/100 g). Fibre rich diets are essential for digestion and effective removal of waste. Consumption of fruits and vegetables with rich fibres can lower the risk of coronary heart disease, constipation, serum cholesterol, diabetes, hypertension, and breast and colon cancer [35, 36]. The total carbohydrate content was found to be 92.743±0.428 g in A. bunius and 93.123±0.084 g in E. operculata which is higher than the values reported by Gnansounou et al. [37]. The fruits having good composition of carbohydrates are very nutritious for health products and responsible for their high calorific value [38]. The calorific value of E. operculata and A. bunius fruits were 394.586±0.025 kcal/100 g and 384.651±1.296 kcal/100 g, respectively which is higher to that of Grewia sapida fruit (346.34±0.04 kcal/100 g) presented in the previous report [26].

Table 1
Proximate composition of E. operculata and A. bunius fruits per 100 g of DW

Parameters E. operculata A. bunius

Moisture (g) 3.343±0.004^a 4.530±0.351^b

52.530±0.404^a 64.466±0.251^b

Ash (g) 0.343±0.004^a 0.516±0.003^a

Acid insoluble ash (g) 0.235±0.003^a 0.337±0.004^a

Acid soluble ash (g) 0.108±0.005^a 0.178±0.006^a

Crude fat (g) 1.860±0.020^a 0.970±0.026^b

Crude protein (g) 1.323±0.035^a 1.231±0.050^a

Crude fiber (g) 17.566±0.351^a 9.433±0.305^b

Total carbohydrate (g) 93.123±0.084^a 92.743±0.428^b

Dry matter (g) 96.656±0.025^a 95.466±0.351^b

Calorific value (kcal) 394.586±0.025^a 384.651±1.296^b

Parameters	E. operculata	A. bunius
Moisture (g)	3.343±0.004^a	4.530±0.351^b
	52.530±0.404^*a	64.466±0.251^*b
Ash (g)	0.343±0.004^a	0.516±0.003^a
Acid insoluble ash (g)	0.235±0.003^a	0.337±0.004^a
Acid soluble ash (g)	0.108±0.005^a	0.178±0.006^a
Crude fat (g)	1.860±0.020^a	0.970±0.026^b
Crude protein (g)	1.323±0.035^a	1.231±0.050^a
Crude fiber (g)	17.566±0.351^a	9.433±0.305^b
Total carbohydrate (g)	93.123±0.084^a	92.743±0.428^b
Dry matter (g)	96.656±0.025^a	95.466±0.351^b
Calorific value (kcal)	394.586±0.025^a	384.651±1.296^b

^*Moisture content of fresh fruit; DW, dry weight; The results followed by different letters along a row are significantly different from each other at p < 0.05.

3.2 Mineral composition

The mineral contents of E. operculata and A. bunius fruits per 100 g of FD sample are shown in Table 2. The results of mineral analyses in the two wild edible fruits revealed that E. operculata had the sodium content of 4.640±0.046 mg and A. bunius had 5.377±0.032 mg per 100 g which is comparable to that of Ziziphus mauritiana (5.03 mg/100 g) [39]. High content of potassium was found in A. bunius (3043.852±6.088 mg/100 g) and E. operculata (2219.736±6.659 mg/100 g) which is in agreement with results reported by Saka et al. [40] and Amarteifio et al. [41]. Potassium is one of the most essential and major plant nutrients and foods rich in potassium are generally used for the treatment of rheumatoid arthritis and heart disease [42]. In this study, calcium was found to be 714.820±8.578 mg in E. operculata and 787.900±14.182 mg in A. bunius. Leterme et al. [43] reported calcium content of Annona squamosa L. fruit as 991 mg/100 g which is higher in comparison to this report. The recommended daily intake of calcium for adult ranges from 1000 – 1500 mg. The present study showed the satisfactory amount of magnesium and found to be 172.387±0.517 mg/100 g in E. operculata and 250.703±0.251 mg/100 g in A. bunius. Magnesium is very important metal for many enzymatic reactions and magnesium deficiency causes various health disorders including asthma, high blood pressure, angina pectoris, cardiac arrhythmias, coronary artery disease, all types of musculoskeletal disorders, mitral valve prolapse, panic disorder, epilepsy, anxiety, chronic fatigue syndrome and psychiatric conditions [37 , 42]. The iron detected in E. operculata and A. bunius fruits were 8.279±0.033 mg and 7.579±0.015 mg per 100 g, respectively which are comparable to that of Melastoma malabathricum (8.00±0.19 mg) and Calamus guruba (8.50±0.19 mg) reported by Nayak el al. [31]. Iron is required for haemoglobin synthesis in red blood cells which is needed for oxygen transportation to all parts of the body. Iron deficiency causes anemia and immune system dysfunction [26]. The level of copper, zinc and manganese in A. bunius were 1.774±0.060 mg, 2.903±0.012 mg and 7.616±0.023 mg, respectively and in E. operculata were 1.493±0.051 mg, 1.828±0.011 mg and 2.817±0.020 mg, respectively. Nayak el al. [31] reported copper content of Careya arborea as 1.90±0.04 mg which is comparable to the fruits of this study. The zinc level found in A. bunius is close to that of the fruits of blackberry (2.30±0.35 mg/100 g), raspberry (2.97±0.1 mg/100 g) and red currant (2.11±0.54 mg/100 g) reported by Plessi et al. [44] and these values are slightly higher to that of E. operculata. Copper is an essential element of many enzyme systems such as cytochrome oxidase, lysyl oxidase and ceruloplasmin, manganese is important for haemoglobin formation and the deficiency of zinc leads to impaired growth and malnutrition [45]. The level of cobalt in E. operculata and A. bunius fruits were 0.352±0.050 mg/100 g and 0.390±0.019 g/100 g, respectively. Cobalt is an essential part of vitamin B₁₂ also known as cyanocobalamin, and its deficiency can cause pernicious anemia. As human body is not capable to synthesize vitamins, the consumption of diets containing these compounds is essential.

Table 2
Mineral contents of E. operculata and A. bunius fruits

Minerals E. operculata A. bunius

(mg/100 g FD) (mg/100 g FD)

Sodium 4.640±0.046^a 5.377±0.032^b

Potassium 2219.736±6.659^a 3043.852±6.088^b

Calcium 714.820±8.578^a 787.900±14.182^b

Magnesium 172.387±0.517^a 250.703±0.251^b

Iron 8.279±0.033^a 7.579±0.015^b

Zinc 1.828±0.011^a 2.903±0.012^b

Copper 1.493±0.051^a 1.774±0.060^a

Manganese 2.817±0.020^a 7.616±0.023^b

Cobalt 0.352±0.050^a 0.390±0.019^a

Minerals	E. operculata	A. bunius
Sodium	4.640±0.046^a	5.377±0.032^b
Potassium	2219.736±6.659^a	3043.852±6.088^b
Calcium	714.820±8.578^a	787.900±14.182^b
Magnesium	172.387±0.517^a	250.703±0.251^b
Iron	8.279±0.033^a	7.579±0.015^b
Zinc	1.828±0.011^a	2.903±0.012^b
Copper	1.493±0.051^a	1.774±0.060^a
Manganese	2.817±0.020^a	7.616±0.023^b
Cobalt	0.352±0.050^a	0.390±0.019^a

FD, freeze dried; The results followed by different letters along a row are significantly different from each other at p < 0.05.

3.3 Phytochemical screening

In this study, five types of solvents including both polar and non-polar viz. methanol, chloroform, hexane, acetone and water were used to get fruit extracts and to screen the presence of different phytochemicals in the solvent extracts. The results of phytochemical screening of different solvent extracts from the fruits of E. operculata (Table 3) and A. bunius (Table 4) showed the presence of many phytochemicals. Both the fruits indicated the presence of alkaloids, saponins and carbohydrates (Fehling’s test) in all the five extracts. However, A. bunius showed the presence of steroids in all the extracts whereas in E. operculata, steroids were not detected in acetone extract. Cardiac glycoside in E. operculata was not detected in water extract, while in A. bunius, it was detected in methanol, chloroform, and acetone extracts. In E. operculata, methanol, acetone and water extracts showed positive test for anthraquinones and coumarins while in A. bunius, anthraquinone was not detected in chloroform extract and coumarin was not detected in methanol, chloroform and hexane extracts. Positive test for tannins was observed in the acetone extract of E. operculata and in the methanol and acetone extracts of A. bunius. Chloroform and acetone extracts showed negative results for flavonoids in E. operculata and found to be present in all other three extracts. Chloroform and hexane extracts showed negative results for flavonoids in A. bunius. Chloroform, hexane and water extracts indicated positive results for starch in E. operculata, while starch was detected in all the five extracts in A. bunius. Methanol, hexane, and acetone extracts were found to contain anthocyanins in E. operculata and in A. bunius, only hexane and acetone extracts showed the presence of anthocyanins. Both the fruits showed positive results for proteins (Millon’s test) in methanol and water extracts and phlobatannins were detected in acetone and water extracts of both the fruits. Lignin was detected in four different solvent extracts except methanol in E. operculata, whereas in A. bunius, chloroform, acetone and water extracts showed the presence of lignin. Phytochemicals found in the plant materials are known to possess many biologically active compounds and they are responsible for several biological activities such as antioxidant, antimicrobial, antifungal, antiinflammatory, and anticancer activities [8 , 46].

Table 3
Qualitative phytochemical analysis of E. operculata fruit with different solvent extracts

Phytochemical Test Methanol Chloroform Hexane Acetone Water

constituents

Alkaloids Wagner’s reagent + + + + +

Dragendroff’s reagent + + + + +

Saponins Froth test + + + + +

Cardiac glycosides Keller-Killiani’s test + + + + –

Steroids (Terpenoids) Liebermann-Burchard test + + + – +

Salkowski’s test + + + + –

Anthraquinones Modified Borntrager’s test + – – + +

Coumarins + – – + +

Phenols FeCl₃ test + + + + +

Tannins Gelatin test – – – + –

Flavonoids Shinoda’s test + – + – +

Carbohydrates Molisch’s test + + – + –

Fehling’s test + + + + +

Starch Iodine test – + + – +

Anthocyanins + – + + –

Proteins Ninhydrin test – – + – –

Millon’s test + – – – +

Phlobatannins – – – + +

Lignin – + + + +

Phytochemical	Test	Methanol	Chloroform	Hexane	Acetone	Water
Alkaloids	Wagner’s reagent	+	+	+	+	+
	Dragendroff’s reagent	+	+	+	+	+
Saponins	Froth test	+	+	+	+	+
Cardiac glycosides	Keller-Killiani’s test	+	+	+	+	–
Steroids (Terpenoids)	Liebermann-Burchard test	+	+	+	–	+
	Salkowski’s test	+	+	+	+	–
Anthraquinones	Modified Borntrager’s test	+	–	–	+	+
Coumarins	+	–	–	+	+
Phenols	FeCl₃ test	+	+	+	+	+
Tannins	Gelatin test	–	–	–	+	–
Flavonoids	Shinoda’s test	+	–	+	–	+
Carbohydrates	Molisch’s test	+	+	–	+	–
	Fehling’s test	+	+	+	+	+
Starch	Iodine test	–	+	+	–	+
Anthocyanins	+	–	+	+	–
Proteins	Ninhydrin test	–	–	+	–	–
	Millon’s test	+	–	–	–	+
Phlobatannins	–	–	–	+	+
Lignin	–	+	+	+	+

(+), present; (–), absent.

Table 4

Qualitative phytochemical analysis of A. bunius fruit with different solvent extracts

Phytochemical	Test	Methanol	Chloroform	Hexane	Acetone	Water
constituents
Alkaloids	Wagner’s reagent	+	+	+	+	+
	Dragendroff’s reagent	+	+	+	+	+
Saponins	Froth test	+	+	+	+	+
Cardiac glycosides	Keller-Killiani’s test	+	+	–	+	–
Steroids (Terpenoids)	Liebermann-Burchard test	+	+	+	+	+
	Salkowski’s test	+	+	+	+	–
Anthraquinones	Modified Borntrager’s test	+	–	+	+	+
Coumarins	–	–	–	+	+
Phenols	FeCl₃ test	+	–	+	–	+
Tannins	Gelatin test	+	–	–	+	–
Flavonoids	Shinoda’s test	+	–	–	+	+
Carbohydrates	Molisch’s test	+	+	–	+	–
	Fehling’s test	+	+	+	+	+
Starch	Iodine test	+	+	+	+	+
Anthocyanins	–	–	+	+	–
Proteins	Ninhydrin test	–	–	+	–	–
	Millon’s test	+	–	–	–	+
Phlobatannins	–	–	–	+	+
Lignin	–	+	–	+	+

(+), present; (–), absent.

3.4 Antioxidant properties

Most of the polyphenol and flavonoid compounds found in plants are soluble in methanol which is a polar solvent and these compounds display various biological properties including the antioxidant activity [8, 26]. In this study, the methanol extracts of E. operculata and A. bunius FD fruits were investigated for antioxidant activities using DPPH, ABTS, H₂O₂ and FRAP methods. DPPH is a stable radical species with a maximum absorption at 517 nm that can readily undergo scavenging by antioxidants [26]. This assay has been used commonly to investigate the capacity of compounds as free radical scavengers or hydrogen donors and to assess the antioxidant property of food and plant extracts [47]. The DPPH free radical scavenging activities of different concentrations of methanol extracts of E. operculata and A. bunius fruits, and standard ascorbic acid are presented in Table 5 and it was observed that the radical scavenging capacity increased with concentration of sample. At 500 μg/mL concentration, E. operculata fruit extract (89.651±0.552%) showed higher percentage of inhibition than A. bunius fruit extract (61.413±0.398%). While standard ascorbic acid showed 95.066±0.45% inhibition at the same concentration. The IC₅₀ values of A. bunius, E. operculata and ascorbic acid obtained in DPPH assay were 395.002±3.605 μg/mL, 92.330±4.163 μg/mL, and 16.666±2.516 μg/mL respectively. A lower IC₅₀ value of sample exhibits higher antioxidant capacity. The IC₅₀ value of M. calabura fruit reported by Preethi et al. [48] was 90±0.04 μg/mL which is close to that of E. operculata fruit. A. bunius fruit extract exhibited similar IC₅₀ value to that of Punica granatum fruit (398.54±47.6 μg/mL) reported by Khomdram et al. [47]. ABTS free radical scavenging activities in methanol extracts of E. operculata and A. bunius fruits and standard ascorbic acid are shown in Table 5 and this assay also showed antioxidant activities in a concentration-dependent manner. E. operculata displayed 90.17±0.655% inhibition at concentration of 250 μg/mL with an IC₅₀ value of 52.66±1.154 μg/mL and A. bunius showed 80.743±0.895% inhibition at the same concentration with IC₅₀ value of 105.331±3.055 μg/mL which indicated that the fruit extract of E. operculata had better antioxidant capacity than A. bunius. In our previous study, a wild edible fruit (Grewia sapida) reported from Assam of North-East India exhibited an ABTS IC₅₀ value of 134.33±4.041 μg/mL [26]. H₂O₂ scavenging activities in methanol extracts of E. operculata and A. bunius fruits are presented in Table 5. The H₂O₂ IC₅₀ value for methanolic extract of E. operculata fruit was found to be 20.566±0.208 μg/mL and that of A. bunius was 24.366±0.057 μg/mL, while the standard ascorbic acid showed an IC₅₀ value of 19.766±0.152 μg/mL. Table 6 shows that E. operculata fruit (281.583±8.799 μM TE/g DE) had stronger ferric reducing power than A. bunius fruit (61.583±3.818 μM TE/g DE). It is interesting to note that all the four assays (DPPH, ABTS, H₂O₂ and FRAP) employed for determination of antioxidant properties revealed that the methanol extract of E. operculata fruit exhibited better antioxidant activities than A. bunius fruit extract.

Table 5
DPPH, ABTS and H₂O₂ scavenging activities of methanolic extracts of E. operculata and A. bunius fruits

E. operculata A. bunius Ascorbic acid^**

Conc. ( μg/mL) Inhibition (%) of fruits for DPPH assay

2 17.973±0.642^a 17.481±0.320^a 15.800±0.556^b

5 22.496±0.960^a 18.903±0.475^b 27.100±0.754^c

10 25.636±0.825^a 19.381±0.161^b 36.433±0.702^c

50 61.960±0.187^a 22.936±0.555^b 93.233±0.404^c

100 80.551±1.011^a 29.373±0.636^b 93.600±0.501^c

200 83.421±0.371^a 31.403±0.475^b 94.166±0.550^c

400 85.012±0.642^a 48.441±0.363^b 94.333±0.650^c

500 89.651±0.552^a 61.413±0.398^b 95.066±0.450^c

IC₅₀ 92.330±4.163^a 395.002±3.605^b 16.666±2.516^c

Inhibition (%) of fruits for ABTS assay

25 28.93±0.351^a 24.771±0.752^b 36.093±0.875^c

50 41.006±0.467^a 36.912±1.193^b 38.520±1.176^c

75 66.033±0.503^a 46.363±0.521^b 55.551±1.023^c

100 78.69±0.739^a 52.483±1.058^b 66.856±0.661^c

150 81.613±0.452^a 63.726±0.295^b 73.506±0.810^c

250 90.170±0.655^a 80.743±0.895^b 79.426±1.168^c

IC₅₀ 52.660±1.154^a 105.331±3.055^b 73.666±3.214^c

Inhibition (%) of fruits for H₂O₂ assay

5 13.243±0.095^a 5.936±0.145^b 10.410±0.307^c

10 27.631±0.645^a 18.006±0.225^b 27.890±0.160^a

15 36.073±0.315^a 27.203±0.155^b 41.940±0.232^c

20 47.566±0.120^a 32.473±0.110^b 51.451±0.122^c

25 62.196±0.245^a 55.013±0.066^b 60.523±0.281^c

IC₅₀ 20.566±0.208^a 24.366±0.057^b 19.766±0.152^c

	E. operculata	A. bunius	Ascorbic acid^**
Conc. ( μg/mL)	Inhibition (%) of fruits for DPPH assay
2	17.973±0.642^a	17.481±0.320^a	15.800±0.556^b
5	22.496±0.960^a	18.903±0.475^b	27.100±0.754^c
10	25.636±0.825^a	19.381±0.161^b	36.433±0.702^c
50	61.960±0.187^a	22.936±0.555^b	93.233±0.404^c
100	80.551±1.011^a	29.373±0.636^b	93.600±0.501^c
200	83.421±0.371^a	31.403±0.475^b	94.166±0.550^c
400	85.012±0.642^a	48.441±0.363^b	94.333±0.650^c
500	89.651±0.552^a	61.413±0.398^b	95.066±0.450^c
IC₅₀	92.330±4.163^a	395.002±3.605^b	16.666±2.516^c
	Inhibition (%) of fruits for ABTS assay
25	28.93±0.351^a	24.771±0.752^b	36.093±0.875^c
50	41.006±0.467^a	36.912±1.193^b	38.520±1.176^c
75	66.033±0.503^a	46.363±0.521^b	55.551±1.023^c
100	78.69±0.739^a	52.483±1.058^b	66.856±0.661^c
150	81.613±0.452^a	63.726±0.295^b	73.506±0.810^c
250	90.170±0.655^a	80.743±0.895^b	79.426±1.168^c
IC₅₀	52.660±1.154^a	105.331±3.055^b	73.666±3.214^c
	Inhibition (%) of fruits for H₂O₂ assay
5	13.243±0.095^a	5.936±0.145^b	10.410±0.307^c
10	27.631±0.645^a	18.006±0.225^b	27.890±0.160^a
15	36.073±0.315^a	27.203±0.155^b	41.940±0.232^c
20	47.566±0.120^a	32.473±0.110^b	51.451±0.122^c
25	62.196±0.245^a	55.013±0.066^b	60.523±0.281^c
IC₅₀	20.566±0.208^a	24.366±0.057^b	19.766±0.152^c

Conc., concentration; IC₅₀ value in μg/mL; **Ascorbic acid was used as standard for DPPH, ABTS, H₂O₂ assays; Results are expressed as mean of 3 replicates±standard deviation; The results with different letters along a row are significantly different from each other at p < 0.05.

Table 6

Ferric reducing antioxidant power (FRAP), TPC, TFC and vitamin C content of the fruits

Parameters	E. operculata	A. bunius
FRAP value ( μM TE/g DE)	281.583±8.799^a	61.583±3.818^b
TPC (mg GAE/g DE)	226.741±2.099^a	119.356±1.395^b
TFC (mg QE/g DE)	108.761±7.015^a	64.323±8.828^b
Vitamin C (mg/100 g FW)	6.60±1.123^a	7.30±1.452^b

DE, dry extract; FW, fresh weight; The results followed by different letters along a row are significantly different from each other at p < 0.05.

3.5 TPC, TFC and vitamin C content

The TPC and TFC in methanol extracts of E. operculata and A. bunius FD fruits are shown in Table 6. The TPC in E. operculata and A. bunius fruits were 226.741±2.099 mg GAE/g DE and 119.356±1.395 mg GAE/g DE, respectively, while the TFC were found to be 108.761±7.015 mg QE/g DE and 64.323±8.828 mg QE/g DE, respectively. E. operculata fruit extract showed higher content of both total phenolic and total flavonoid than that of A. bunius fruit which attributed to better antioxidant capacity of the former fruit. Similarly, the TPC and TFC in the methanol extract of G. sapida fruit reported in the previous study were 294.353±4.696 mg GAE/g DE and 116.95±10.71 mg QE/g DE, respectively [26]. Prakash et al. [49] studied some wild fruits from Sikkim Himalayan region of India and reported the TPC that varied from 7.3 to 119.2 mg GAE/g. While the total phenolic contents of 56 wild fruits from South China ranged from 0.49±0.04 to 54.8±3.05 mg GAE/g wet weight [7]. Saikia et al. [50] reported phenolic content (4.62–14.74 mg GAE/g dry weight) and flavonoid content (0.65 – 7.72 mg QE/g dry weight) in some leafy vegetables which are lower in comparison to this study. Ascorbic acid also known as vitamin C is a water-soluble vitamin and found in many fresh fruits and vegetables. The fresh fruit of A. bunius (7.30±1.452 mg/100 g) showed slightly higher vitamin C content than E. operculata (6.60±1.123 mg/100 g) fruit (Table 6). Earlier study showed vitamin C content of 8.6±0.30 mg/100 g fresh G. sapida fruit [26]. Khomdram et al. [47] reported the vitamin C content in the wild endemic fruits from Manipur (India) that varied from 6.91 mg/100 g in P. armeniaca to 375.68 mg/100 g of fresh weight in P. emblica. The extraction of phenolic and flavonoid contents from plant materials depend on the polarity of solvent used for extract preparation [26]. Fruits are important sources of ascorbic acid, phenolic, flavonoid and many other compounds, and possess beneficial effects on human health as antioxidant and antibacterial agents [18].

3.6 Pearson’s correlation

This study of antioxidant capacity in the fruit extracts displayed a strong positive correlation of DPPH assay with ABTS assay, H₂O₂ assay and Vitamin C significantly at p < 0.01 (Table 7). The study also showed a strong positive correlation of ABTS with H₂O₂ and Vitamin C, H₂O₂ with Vitamin C, FRAP with TPC and TFC, and TPC with TFC. Similar type of study on some wild and cultivated blueberries from Romania was reported by Bunea et al. [51] which is in agreement with this report. Ku et al. [52] also reported a positive correlation of FRAP assay with phenolic and flavonoid contents. A very well-correlation of phenolic and flavonoid compounds with antioxidant capacity of plant extract was established and the involvement of these compounds to the overall antioxidant activity is mainly because of their redox properties and proton donating abilities [18 , 51–55].

Table 7
Pearson’s correlation coefficients of antioxidant activity (DPPH, ABTS, H₂O₂, FRAP), TPC, TFC and vitamin C in the fruits

DPPH ABTS H₂O₂ FRAP TPC TFC Vitamin C

DPPH 1

ABTS 1.000^a 1

H₂O₂ 1.000^a 1.000^a 1

FRAP –1.000^a –1.000^a –1.000^a 1

TPC –1.000^a –1.000^a –1.000^a 1.000^a 1

TFC –1.000^a –1.000^a –1.000^a 1.000^a 1.000^a 1

Vitamin C 1.000^a 1.000^a 1.000^a –1.000^a –1.000^a –1.000^a 1

	DPPH	ABTS	H₂O₂	FRAP	TPC	TFC	Vitamin C
DPPH	1
ABTS	1.000^a	1
H₂O₂	1.000^a	1.000^a	1
FRAP	–1.000^a	–1.000^a	–1.000^a	1
TPC	–1.000^a	–1.000^a	–1.000^a	1.000^a	1
TFC	–1.000^a	–1.000^a	–1.000^a	1.000^a	1.000^a	1
Vitamin C	1.000^a	1.000^a	1.000^a	–1.000^a	–1.000^a	–1.000^a	1

^aCorrelation is significant at p < 0.01.

4 Conclusion

The two wild edible fruits have appreciable proximate and mineral compositions. Phytochemical screening exhibited the presence of various phytochemical constituents which are of biologically and pharmaceutically important. The studies revealed that E. operculata fruit had stronger antioxidant capacity than A. bunius fruit showing better DPPH, ABTS and H₂O₂ scavenging activities, and higher FRAP value, TPC and TFC. TPC, TFC and vitamin C content of fruits established the food properties which are linked to free radical scavenging activities. TPC and TFC showed a strong positive correlation with antioxidant activity assayed by FRAP and a strong positive correlation of antioxidant activity (DPPH, ABTS and H₂O₂) was also observed with vitamin C suggesting that these compounds are the main compounds responsible for the antioxidant property. Hence, the fruits could play a role against the diseases caused by oxidative stress inhibiting the development of various human diseases and further, isolation and identification of bioactive compounds responsible for antioxidant activity is encouraged.

Footnotes

Acknowledgments

The authors are thankful to the Botanical Survey of India, Shillong (Meghalaya) for identification of plants. Thanks also goes to Head(s), Department of Biotechnology, Bodoland University, Kokrajhar and Department of Food Engineering & Technology, Central Institute of Technology, Kokrajhar for providing necessary facilities for this study.

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