Measurement of antioxidant synergy between phenolic bioactives in traditional food combinations (legume/non-legume/fruit) of (semi) arid regions: insights into the development of sustainable functional foods

Abstract

Numerous under-researched edible plants are present in the desert regions of the world. These plants could be potential candidates to ensure food security and provide valuable bioactive compounds through diet. In general, the bioactives present in food manifest synergistic, additive, or antagonistic interactions. The current study investigates such interactions between food combinations traditionally consumed in (semi) arid regions. Five edible plants (representing three food categories) were selected: Prosopis cineraria and Acacia senegal (legume), Capparis decidua and Cordia dichotoma (non-legume), and Mangifera indica (fruit), in which the first four are largely underutilized. The antioxidant capacities of individual plant extracts and their binary mixtures were analyzed by DPPH free radical scavenging and FRAP assays. The total phenolic content (TPC) and total flavonoid content (TFC) were also determined. The highest antioxidant activity was obtained for Prosopis cineraria extract (EC₅₀—1.24 ± 0.02 mg/ml, FRAP value—380.58 ± 11.17 μM/g), while Mangifera indica exhibited the lowest antioxidant activity (EC₅₀—2.54 ± 0.05 mg/ml, FRAP value—48.91 ± 4.34 μM/g). Binary mixture of Prosopis cineraria (legume) and Mangifera indica (fruit) manifested maximum synergy (experimental EC₅₀—0.89 ± 0.01 mg/ml, theoretical EC₅₀—3.79 ± 0.05 mg/ml). Correlation studies [Pearson’s correlation coefficient (r) and Principal component analysis (PCA)] showed a high correlation of TFC with DPPH and TPC with FRAP values. LC–MS analysis of methanolic plant extracts detected 43 phenolic compounds (including phenolic acids, flavonoids, and isoflavonoids), possibly responsible for the observed food synergy. For edible plants of the (semi) arid zones, this study is a first-of-its-kind and provides scientific validation to the traditional wisdom of consuming these foods together. Such indigenous food combinations derived from desert flora could offer valuable insights into development of sustainable functional foods and nutraceuticals.

Graphical Abstract

1 Introduction

The plant-based foods that we eat not only provide macronutrients (proteins and carbohydrates) and micronutrients (vitamins and minerals) but also phytochemicals like phenolics and flavonoids. The latter are bioactive non-nutrient chemicals present in fruits, vegetables, legumes, and other plants, which they often accumulate under different biotic and abiotic stresses. A growing body of evidence suggests that the potential benefits of antioxidant capacity inherent in phytochemicals in fruits and vegetables may be even more significant than currently acknowledged. This is because oxidative stress caused by free radicals is implicated in the genesis of a wide range of non-communicable chronic disorders like cancer, obesity, diabetes, and cardiovascular disease, to name a few [1, 2]. Phytochemicals are classified as phenolics, nitrogen-containing compounds, carotenoids, terpenoids, and sulfur-containing compounds. Phenolic acids and flavonoids (flavone, isoflavone, flavanol, flavonol, flavonone, anthocyanidin) (Fig. 1), are the most studied phytochemicals owing to their vast structural and functional diversity. These phytochemicals and their synthetic derivatives have shown beneficial biological activities, such as anticancer, antifungal, antibacterial, and antioxidant activities [3]. Their antioxidant activity is linked to the structure, length of the side chain, and substitutions on the aromatic ring [4]. However, in the current era where consumption of isolated dietary supplements is more prevalent, it cannot be overemphasized that the same compounds can become toxic or antagonistic if consumed in excess or wrong combinations. In this context, wisdom lies in their intake via the food matrix, where the individual concentrations of the nutraceuticals are balanced.

Fig. 1

Basic flavan structure and chemical structures of flavonoid subgroups. Structures are drawn using Marvin sketch software

In our body, cellular respiration, protein folding, and some metabolic activities constantly produce reactive oxygen species (ROS), such as hydroxyl radicals, superoxide anions, and singlet oxygen, which are essential for various signalling pathways and regulation of physiological activities. But, in the case of redox imbalance, excessive ROS can be harmful and cause oxidative stress, which can trigger inflammation or lead to chronic diseases [5]. According to epidemiological reports, regular intake of fruits and vegetables has been related to a lower risk of diseases, often heralded by this redox imbalance. Phytochemical extracts from vegetables and fruits have potent antioxidant and antiproliferative properties, and the majority of overall antioxidant activity comes from the combination of phytochemicals. Their strong antioxidant activity is often implicated to their additive and synergistic effects [1].

As a concept, food synergy highlights the positive interactions between nutrients and/or nutraceuticals, their absorption, and bioavailability in human body. It emphasizes the interaction of nutrients in various foods rather than a single component present therein. For example, green tea and black pepper were reported to have a synergistic effect, increasing the bioavailability of epigallocatechin gallate (EGCG), a compound present in green tea [6]. It is important to remember that phytochemicals may still cause cytotoxicity. The cytotoxic effects of phytochemicals may be worsened by excessive dosageor incompatible interactions. Because of these reasons, it is preferable to obtain bioactive compounds from whole plant-based foods that contain low-dose phytochemicals and have a generally favorable safety profile [7].

Plants growing in desert regions face harsh environmental conditions like excessive temperatures and low rainfall, and therefore accumulate unique anti-stress metabolites to combat the same [8]. Studies on synergistic interactions between traditional food combinations from the (semi) arid zones are missing. For this, five foods from three different categories, including Prosopis cineraria and Acacia senegal (legumes), Capparis decidua and Cordia dichotoma (non-legume), and Mangifera indica (fruit), were selected (Fig. 2). These plants are found in different regions worldwide and fruits/vegetables derived from them are dried/processed, and traditionally consumed in the arid and semi-arid parts of Indian subcontinent and Western Asia in different combinations, like ker-sangri (Capparis decidua-Prosopis cineraria), ‘Panchkuta’ (combination of all five above-mentioned plants), etc. Indigenous communities (such as the Bishnoi in Rajasthan, India) have been known to protect these plants/trees, particularly Prosopis cineraria, since ancient times. However, apart from corroboration through ethnic dietary practices, there is no scientific evidence till date on whether these foods have any synergistic effects. The objective of the current study is to investigate interactions between phenolic bioactives in the food matrix in order to scientifically validate the antioxidant synergy in traditional food combinations of the (semi) arid regions, by employing multi-mechanistic antioxidant assays.

Fig. 2

Dried edible parts of the five selected semi (arid) zone plants from India

2 Materials and methods

2.1 Chemicals and reagents

DPPH, ascorbic acid, quercetin, ferric chloride (FeCl₃), sodium hydroxide (NaOH), sodium nitrite, and sodium carbonate were purchased from HiMedia (Mumbai, India), methanol, aluminium chloride, sodium acetate, and Folin–Ciocalteu reagent (FCR) were obtained from Sigma Company (United States), glacial acetic acid and 2,4,6-tripyridyl-s-triazine (TPTZ) were purchased from SRL (Mumbai, India).

2.2 Sample collection and extraction

Commercially available dried pods of Prosopis cineraria (PC; Local name in Rajasthan: Sangri), berries of Capparis decidua (CD; Local name in Rajasthan: Ker), seeds of Acacia senegal (AS; Local name in Rajasthan: Kumatiya), the fruit of Cordia dichotoma (CDI; Local name in Rajasthan: Lasora), and pulp of Mangifera indica (MI; Mango) were purchased from a local grocery store (Nagaur district, Rajasthan, India; N 27° 11′ 55.0644″, E 73° 44′ 4.9848″).

2.2.1 Sample preparation and extraction

The dried edible parts were powdered using a Waring blender. For each plant, 10 g of the dry powder was extracted with 80% methanol (1:5 w/v) at room temperature (1 h) in an orbital shaker incubator. The samples were further centrifuged at 3000×g for 10 min. The supernatant was filtered with a filter paper (Whatman). This step was performed twice, followed by concentrating the pooled supernatants to dryness using a rotary evaporator (Aditya Scientific, India). The resulting extracts were stored at 4 °C for further analysis.

2.3 Evaluation of antioxidant activity

Different concentrations of methanolic extracts were prepared to evaluate their antioxidant activities. For binary combinations, the food extracts were mixed in a 1:1 (v/v) ratio. Individual extracts were twofold diluted before testing.

2.3.1 Total phenolic content (TPC)

The TPC was determined by the Folin–Ciocalteu method described by Tsao et al. [9] and Wang et al. [10] with some modifications. Briefly, 1 ml of the FCR (Folin–Ciocalteu Reagent) and 0.8 ml of 7.5% sodium carbonate were mixed with 0.2 ml of the extract. After gently shaking, the mixture was incubated for 30 min at room temperature, and the absorbance was recorded at 765 nm. A standard curve of gallic acid was prepared (50–250 μg/ml), and the TPC values were expressed as micrograms of gallic acid equivalents (GAE) per gram of sample. All tests were performed in triplicates.

2.3.2 Total flavonoid content (TFC)

The TFC was calculated by the Aluminum chloride colorimetric assay described by John et al. [11]. Briefly, in a flask containing 4 ml distilled water, an aliquot (1 ml) of extracts or standard solutions of quercetin (200–1000 μg/ml) was added. To the flask was added 0.30 ml 5% NaNO₂, followed by 0.30 ml 10% AlCl₃ after 5 min. Further, 2 ml of 1 M NaOH was added after five minutes, and the volume was adjusted to 10 ml with distilled water. After shaking absorbance of the sample was read at 510 nm. The TFC values were expressed as mg of quercetin equivalents (QE) per g of sample. All tests were performed in triplicates.

2.3.3 DPPH free radical scavenging assay

The DPPH assay was performed as described by Hidalgo et al. [12]. A 100 μM DPPH solution was prepared in methanol, and to 290 μl of this solution, 10 μl of individual food extract or extract combination was added. The concentration of methanolic extracts ranged between 0.5 and 2.5 mg/ml (final concentration). The above reactions were carried out in a 96-well microplate, followed by incubation in the dark at room temperature for 1 h, and absorbance was measured at 517 nm using a microplate reader (Thermo Scientific Multiskan GO). The percentage DPPH radical scavenging activity was calculated using the following equation:

$$Inhibition\;\% = \frac{{A_{c} - A_{s} }}{{A_{c} }},$$

where A_c represents the absorbance of the control while A_s represents that of the sample. Solution devoid of the sample was taken as control. The results were expressed as EC₅₀ (μM) obtained by plotting a graph between concentration and inhibition percentage.

2.3.4 FRAP assay

The FRAP assay was performed using the protocol described by earlier researchers [

Data availability

The datasets generated in present study are available from the corresponding author on reasonable request.