Preliminary qualitative phytochemical screening of fruits would be a useful measurement in quantitative estimation of chemical constituents in plant material leading to assess their potential as sources of pharmacologically active chemicals. In the present research, preliminary phytochemical screening tests revealed the presence of polyphenols, tannins, flavonoids, saponins and alkaloids in different levels in the tested underutilized fruits. Polyphenols are a major group of phytochemicals with the ability to act as free radical scavengers to combat oxidative stress and prevent cellular damage caused by free radicals. Antioxidant activity of polyphenols in fruits is beneficial in preventing certain chronic diseases like coronary heart diseases, cancers, and diabetes [14]. Fruits containing tannins not only have free-radical scavenging properties but also possess antimicrobial, antiviral, and anti-inflammatory properties [15]. Saponins, commonly found in many plants, are believed to have a wide range of biological activities, including antioxidant, anticarcinogenic, and immune-stimulant properties hence have the potential to treat several diseases [16]. Alkaloids in fruits have properties, including antibacterial, anti-inflammatory, and analgesic effects [10].
The solvents used in extracting plant secondary metabolites affect the efficiency of extraction as different compounds have varying polarities and solubility properties [17]. Therefore, the selection of an appropriate solvent for the extraction of bioactive compounds in fruit samples is challenging. In this study, three solvents, methanol, acetone and water were used for extraction.
This research has proven the possess of high total phenolic content in 10 studied underutilized native fruit species compared to the commercially available most common fruits that are imported to Sri Lanka. TPC values of red apple, red grape and orange are 73.96, 80.28, 77.23 mg GAE/100 g FW, respectively [18]. Australian grown ripe Cavendish banana pulp has a much lower TPC value of 0.43 mg GAE/g FW [19]. Mango often recognized as “king of the fruits” has a TPC range of 1.39 − 0.32 mg GAE/g FW (Liu, 2013). Date fruit cultivars have comparatively high TPC values ranging between 100–350 mg GAE/g FW [20]. Interestingly in the present research P. pussilla a wild date species exhibited the highest TPC content of 169 mg GAE/g FW in acetone fruit extracts. Fruits of S. caryophyllatum had TPC values ranging between 1.72–8.92 mg GAE/g and aligned with previously reported findings [21].
The yield of total phenols can vary depending on the extraction method and the choice of the solvent. Additionally, factors such as season, genetics, and agronomic conditions, maturation stages, temperature and rainfall can influence TPC in plant tissues [22].
The total flavonoid content of the 10 fruit extracts was determined using Aluminium chloride method that based on the reaction of aluminum chloride with the carbonyl group of flavonoids to form stable complexes. Flavonoids are a class of compounds including flavones, flavanols and condensed tannins. These compounds are known for their potential health benefits, including protection against diseases associated with oxidative stress. Epidemiological studies suggest that the consumption of flavonoid-rich foods can offer protection against diseases related to oxidative stress [23]. Total flavonoid contents of fresh fruit extracts ranged between 0.01–1.00 mg QE/g FW. A study in Burkina Faso, flavonoid contents of banana varieties varied from 1.7 to 116.05 mg QE/100 g for methanolic extracts and from 5.3 to 155.9 mg QE/100 g for acetone extracts. It was observed that acetone was more effective in extracting flavonoids compared to methanol. Present research also showed that flavonoids in the studied fruits are more extractable by acetone than methanol and the findings are in accordance with previous reports [2]. In early research it was reported that flavonoids have low solubility in aqueous media, and this can be a reason for low TFC obtained in the water extracts [24].
There are numerous methods that have been established for quantitative measuring of the antioxidant capacity of food and biological samples [25]. As there are various mechanisms for inhibiting oxidation, it is important to use different assays to gain a more comprehensive understanding of the antioxidant properties of samples [26]. Hence the antioxidant activities of the selected 10 underutilized fruits were analyzed by means of the free radical scavenging capacity (DPPH), the ferric reducing antioxidant power (FRAP) and the ABTS radical cation scavenging capacity (ABTS) for the 10 fruit extracts.
DPPH assay is a widely used method for determining the antioxidant activity of substances including fruits [2]. It measures the ability of antioxidants to donate hydrogen atoms to the stable free radical 2,2-diphenyl-1-picrylhydrazyl, thereby reducing it [27].. The antioxidant activity of 10 different fruits was tested and the DPPH free radical scavenging activity values ranged between 0.69–280.31 TE µ mol/g FW for the three solvents used in this research. The results showed that acetone extracts generally had higher radical scavenging capacity compared to methanol extracts, with water extracts showing the lowest activity. Similar studies on the antioxidant activity of fruits using the DPPH assay have been conducted in Turkey, methanol extracts of five black berry cultivars have shown higher values than aqueous extracts [28]. In Burkina Faso acetone extracts of 15 wild edible fruits exhibited high DPPH scavenging activities. In Ecuador guava, strawberry, passion fruit and mango had DPPH radical scavenging capacity values of 30, 11, 0.5 and 3.1 TE µ mol/g FW, respectively. The 10 wild fruit species in the present study had greater values than these common fruits suggesting higher antioxidant potential.
FRAP is a method used to directly measure the reducing potential of antioxidant compounds in a sample. It involves reacting antioxidants in a sample with a ferric tripyridyltriazine (Fe3+-TPTZ) complex which results in the formation of coloured ferrous tripyridyltriazine (Fe2+-TPTZ) [12]. FRAP values of the fresh fruit extracts studied in this research were found to range from 1.29–294 FeSO4 µmol/g FW. It was observed that the acetone extracts have higher FRAP values in most of the tested fruits and in agreement with previously reported findings[2]. The antioxidant activity of regularly consumed fruits, apple, banana and grapes was studied using FRAP assay and obtained values for methanolic (50%) extracts as 3.94, 1.64 and 8.29 FeSO4 µmol/g FW, respectively [29]. Interestingly FRAP values of the methanolic extracts of most of the fruits studied in the present research were higher than the above findings. Several fruit cultivars in Sri Lanka were tested for the antioxidant activity and has proved that the FRAP values were higher in methanolic fruit extracts (80%) of the underutilized fruits Phyllanthus emblica, Averrhoa carambola and Annona squamosa by having FRAP values of 1022.05, 46.75 and 41.50 FeSO4 µmol/g FW, respectively [30].
ABTS assay is described as an excellent tool for measuring the antioxidant activity of hydrogen-donating antioxidants [31]. The antioxidant capacity of the fruits studied using ABTS assay in this research, ranged from 2.04–241.16 TE µ mol/g FW. Similar to FRAP assay, in the ABTS assay, acetone extracts of most of the fruits have higher compared to aqueous and methanol extracts. Further, it was reported that ABTS activity of water and methanolic extracts of raspberry cultivars in Turkey with values ranging between 64.36–83.00 TE µ mol/g FW and 72.92–117.07 TE µ mol/g FW, respectively [28]. In another study on ABTS antioxidant activity of several fruits including apple (red delicious), avocado, banana, orange (South Africa) and grape (USA), obtained values were 4.98, 1.16, 3.44, 4.90 and 1.23 TE µ mol/g FW, respectively [18]. Interestingly, those values were lower than the ABTS antioxidant activity of most of the wild indigenous fruits studied in the current research.
The antioxidant activity of most polar solvent extracts (such as water, methanol and acetone) is relatively higher than those of non-polar solvent extracts. The type and polarity of the extracting solvent can significantly impact the antioxidant activity of a sample. Some of the tested fruits have high Trolox equivalent values indicating strong antioxidant activity. However, it is noted that their total phenolic content, measured as GAE, is low. This suggests that these fruits may contain other potent phenolic antioxidants that contribute to their high antioxidant activity, even if their total phenolic content is not particularly high.
Ascorbic acid is considered as an antioxidant and is known for its role in preventing oxidative damage in the body. A deficiency of ascorbic acid can lead to health problems and is recommended daily intake of vitamin C as 75 mg for women and 90 mg for men [32]. The 10 fruit species studied in the present research contained varying amounts of ascorbic acid ranging from 21.72 to 489.03 mg/100g FW. These fruits have shown the potential to be used as source of natural ascorbic acid. Ascorbic acid content of the studied fruits is higher compared to that of commonly available fruits in Sri Lanka, such as Mangifera indica (30.8 mg / 100 g FW), Ananas comosus (15.1 mg/100 g FW), Musa paradisiaca AAB “Mysore” (2.3 mg/100 g FW), Persea americana (5.0 mg/100 g FW) and Nephelium lappaceum (18.5 mg/100 g FW) [33]. Wild edible fruits from the Indian Himalayan region such as Phyllanthus emblica (3315 mg/100 g FW) followed by Morus alba (2953 mg/100 g FW), Ficus palmata (727 mg/100 g FW) and Terminalia chebula (626 mg/100 g FW) have showed high ascorbic acid contents [34].
The present study reveals that indigenous fruit species studied, P. pussilla, A. ghaesembilla, A. alexiteria and S. caryophyllatum, have significant antioxidant properties and can potentially be used in various industries such as natural food colours, cosmetics and pharmaceuticals. In proximate analysis, nutritional composition, moisture, ash, protein, carbohydrates, energy and fat percentages of 10 underutilized fruit species were determined. In a previous study conducted for six underutilized Arecaceous fruits in Brazilian region (Acrocomia intumescens, Pinanga kuhlii, Ptychosperma macarthuri, Syagrus cearensis, Syagrus coronata, and Veitchia merrillii)), revealed the moisture level of fruit pulp ranged from 60–75%, except for P. kuhlii (22.9%) and carbohydrate contents ranged from 1.5–20.6% [35]. In another study involved in berry fruits reported a varying moisture content ranging from 86.43% (cherry) to 92.68% (strawberry), ash content varied from 0.08% (blueberry) to 0.42% (cherry), fat content ranging from 0.19% (blueberry) to 0.42% (blackberry), and protein content varying from 0.48% (blueberry) to 1.27% (blackberry) [36]. In contrast, the fruits tested in the present study are noted to have low moisture content and high levels of ash, carbohydrate and protein. This combination of low moisture content can contribute to longer storage periods without perishing, making these fruits suitable for extended shelf life.
Furthermore, research on antioxidant properties of underutilized fruits in Sri Lanka is rare indicating lack of scientific data on bioactivity of secondary metabolites [33]. The present research represents a significant effort on this aspect, as it is the first attempt to investigate the total phenolic contents, flavonoid contents and antioxidant activities of several underutilized fruits such as P. pussilla, A. ghaesembilla, A. alexiteria and Z. oenoplia using three different solvent extracts. The research confirmed that these underutilized fruits are valuable in their nutritional and health promoting properties. The fruit species studied in this research have high potential to be commercialized in Sri Lanka, undoubtedly could help increase food and nutritional security, especially in urban and rural communities and promote local cultivation and consumption. Dissemination of knowledge on health promoting properties of these underutilized fruits could encourage their cultivation, commercialization and consumption. Moreover, this envisioned the potential strategy to address the growing issue of non-communicable diseases in Sri Lanka.