Appraisal of wild fig fruits for nutra-pharmaceutical and biological attributes

2.1 Pretreatment, extraction and fractionation

The ripened fruits of wild fig were collected from Soon valley, district Khushab, Punjab, Pakistan (geographical coordinates: 32^o58^/N 72^o15^/E, lowest temperature: 1 °C, highest temperature: 36 °C and annual rainfall: 50 cm). Prior to experimental procedure, collected fruit samples were authenticated by a taxonomist, then washed with tap water and shade-dried. These dried fruits (10.0 g) were milled and immersed in different solvents (100 mL) of varying polarity including absolute methanol, aqueous methanol (80:20, methanol:water), absolute ethanol, aqueous ethanol (80:20, ethanol:water), absolute acetone and aqueous acetone (80:20, acetone:water) for five days to extract bioactives (Ahmad et al., 2011).

After separating insoluble residual biomass through filtration (Whatman, 8µ), recovered extracts were freed from solvents under vacuum using rotovap (EYELA, Tokyo, Japan) to have CCEs which were further fractioned to produce PRFs (Maheshwari et al., 2011). Briefly, each CCE (5 g) was reconstituted in distilled water (100 mL) and processed for sequential extraction with n-hexane (100 mL) to eliminate non-polar components while ethyl acetate (100 mL) was used to purify phenolic bioactives. The additional solvent from each ethyl acetate extract was detached under vacuum using rotavapor to yield phenolic rich fraction (PRF). The recovered CCEs/PRFs were stored at −20 °C.

2.2 Total phenolic content (TPC)

The CCEs/PRFs of tested fruits were analyzed for TPC using Folin reagent method (Singleton and Rossi, 1965). TPC in different CCEs/PRFs were presented as gallic acid equivalents (GAE g per 100 g dry matter). Standards and samples were run in triplicate under same experimental protocols.

2.3 Total flavonoids content (TFC)

The CCEs/PRFs of tested fruits were analyzed for presence of TFC (Zhishen et al., 1999, Zhuang et al., 1992). TFC was given as catechin equivalents (CE g/100 g of dry matter). Standards and samples were run in triplicate under same experimental protocols.

2.4 Antioxidant properties

Antioxidant properties of different CCEs/PRFs, derived from the tested fruits, were assessed by employing various in-vitro antioxidant assays such as radical scavenging ability, reducing capacity and inhibition of peroxidation in lipid. Tepe et al. (2005) was used for DPPH radical scavenging activity (as IC₅₀ value). Reducing capacity was calculated using a protocol described by Yen et al. (2000). Tested extracts were appraised to retard the formation of peroxide in lipids following a method described by Ahmad et al. (2011). This inhibition potential directly correlated with antioxidant capacity of various CCEs/PRFs. All treatments and positive control were run under same experimental protocols.

2.5 Antimicrobial activity

Antimicrobial attributes of tested CCEs/PRFs were performed out against different infective microbes (Bacillus cereus, Escerichia coli, Staphylococcus aureus, Aspergilus niger, Aspergilus flavus and Fusarium oxysporum) using micro dilution broth and disc diffusion methods (NCCLS, 1999, 1997). Bacterial and fungal strains were cultured on nutrient agar (37 °C) and potato dextrose agar (29 °C), respectively. Standard drugs and negative control were also run under same experimental protocols. The growth inhibition zones (mm) of each tested microbe was measured.

Microbial (fungus and bacteria) cultures were grown on nutrient broth (37 °C) and sabouraud dextrose broth (29 °C), respectively, to assess minimal inhibitory concentration (MIC). Sterility control (CCE/PRF + NB + Tween 80), positive control (microbial culture only) and growth control (NB + Tween 80) were also executed under same experimental protocols. MIC value of CCE/PRF can be calculated by finding its lowest concentration that inhibited microbial growth completely.

2.6 Quantification of minerals

The evaluation of valuable minerals in tested fruits was done using inductively coupled plasma-optical emission spectroscopy (ICP-OES) (Link et al., 1998). USEPA method (3051A) was employed for the digestion of sample with a digestion mixture (concentrated HCl: concentrated HNO₃, 1:3) in microwave oven at 182 °C for 9.5 mins followed by cooling, filtration, centrifugation and dilution of final reaction mixture with water to required volume for ICP-OES analysis. High-purity water (18 MΩ cm) was used to prepare standard and sample’s solutions.

2.7 Analysis of organic acids and individual sugars

According to Mahmood et al. (2012), tested fruits were appraised for the presence of organic acid and individual sugars. Briefly, the fruit juice was obtained by blending fresh aliquot (10 g) of fruits with distilled water (20 mL) in electrical blender (Deuron, GL-109) which was spun (3000 g) and then filtered (0.25 µm) to remove the suspended debris. Organic acid profiling of tested fruits juice was carried out by analyzing on HPLC (Varian Pro star, USA) equipped with UV–vis detector and RP-C₁₈ column (250 × 2.0 mm × 1/4″) while individual sugars were estimated by analyzing on HPLC (Shimadzu, Japan) where amino packing column and refractive index detector (10A) were employed. Water was used as diluent in sugar analysis.

2.8 Other biological activities

The tested CCEs/PRFs were evaluated for inhibition of biofilm produced by different bacterial strains (S. aureus and E. coli) (Stepanović et al., 2000). Haemolytic and antithrombotic capacity of tested extracts/fractions were estimated by spectrophotometer method (Yang et al., 2005) and Prasad et al., (2006), respectively.

2.9 Statistical analysis

All the treatments were conducted in three replicates unless stated otherwise and data thus produced was statistically analyzed by analysis of variance (ANOVA) using STATISTICA 5.5 (Stat SoftInc, Tulsa, Oklahoma, USA) software. P ≥ 0.05 was taken as probability value to indicate statistically significant difference.

3.1 Extraction yield

Fruits of wild fig were extracted with a range of extracting solvents to recover extractable bioactives of varying polarity. The yield of CCEs (g/100 g) were determined to vary considerably in the range of 3.88–53.25 %. Hydroxyethanol recovered the highest yield (53.25 %) and the lowest yield (3.88 %) was extracted with absolute acetone. The tested fruit’s CCE are in crude form and thus contain some moieties of interfering components that offer resistance to detect and consequently alter their antioxidant properties. Therefore, these interfering impurities were removed from CCEs by sequential liquid–liquid extraction (LLE) to get PRFs whose yield was found to range from 2.70 g/100 g to 19.00 g/100 g of CCE. The maximum yield (19.00 %) was recovered from ethanolic CCE while hydroxymethanolic CCE produced the lowest yield (2.70 %) of PRF (Table 1).

Hydroxyethanol recovered higher amount of bioactives from tested fruits exhibiting its superior efficacy as compared to other extracting solvents. The yield of bioactives using different solvents was varied significantly (p < 0.05). Manzoor et al., (2013) and Shabir et al., (2011) showed higher efficiency of hydroxymethanol and hydroxyethanol to extract bioactives from different plant matrices. In this analysis, the efficiency of hydroxymethanol and hydroxyethanol was found to be better than other extracting solvents for optimum recovery of antioxidant molecule from tested fruits.

Soni et al. (2014) recovered the yield (11.47 %) of crude extract from the fruits of fig using methanol which is in lesser quantity compared to methanolic extract of current analysis. This difference in extraction yield has been attributed to various factors including agroclimatic conditions, type of soil and content of bioactive components (Mahmood et al., 2012).

3.2 Total phenolic content (TPC) and total flavonoids content (TFC)

The antioxidant activity of various plants is attributed to the natural occurrence of physiologically active components including flavonoid and phenolic components in them (Katalinic et al., 2006). Due to antioxidant activity, plants have extensive application in food industry. TPC and TFC in tested CCEs/PRFs were calculated to be varied from 0.07 to 1.87 % GAE and 0.01 to 0.13 % CE, respectively. Hydroxyethanol-derived CCE exhibited maximum TPC (1.87 % GAE) and TFC (0.13 % CE) and acetone-derived CCE displayed the lowest of TPC (0.07 % GAE) and TFC (0.01 % CE). (Table 1).

As a result of fractionation of tested fruit’ crude extracts, impurities were removed to produce PRFs in which TPC and TFC varied in the range of 2.24–3.80 GAE (%) and 0.35–0.53 CE (%), respectively. The maximum TPC (3.80 % GAE) and TFC (0.53 % CE) was exhibited by PRF derived from hydroxyethanolic CCE, while the lowest TPC (2.24 % GAE) and TFC (0.35 % CE) was displayed by PRF derive from hydroxymethanolic CCE, respectively. TPC was considerably (p < 0.05) varied in CCEs/PRFs, extracted with different solvents from tested fruits. In this analysis, phenolic components in PRFs were found to be significantly enhanced (3–4 times) than their corresponding CCEs which is ascribed to the successful fractionation CCEs through LLE.

The determination of TPC and TFC from tested fruits was scarce. The results of TPC of wild fig fruit was higher when compared to those of various cultivars of fig fruits by, (Nakilcioğlu and Hışıl, 2013), (Çalişkan and Aytekin Polat, 2011), (Bey et al., 2013) and (Soni et al., 2014), nevertheless, lesser to results of Pourghayoumi et al. (2012). TFC in fig fruits was in lesser quantity than the results reported by (Nakilcioğlu and Hışıl, 2013). These differences in concentration of TPC and TFC in various plant matrices can be associated to different factors including genetics, harvesting season, maturity stage and agro-climatic regions.

3.3 Reducing capacity

The CCEs/PRFs, recovered with different extracting solvents from fig fruits, were explored for their ability to reduce iron (III) into iron (II) by phenolic compounds in them which have been documented to behave like reducing agents to convert ferric into ferrous (Joshi et al., 2010). Therefore, reducing ability (antioxidant attributes) of plant matrices might be correlated to phenolic identities in them (Zou et al., 2004). By enhancing the concentration of CCEs/PRFs, a regular increase in its reducing capacity was recorded (Table 2). There was a notable (p < 0.05) difference in the reducing capacities of tested CCEs/PRFs. Similar trends of enhanced reducing capacity with increasing concentration of extracts was also recorded by Ahmad et al., (2011).

3.4 Free radical scavenging ability

DPPH is stable free radical whose λ^max falls in visible region (515–528 nm) that turns to yellow by accepting hydrogen from phenolic component present in plant extract. Therefore, free radical scavenging capacity has been documented to be enhanced with the increase of bioactives in the extract (Larrauri et al., 1999). Wild fig’s CCEs/PRFs were explored for their ability to scavenge free radical and the results thus obtained were presented as IC₅₀ values that ranged from 0.15 to 1.96 mg/mL and 0.04 to 0.23 mg/mL, respectively (Table 2).

The hydroxyethanol-derived CCE/PRF showed lowest IC₅₀ value (0.15 mg/mL, 0.04 mg/mL) representing their maximum radical scavenging capacity compared to other extracts/fractions. BHT was also run along with other treatments and its radical scavenging ability was found to be higher than those of investigated CCEs/PRFs. PRFs of tested fruits exhibited higher (p < 0.05) free radical capacity (0.04–0.23 mg/mL) compared to CCEs (0.15–1.96 mg/mL) showing their higher antioxidant attributes which is ascribed to greater amount of phenolics in them as a result of successful fractionation of CCEs. The capacity of plant extract to scavenge free radicals has been found to be directly associated with their phenolic content (Siddhuraju et al., 2002).

Wild fig fruit’s CCEs/PRFs were evaluated for the first timeevaluated to scavenge free radical. The antioxidant properties (IC₅₀ value) of tested fruit was lower compared to the results of Uddin, (2013), Faleh et al. (2012), Nakilcioğlu and Hışıl. (2013) and Pourghayoumi et al. (2012). These differences in DPPH radical scavenging ability of various plant matrices can be associated to different factors including genetics, harvesting season, maturity stage and agro-climatic regions.

3.5 Inhibition of lipid peroxidation

The oxidation of lipids produces peroxides which combine with thiocyanate to give a complex (λ^max = 500 nm). The tested CCEs/PRFs were appraised to hinder the formation of peroxide in lipids which were found to vary over the range of 37.05–65.88 % and 70.46–82.22 %, respectively. Hydroxyethanol-derived CCE/PRF inhibited the formation of peroxides to the highest levels 65.88 % and 82.22 %, respectively, while the minimum potential was observed for acetone-derived CCE (37.05 %) and ethanolic PRF (70.46 %). Hydroxyethanolic CCE/PRF of tested fruits were found to hinder the formation of peroxides in linoleic acid representing their higher antioxidant properties which is positively correlated with occurrence of higher amount of phenolics in them. Positive control (BHT) was also processed with other treatments to retard the formation of peroxides in linoleic acid which was found to be higher (92.01 %) than other tested CCEs/PRFs of tested fruits. PRFs exhibited superior inhibition potential than those of CCEs, which is an indication of successful fractionation of CCEs to purify phenolic identities. The findings of this study could not be compared with others due to unavailability of reports in the literature.

3.6 Antimicrobial attributes

The occurrence of potent antimicrobial compounds in plants makes them applicable in folk medicinal system to cure different ailments (Ahmad et al., 2011; Hussain et al., 2011, 2008). In order to detect these potential medicinal compounds, antimicrobial properties of CCEs/PRFs were determined by performing disc diffusion plate assay and measurement of MIC values (Table 3). The lowest concentration of bioactive component with minimum microbial growth is referred as MIC (Walsh et al., 2003).

The tested CCEs/PRFs were assessed to be most effective against S. aureus with bigger inhibition zone (7–17 mm and 15–18 mm) and crossponding lower MIC values (182–247 µg/mL and 168–193 µg/mL), respectively. Among others, hydroxymethanolic CCE/PRF showed lowest MIC value (182 µg/mL and 172 µg/mL) representing their maximum antibacterial properties against S. aureus, while acetone derived extracts showed minimum antibacterial attributes with higher MIC value (223–258 µg/mL). Positive control was also processed under similar set of experimental conditions of treatments and exhibited higher (p < 0.05) antibacterial potential compared to tested CCEs/PRFs.

The tested CCEs/PRFs were most effective against F. oxysporum with bigger zone of inhibition (6–17 mm and 11–18 mm) and corresponding lower MIC values (187–273 µg/mL and 169–209 µg/mL), respectively. CCEs/PRFs showed lowest antifungal properties against A. flavus with smaller zone of inhibition (6–12 mm; 11–14 mm) with greater MIC (206–280 µg/mL; 201–245 µg/mL), respectively (Table 4). In comparison to others, hydroxyethanolic CCE/PRF exhibited maximum antifungal attributes indicating higher concentration of intrinsic bioactive compounds. Various biological properties including antioxidant and antimicrobial potential of different plants have been linked with the natural occurrence of bioactives in them (Ahmad et al., 2011).

3.7 Inhibition of biofilm formation

Different bacterial strains have been known to exhibit biofilm formation for their improved resistance against a variety of antibiotics which is serious environmental and health concerns. Isolation of different medicines from plant matrices have gained the attention of scientists to control various infectious diseases caused by multiple-drug resistant bacterial strains (Prabu et al., 2006).

In this experiment, tested CCEs/PRFs were applied against E. coli and S. aureus to retard biofilm formation and their efficacy was found to vary from 26.15 to 78.34 % and 63.29 to 80.71 %, respectively (Table 5). The maximum biofilm inhibition potential (76.16–78.34 % and 72.67–80.71 %) was expressed by CCE/PRF, recovered with hydroxyethanol, and acetone-derived CCE (26.15–45.93 %)/methanolic PRF (63.29–66.13 %) showed the lowest potential to inhibit biofilm formation compared to other extracts/fractions. Hydroxyethanolic CCE/PRF showed better biofilm inhibition potential which might be associated with the occurrence of higher phenolic contents. Due to the pre-concentration, PRFs exhibited superior biofilm inhibition potential than CCEs. The rifamycin (reference drug) showed higher inhibition potential (87.91–89.43 %) compared to tested CCEs/PRFs. The findings of this study could not be compared with others due to unavailability of reports in the literature.

3.8 Thrombolytic efficiency

The use of synthetics drugs to cure the problem of thrombus in living organisms has some severe health concerns. Therefore, a variety of medicinal plants have been explored to exhibit their potential to treat thrombus which might be associated to the occurrence of potent biological active components (Mclaughlin et al., 1998, Maheshwari et al., 2011).

Tested CCEs/PRFs were evaluated for thrombolytic potential which ranged from 38.74 to 55.21 % and 43.51 to 66.52 %, respectively (Table 5), where hydroxyethanolic CCE (55.21 %) and PRF (66.52 %) showed highest thrombolytic efficiency and the lowest thrombolytic activity was exhibited by acetone-derived CCE (38.74 %) and methanolic PRF (43.51 %). Due to its enhanced phenolic contents, the thrombolytic ability of hydroxyethanolic CCE/PRF was also higher when compared to other extracts/fractions. Furthermore, thrombolytic activity of PRFs has been noted to be higher in comparison to their crossponding CCEs which might be attributed to purification/fractionation of CCEs. Standard drug (streptokinase) was also processed along with other treatments and its thrombolytic potential (87.91–89.43 %) was higher than tested CCEs/PRFs of wild fig fruits. The findings of this study could not be compared as there is no report available on thrombolytic activity of wild fig fruits.

3.9 Haemolytic capacity

Erythrocytes are one of the most important types of blood cell which are used to deliver oxygen and drug molecules to different body tissues (Hamidi and Tajerzadeh, 2003). Oxidative stress has been found to produce hemolysis by damaging the erythrocyte (Ko et al., 1997). Tested CCEs/PRFs were examined for hemolysis which was found to be in the range of 2.17 to 3.32 % and 2.51to 2.72 %, respectively, where lowest hemolysis (2.17 %) was exhibited by ethanolic CCE and the highest (3.32 %) was displayed by acetone-derived CCE, compared to other tested CCEs. Among other PRFs, the minimum hemolysis (2.51 %) was recorded for methanolic PRF and highest hemolysis (2.72 %) was showed by hydroxymethanolic PRF (Table 5).

The damaging effect of tested PRFs on erythrocytes was found to be 1–2.50 times lower when compared to those of CCEs which might be linked with improved phenolic content. The hemolytic potential of PBS (1.54 %) was observed to be lower than CCEs/PRFs of tested fruits.

3.10 Individual sugars and organic acid profiling

Organoleptic characteristics of different plant-derived food is attributed to its individual sugars and organic acids. The concentration of organic acids and individual sugars were found to fluctuate in different phases of maturation (Zhang et al., 2021).

The fruits of wild fig were evaluated for individual sugars and organic acids. Glucose (5.63 %) was detected as the most dominant individual sugar followed by fructose (0.98 %) (Fig. 1). According to the results of present analysis, acetic acid was recorded as major organic acid (2.40 mg/100 g DW) with relatively lower concentration of citric, malic, gluconic and oxalic acids (Tables 6). The varying concentration of organic acids has been documented in different plant based foods due to varietal and difference in weather conditions of experimental sites (Poyrazoğlu et al., 2002). The findings of current assay recommended the consumption of tested fruits as a excellent source of organic acids and natural sugars.

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Fig. 1

Estimation of individual sugars in Fig fruit.

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Slatnar et al. (2011) analyzed fruits of fig cultivars from Slovenia to contain malic (0.52–9.07 g/kg) and citric acids (1.36–10.54 g/kg), respectively. Malic acid (9.89–32.64 %), oxalic acid (1.11–8.54 %) and citric acid (53.06–84.97 %) were found to be present as principal organic acids in Japanese fig cultivars fruit’s juice (Shiraishi et al., 1996).

3.11 Estimation of minerals

The tested fruits were appraised for the presence of useful minerals. Potassium (11339.22 mg/kg) and calcium (4193.69 mg/kg) were detected as major minerals and substantial concentration of other high-value minerals including sodium, magnesium, phosphorus and iron were also present in tested fruits (Tables 7). Sodium and potassium have been reported to be present in the body at suitable concentration for optimum blood pressure (NRC, 1989). The findings of this analysis support the optimum use of tested fruits as a potential source of nutrients for smooth functioning of different physiological processes. Valvi and Rathod, (2011) reported the occurrence of potassium (1922 mg/100 g) as principal mineral along with significant amount of other important minerals including calcium, sodium, iron and magnesium in Indian fig fruit cultivars.

3.12 Correlation analysis

According to Pearson method (P = 0.001), a strong positive correlation was observed between bioactive components (TPC and TFC) and its biological potential including antimicrobial, inhibition capacity, thrombolytic and biofilm activities (P = 0.614–0.867, 0.804, 0.785 and 0.324–0.787), (P = 0.586–0.891, 0.710, 0.782, 0.245–0.685), however, strong negative correlation was also recorded for haemolysis and IC₅₀ value (P = -0.523, −0.762), (P = -0.386, −0.674), respectively (Tables 8, 9). Our research group (Ahmad et al., 2016) reported the presence of eight phenolic acid and flavonoids in tested fruits, therefore the tested biological attributes are directly associated with its TPC and TFC.