Statement of Novelty

Pomegranate juice is widely consumed throughout the world. However, it is not possible to say the same trend for pomegranate waste materials. Therefore, it should be demonstrated that the biological activities of these materials are superior to pomegranate juice to increase the demand for these materials in different industries. In this study, functional properties of pomegranate waste materials including pomegranate peel, pomegranate mesocarp, and pomegranate seed were compared to pomegranate juice.

Introduction

The edible parts of pomegranate fruit (Punica granatum) contain bioactive natural compounds including punicalagins, ellagic acid, anthocyanins, flavonoids, and a wide array of phenolic compounds [1]. The bioactive powers of the pomegranate aril and juice have been attributed to their broad-spectrum natural compound content. There are many studies in the literature reporting the health-benefiting properties of regular pomegranate consumption. It was shown that pomegranate consumption is associated with a lower risk of cardiac diseases, atherosclerosis, tumor formation, and cancer development. The animal studies strongly indicated that rats have a longer life span when they are fed with pomegranate [2] and cell culture studies revealed a strong relationship between healthy cell growth and administration of pomegranate-derived products [3, 4]. In vitro studies also emphasized that pomegranate and its derived products are a precious source of antioxidants, antidiabetics, and antimicrobials structures [5, 6]. These studies were carried out by using more than a single method exhibiting different actions and/or microorganisms having a different cell structure. Because a single method/microorganism in in vitro conditions is not enough to understand exactly the functional property of bioactive compounds. For example, the ant-oxidative effects of bioactive compounds are associated with their different behavior such as reducing agents, hydrogen donors, transition metal chelators [7]. Therefore, it is essential to investigate their potential antioxidant, antidiabetic, and antimicrobial activities with different assays/microorganisms having various mechanisms in order to extrapolate the different behaviors of bioactive compounds. Four different antioxidant activity methods (DPPH, ABTS, FRAP, CUPRAC) were used in this study. The antioxidant assays are evaluated in 2 groups based on reaction mechanisms, namely hydrogen atom transfer and single electron transfer. Radical scavenging (DPPH and ABTS) and reducing antioxidant power (FRAP and CUPRAC) assays are included in single electron transfer methods. These methods investigate the capability of a bioactive compound to transfer an electron to abate any structures (metals, radicals etc.) [8]. The aim is the same for radical scavenging and reducing antioxidant power assays and they give information about the ability of functional compounds to scavenge/break free radicals chains (limit/stop the peroxidation chain reactions) in food and biological systems. In other words, they put forth the ability of functional compounds to break the free radicals chain by donating a hydrogen atom. These explanations clearly showed the importance of selected assays in this study to understand the effects of pomegranate tissues and juice on free radicals chains. Additionally, 2 antidiabetic activity methods (α-amylase, α-glycosidase), and 6 microorganisms for antimicrobial assay were used in this study.

Pomegranate is an industrial fruit crop and currently is being processed into juice and concentrate and there are more than 400 products in the market that contain pomegranate parts or pomegranate derived products. A significant amount of waste comes out during the processing of pomegranate fruits. Pomegranate waste is composed of mesocarp tissue (PM), pomegranate peel (PP), and pomegranate seeds (PS). These wastes represent about 60% of the total fruit weight. A large portion of pomegranate wastes is disposed of without being included in any process. Therefore, environmental and economical concerns arise as the large amount of these wastes generated during pomegranate processing. Additionally, fruit processing establishments suffer considerable waste disposal expenditures. It has been emphasized in the literature that transforming such materials into value-added products is essential and different studies have been conducted regarding this phenomenon for years. These studies mostly cover the extraction of polyphenolic materials having biological properties including antioxidant, antidiabetic, and antimicrobial effects from waste materials with appropriate techniques. The extraction of polyphenolic antioxidants from orange peel [9], lime peel [10], peach waste [11], potato peel [12], soybean processing waste [13], and strawberry and raspberry waste [14] was reported by previous studies. These extracts have the potential for incorporation into different industries such as food, cosmetics, and pharmacology. Similarly, the use of pomegranate tissues for the manufacture of added value products may display economical benefits for the processing plants. Previous studies on pomegranate mainly concentrated on the phenolic content of edible parts, and only some studies reported the antioxidant and antimicrobial activity of phenolics extracted from other pomegranate tissues [15]. Therefore, a comparison of the biological activity of different parts of the fruit with edible parts of the pomegranate may give one insight to evaluate the nutritional value of pomegranate wastes. The pomegranate parts under investigation in this study included pomegranate peel (PP), pomegranate seed (PS), pomegranate mesocarp (PM), and pomegranate juice (PJ). Total phenolic content (TPC), total flavonoid content (TFC), hydrolyzable tannin content (HTC), antioxidant activity (DPPH, ABTS, FRAP, CUPRAC), antidiabetic activity (α-amylase, α-glycosidase), antimicrobial activity (Gram( +)—Gram(−) bacteria, yeast, fungi) of the waste tissues were determined and compared with the PJ. The correlation between biological activity and natural compound content was also evaluated and the individual phenolic compounds present in the distinct tissues and the juice were also identified.

Materials and Methods

Materials

Pomegranate (Punica granatum L., cv. Hicaz) cultivar widely grown in Turkey was purchased from a local grower in Şanlıurfa Province of Turkey. All solvents and chemicals used in spectrophotometric and chromatographic analysis were of standard analytical grade and supplied from either Sigma (St. Louis, MO, USA) or Merck (Darmstadt, Germany) unless otherwise stated.

Phenolics Extraction From Pomegranate Waste Tissues

After peeling the fruit, pomegranate tissues were separated manually. PJ in glass bottles was kept in a freezer at − 20 °C for no longer than 2 months until analyzed. Before use PJ for the experiments, it was centrifuged at 1420 g for 10 min to separate water-insoluble particles. PP, PM, and PS were dried by open-air avoiding from sunlight at room temperature for 5 days, and then pulverized by a Waring commercial lab blender (Conair, Stamford, CT, USA). The pulverized waste tissues were passed through a number of 60 mesh. These tissues were defatted by using hexane as extraction solvent as oils could influence adversely for the extraction of phenolics. For this purpose, 10 g pulverized tissues were mixed with 100 mL hexane in a round bottom flask at 200 rpm in a shaker at room temperature for 30 min. The solvent was removed from the tissues by using a filter paper. The residue was re-defatted in the same conditions. The defatted tissues were dried in an oven at 40 °C and kept in a freezer at − 20 °C for no longer than two months until analyzed. The phenolic extraction procedure used was as follows: 1 g of pulverized defatted pomegranate waste tissues were mixed with 10 mL of the ethanol–water (42–58; v/v) (the ratio of ethanol to water was adapted from pretreatment studies) in a shaker for 30 min at room temperature. After centrifugation at 1420 g for 5 min, the resulting supernatant was filtered through a Whatman No. 1 filter paper. The remaining residue was exposed twice to the extraction process in the same conditions. The extracts were pooled and kept in a freezer at − 20 °C until analyzed.

The Total Content of Phenolic, Flavonoid, Hydrolysable Tannin, and Anthocyanin

The total content of phenolics [16], flavonoids [17], hydrolyzable tannin [18], and anthocyanins [19] in tissues were determined by using a spectrophotometer.

Phenolic Fractions

The tissues were analyzed for their phenolic fractions by using Nexera Shimadzu UHPLC LC–MS/MS (Shimadzu, Kyoto, Japan) coupled to a binary gradient pump, an autosampler (SIL-20AC), a degasser (DGU-20A3R) and a column thermostat (CTO-10ASVP). Chromatographic separations were performed on an Intersil ODS-4 analytical column (3 × 100 mm, particle size 2 μm) at 40 °C using a mixture of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in methanol) as mobile phase at the flow rate of 0.3 mL/min. The elution condition was as follows from 0 to 4 min 5% B, from 4 to 7 min 95% B, from 7 to 7.01 min 95% B and at 7.01 min back to the initial conditions of 5% B. The injection volume was 2 μL. For quantification of phenolics in tissues, calibration curves for each available phenolics were as follows: gallic acid (Y = 65,3835x − 2699,84 R2 = 0.998); catechin (Y = 79,2933x − 2406,22 R2 = 0.999); caffeic acid (Y = 124,785x − 487,132 R2 = 0.995); oleuropein (Y = 25,9240x − 558,916 R2 = 0.998); resveratrol (Y = 46,4361x − 1314,61 R2 = 0.997); p-coumaric acid (Y = 13,1516x + 717,421 R2 = 0.994); ellagic acid (Y = 5,259x ± 1167,31 R2 = 0.994); vanillic acid (Y = 48,0522x − 876,904 R2 = 0.997); salicylic acid (Y = 746,369x + 6072,41 R2 = 0.998); 4-hydroxybenzoic acid (Y = 735,804x − 498,102 R2 = 0.998). MS detection was performed by using a Shimadzu LCMS-8030 quadrupole mass spectrometer operating in both positive and negative the electrospray ionization (ESI) modes with the following parameters: DL temperature, 260 °C; heat block temperature, 400 °C; interface voltage, 4.5 kV; nebulizing gas flow, 3 L/min; drying gas flow, 15 L/min. Data processing and acquisition were evaluated by using LabSolutions software (Shimadzu, Kyoto, Japan) [20].

Antioxidant Activity

Radical scavenging activity was performed by using 1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2 azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS) [21]. The reducing power was carried out by using cupric ion reducing (CUPRAC) [22] and ferric ion reducing antioxidant power (FRAP) [23]. These assays were conducted with slight modifications and results were expressed as µmol Trolox/g for all assays.

For DPPH analysis, 0.1 mL serial dilution of extracts (0.1–100 mg/mL) or Trolox (20–1000 μmol/L) were taken into different test tubes. A 3.9 mL methanolic DPPH solution (0.025 mg/mL) was added to these tubes and vortexed at 15 s. After the vortexed mixture was incubated at room temperature for 30 min, the absorbance was read at 517 nm by using a UV–Vis spectrophotometer (Model UV-1280, Shimadzu Corp., Kyoto, Japan).

For ABTS analysis, ABTS stock solution was prepared by dissolving ABTS (0.96 mg) in 5 mL potassium persulfate (2.45 mM) and 20 mL distilled water. This solution was incubated at room temperature for 16 h and diluted with 0.2 M sodium phosphate buffer for adjusting 0.700 ± 0.02 the absorbance at 734 nm. ABTS stock solution was addd to the different test tubes containing 60 μL diluted extracts (0.1–100 mg/mL) or Trolox (0.1–2 mM) by using the same buffer and incubated at room temperature for 6 min. The absorbance was measured at 734 nm.

For CUPRAC analysis, the mixture of 1 mL 0.01 M copper(II) chloride, 1 mL 7.5 × 10−3 ethanolic neocuproine solution and 1 mL 1 M ammonium acetate solution (pH 7) was prepared and added a volumetric flask containing 0.1 mL serial dilution of extracts (0.1–100 mg/mL) and 1 mL distilled water. After incubation at room temperature for 30 min, the absorbance was determined at 450 nm.

For FRAP analysis, FRAP buffer containing 25 mL 30 mM acetate solution, 2.5 mL 10 mM 2,4,6-Tris(2-pyridyl)-s-triazine, and 2.5 mL 20 mM iron(II) chloride was prepared and mixed with 150 μL serial dilution of extracts (0.1–100 mg/mL) or (40–300 μmol/L). After incubation at room temperature for 30 min, the absorbance was recorded at 593 nm.

Antidiabetic Activity

Alpha-glucosidase and alpha-amylase activities were evaluated by using a combination of enzymatic and colorimetric methods [24]. Results were expressed as IC50 (the concentration of extract required to obstruct 50% of enzyme activity).

For the α-glucosidase analysis, the mixture of 1250 μL potassium phosphate buffer (pH 6.8), and 50 μL α-glycosidase enzyme solution was added into a screw-capped vial containing 50 μL diluted extract (0.1–100 mg/mL) at 37 °C in a water bath. The reaction was started with the addition of 125 μL 10 mM 4-nitrophenyl α-d-glucopyranoside at 5th min. Two mL 0.1 M sodium carbonate was pipetted into this mixture at 20th min in order to terminate the reaction and the absorbance of the resulting pale yellow color was read at 400 nm.

For α-amylase analysis, 1 mL diluted extract (0.1–100 mg/mL), 1 mL starch solution (1%, w/v), and 1 mL 20 mM sodium phosphate buffer (pH 6.9) were pipetted into a screw-capped vial. This mixture was incubated at 37 °C in a water bath for 5 min and then 1 mL α-amylase solution was added. The reaction was stopped by adding 1 mL color reagent (5.31 M sodium potassium tartrate solution prepared with 2 M NaOH and 96 mM 3,5-dinitrosalicylic acid solution). After incubation in a boiling water bath for 5 min, the absorbance of the the resulting orange-yellow to red mixture was recorded at 540 nm.

The extract and enzyme for α-glucosidase and α-amylase analyses were not added to the control and blank samples, respectively.

Antimicrobial Activity

The microorganisms used in this study included Escherichia coli (ATCC 8739), Staphylococcus aureus subsp. aureus (ATCC 6538), Pseudomonas aerugonisa (ATCC 9027), Enterococcus faecalis (ATCC 29,212), Candida albicans (ATCC 10,231), and Aspergillus brasiliensis (ATCC 16,404). The antimicrobial potential of pomegranate tissues was detected by using the agar-well diffusion method. One hundred twenty microliters of PJ and pomegranate waste extracts adjusted to 200 mg/mL concentration by a rotary evaporator were filled in the holes with the size of 9 mm. The microbial count was performed at 0.5 McFarland standard equivalent to give a concentration of 1 × 107 (bacteria), 1 × 105 (fungi), and 1 × 105 (mold) per milliliter. The dilution technique was used to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The pomegranate tissues extracts were serially diluted in the range of 6.25 mg/mL to 200 mg/mL in the plates with bacteria, fungi, and mold grown at 37 °C, 24 °C, and 25 °C, respectively [20].

Data Processing

All the assays were performed at least in triplicate and the results were expressed as mean ± standard error of the mean. The differences between the means were determined by using One-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test at the confidence level of 95% (p ≤ 0.05). Pearson correlation coefficient and principal component analysis (PCA), and as pattern recognition tools were employed to the data. All analyses were carried out by using the Statistical Package for the Social Sciences (SPSS) software (version 22.0 for Windows, SPSS Inc., Chicago, IL, USA).

Results and Discussion

Total Phenolic, Flavonoid, Hydrolysable Tannin and Anthocyanin Content of Pomegranate Waste Tissues and Juice

PJ and the other waste tissues were rich sources of natural compounds. According to the data obtained in this study, PP contained the highest amount of phenolics, flavonoids, and tannins, and followed by mesocarp, juice, and seed. The phenolic content was approximately fivefold higher in the peel (66.1 mg GAE/g), and fourfold higher in the mesocarp (52.2 mg GAE/g) in comparison to the phenolic content of the juice (12 mg GAE/g) (p < 0.05). PS (2.2 mg GAE/g) displayed a much lower phenolic concentration among the assayed samples. The same trend was valid for the flavonoids and hydrolyzable tannins as well (Table 1). Peel and mesocarp tissues included plenty amount of flavonoid and tannin in comparison to seed and juice. However, it should be noted that PJ contained the highest anthocyanin content among the assayed samples. Additionally, no detectable anthocyanin was found in the seeds (p < 0.05).

Table 1 Total phenolic, total flavonoid, hydrolyzable tannin, and total monomeric anthocyanin contents of peel, mesocarp, seeds, and juice
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Phenolic Composition of Pomegranate Waste Tissues and the Juice

Table 2 shows the composition of phenolic compounds present in the pomegranate waste parts and the juice. Gallic acid, catechin, resveratrol, ellagic acid, vanillic acid, 4-hydroxybenzoic acid were found in all of the assayed samples but their quantities were different from each other. The LC–ESI–MS/MS analysis revealed that PP and PM contained the highest amount of phenolic compounds including ellagic acid and catechin. The dimeric form of gallic acid [25] and hydrolysis product of ellagitannins is known as ellagic acid that is classified within the hydroxybenzoic acid group. Ellagic acid is shown to be biosynthesized in the shikimic acid pathway as part of the secondary metabolism [26]. It was reported that ellagic acid exerts cytotoxic action against human promyelocytic leukemia sensitive cell line and its drug-resistant sublines [25]. Ellagic acid improves cholesterol metabolism by inducing upregulation of the low-density lipoprotein receptor (LDLR) gene and reduces apolipoprotein B by downregulating the expression of microsomal triacylglycerol transfer protein [27]. Catechin is distributed in many plant species such as tea leaves, fruits, herbs, and vegetables [28] which is a strong antioxidant compound and involves in anti-cancer, anti-inflammatory, anti-diabetic activities in living organisms [29]. It can be concluded from Table 2 that especially PP contains certain health-benefiting natural phenolic compounds at a higher quantity than the amounts measured in the juice and the same fashion was true for the mesocarp tissue against the juice as well. It is noteworthy that mesocarp tissue possessed a strong anti-diabetic activity compared to the juice (p < 0.05) and could be connected to its richer phenolic content as compared to the juice. The results obtained in this study indicated that PP displayed a stronger antioxidant and antimicrobial activity compared to the other examined waste tissues and the PJ. These phenomena could be linked to its abundant phenolic content.

Table 2 Phenolic fractions of pomegranate tissues
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Antioxidant Activity of Pomegranate Waste Tissues and Juice

The compounds with antioxidant potential have the ability to protect the human body against detrimental effects of free radical species, slow down the aging process and assist to prevent tumor formation and so it is significant to assess the antioxidant activity of plant tissues while evaluating their biological activity [30]. Therefore, many studies in the scientific literature have been conducted with respect to the antioxidant activity of different plants [31,32,33]. The antioxidant potential of pomegranate fruit waste parts and the juice was measured through four different methods including DPPH, ABTS, FRAP, CUPRAC (Fig. 1). The ABTS and DPPH scavenging activity of the pomegranate waste tissues and the juice was varied significantly (p < 0.05). The free radical scavenging activities of both peel and mesocarp were more remarkable when compared to the juice. The DPPH free radical scavenging activity of one gram PP and PM was 850.1 ± 0.8 and 485.6 ± 1.5 µmol Trolox, respectively while the DPPH free radical scavenging activity of PJ was 115.7 ± 2.6 µmol Trolox/g. The lowest antioxidant activity in terms of DPPH assay was found in PS (55.5 µmol Trolox/g). PP contains higher antioxidant structures compared to PS in DPPH test [34]. The highest DPPH radical scavenging activity was determined in PP followed by PS and PJ [35]. On the other hand, the DPPH results for PP were lower than with data reported by Kharchoufi et al. [15]. The authors recorded 4081.4 (methanolic extract) and 3497.0 (aqueous extract) µmol Trolox/g. As for ABTS results, the antioxidant activity was 775.9, 745.6, 37.9, and 50.4 µmol Trolox/g for PP, PM, PS, and PJ, respectively. Elfalleh et al. reported that ABTS radical scavenging activity of PP was 75 μmol Trolox/g and the results were higher than that of PS and pomegranate leaf [36] but lower than our data. In another study, ABTS radical scavenging activity was found as 1361.9 μmol Trolox/g for PP and 2887.1 μmol Trolox/g for PM [37]. DPPH and ABTS assays rely on an electron transfer reaction and require measurement of the color change as a result of reduction of colored oxidant [38]. The ABTS and DPPH analyses are commonly utilized to measure the antioxidant capacities of the plant extracts. The phytochemical composition of the plant extracts is an important factor for determining the appropriate method to select for the reliable evaluation of the antioxidant capacity [39]. We found a strong correlation between antioxidant capacity determined by DPPH and that by ABTS assay with a Pearson correlation coefficient of r = 0.925. Similar findings are reported by previous studies [38]. They found that antioxidant capacity by ABTS strongly correlates to that by DPPH (p = 0.949, p < 0.001). The present study was also tested the antioxidant the reducing power of pomegranate waste tissues and the juice by using FRAP and CUPRAC methods. The correlation between reducing power capacity of FRAP and that of CUPRAC was strong and positive (r = 0.925). As shown in Fig. 1c, d, antioxidant capacities determined by CUPRAC and FRAP displayed significant variations among pomegranate waste parts and the juice. Again their ranking was the same as what we observed in ABTS-DPPH testing and the highest antioxidant capacity was found in PP (FRAP: 755.4 μmol Trolox/g; CUPRAC: 954.4 μmol Trolox/g) and PM (FRAP: 551.4 μmol Trolox/g; CUPRAC: 416.8 μmol Trolox/g) as determined by FRAP and CUPRAC. The lowest FRAP antioxidant capacity was measured in the seed (9.4 μmol Trolox/g) and the lowest CUPRAC antioxidant capacity was measured in the juice (0.15 μmol Trolox/g). It was reported that the PP displays superior antioxidant activity compared to PS and pomegranate pulp as demonstrated by using FRAP assay [40]. Similarly, a previous study confirmed that the antioxidant compounds were noticeably higher in PP than that of PS and PJ [35]. The FRAP assay values of PP obtained from 12 cultivars varied between 1604.8 and 2079.5 μmol Trolox/g [41].

Fig. 1
figure 1

Antioxidant activities a DPPH, b ABTS, c FRAP, d CUPRAC of pomegranate peel, mesocarp, seed, and juice. Different letters (a–d) mean differ significantly for p < 0.05

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Alpha-amylase and Alpha-glucosidase Inhibitory Activities of Pomegranate Waste Tissues and the Juice

Diabetes is one of the prevailing health burdens over the world and persistent hyperglycemia is related to kidney damage, cardiac problems, neuropathy, and diabetic retinopathy [42, 43]. Hyperglycemia is a term that stands for high blood sugar levels and in that condition, an excessive amount of glucose circulates within the blood plasma. Alpha-amylase and alpha-glucosidase are the two main enzymes responsible for the release of oligosaccharides-monomeric sugar units from dietary complex carbohydrates and increase the postprandial glucose level in diabetic patients. Therefore, α-amylase and α-glucosidase inhibitors have the potential roles in controlling of blood plasma glucose levels [44]. Cohort studies strongly indicate the role of plant-based dietary habits in reducing the risk of diabetes mellitus [45] and so WHO suggest daily consumption of plant-based foods with carbohydrates inhibition potency for the management of the diabetic [39]. Many plants were screened to reveal their anti-enzyme potential [46, 47]. The enzyme inhibitory activities of pomegranate waste extracts and the juice were assessed in the present study. The results showed that α-glucosidase and α-amylase activities between pomegranate waste extracts and the juice were significantly different (p < 0.05). In general, all the assayed pomegranate tissues displayed stronger inhibitory activity on α-glucosidase when compared to their inhibitory activity on α-amylase, which is obviously associated with lower IC50 values (Table 3). The strongest α-glucosidase and α-amylase activities were found in PM extracts. The IC50 value of acorbase as positive control was determined in our previous study [20]. The effect of all assayed tissues on α-glucosidase was also higher than that of acarbose but acarbose showed the highest α-amylase enzyme activity. All the assayed waste tissues exhibited higher carbohydrase inhibitory activity than that of PJ possessed.

Table 3 IC50 values of pomegranate peel, mesocarp, seeds, and juice
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Antimicrobial Activities of Pomegranate Waste Tissues and the Juice

The antimicrobial activities of pomegranate waste parts and the juice are given in Fig. 2. The inhibition zone that was created by the extracts varied between 1.3 and 2.9 cm (Fig. 2c). The PS did not display any antimicrobial activity as there was no visible inhibition zone around the seed extracts applied wells. However, Gaber et al. (2015) [48] reported that DMSO, ethanol, and methanol extracts of the pomegranate seed showed antimicrobial activity against E.coli, S.aureus, Aeromonas hydrophila, and Pseudomonas sp. The differences between the results we presented in this study and those that of reported by them may arise from the extraction technique and the extraction solvent that was used to recover the antimicrobial compounds. PP displayed antimicrobial activity against all of the examined reference strains except for A. brasiliensis. The maximum observed antimicrobial activity of the peel extract was against E.coli and the minimum activity was against the C.albicans. Kharchoufi et al. [15] investigated the antimicrobial activity of methanolic and aqueous extracts of pomegranate peel. Methanolic and aqueous extracts brought about varying degrees of antimicrobial activity and indicated that extraction solvents have a profound effect on the biological activity of the extracts. For example, the methanolic extract had an antimicrobial effect against Saccharomyces cerevisiae while aqueous extract did not have any measurable antimicrobial activity against the same assayed microorganism. Extraction solvent may remove the different natural compounds at a distinct concentration from the plant tissues which altogether establish specific antimicrobial strength. PM extract also displayed the highest microbial activity against E. coli and the lowest against the C.albicans and in a similar fashion to the other pomegranate parts there was no visible activity against A. Brasiliensis. The antimicrobial activity of the PJ was different than the antimicrobial activities of the PP and the PM. There was no detectable antimicrobial activity of pomegranate juice against C. albicans and A. brasiliensis. The maximum activity of the juice was against S. aureus and followed by E. coli, P. aeruginosa ve E. faecalis. Similar to our findings Hama et al. [49] also reported the antimicrobial activity of pomegranate juice against S. aureus, E.coli ve P.aeruginosa. One may conclude that PP extract contained the maximum inhibitory activity against the growth of S.aureus, E.coli, P.aeruginosa, E. faecalis, and C. albicans among the assayed samples. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of pomegranate waste extracts and the juice were also shown in Fig. 2a, b. Serial dilutions of pomegranate waste tissue extracts and the juice were employed to determine MIC-MBC values. There were no visible zones in mold cultivated petri dishes. We had elevated the extract amount to 120 µL at 400 mg/mL concentration and still, there was no visible zone and so concluded that pomegranate extracts did not display inhibitory activity against A. brasiliensis at the tested levels. The MIC and MBC values obtained for the C. albicans were quite higher when compared to those of obtained for the bacterium in the current study. The lowest MIC and MBC values were determined in the peel against E. faecalis as < 6.25 mg/mL and 6.25 mg/mL, respectively and indicated its most effective activity among all the tested microorganisms. The maximum MIC and MBC concentrations were determined against C. albicans that is invasive yeast revealed the limited ability of pomegranate waste extracts and the juice against the activity of this microorganism.

Fig. 2
figure 2

Antimicrobial activities a MIC, b IZ, c MBC of pomegranate tissues, d the MIC analysis of mesocarp extract on E.faecalis. Black bar PP, dark grey PM, light grey PJ

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Multivariate Analysis of Phytochemical Content and Biological Activity of Pomegranate Extracts

There are many different polyphenol groups (phenolic acids, flavonoids, and tannins) that have biological activity including antioxidant, antidiabetic, and antimicrobial activities. However, it is not known exactly which groups contribute to which biological activities. In this study, the correlation analysis between phenolic groups (TPC, TFC, HTC, specific phenolics) and biological properties (antioxidant, antidiabetic, and antimicrobial activities) was performed through Pearson Correlation Coefficient. The antioxidant activities analyzed by the radical scavenging activity (DPPH and ABTS) and reducing power activity (FRAP and CUPRAC) showed strong positive correlate (r ≥ 939) with TPC, TFC, and HTC, although the functioning of the methods are different from each other [50]. The results clearly demonstrated that antioxidant activity could be associated with the main phytochemicals (phenolics) having redox potential [51]. This approach is in accordance with previous findings [52, 53]. However, antioxidant activity has less correlation with total phenolic content in Hibiscus sabdariffa [54]. The authors reported that antioxidant activity of Hibiscus sabdariffa is due to its individual phenolic compounds rather than its total polyphenols. Therefore, it is not adequate to examine only the correlation between the total content of polyphenols and biological activities in scientific studies.

Among all individual phenolics determined in PP, PM, PS and, PJ, ellagic acid contents exhibited the highest correlation degree with antioxidant activity and the correlation values were found as 0.993, 0.933, 0.960, and 0.992 for DPPH, ABTS, FRAP, and CUPRAC respectively. Ellagic acid might be evaluated a principally rich source of anti-oxidative agent [55, 56]. The enzyme inhibition activity against α-glucosidase and α-amylase enzymes of PP, PM, PS extracts and PJ was noteworthy negative correlation with the total content of phenolics, flavonoids, and hydrolysable tannins (Pearson’s correlation coefficient α-glucosidase: r ≤ 0.826; α–amylase: r ≤ 0.553) although there was a highly correlation with gallic acid content (Pearson’s correlation coefficient α-glucosidase: r = − 0.994; α-amylase: r = − 0.892). The increase of gallic acid content corresponds to the decrease of IC50 (the increase of enzyme inhibition). Gallic acid has an inhibitory activity against α-glucosidase and α-amylase enzymes in a dose-dependent manner [57]. The antidiabetic activity of PP, PM, PS and PJ appears to be strongly influenced by the individual phenolics compared to the total content of polyphenols. The antidiabetic activities of herbal extracts relate to their individual phenolics [58, 59].

Pearson correlation coefficient indicated negative correlation between microbial resistance and the total content of phenolics, flavonoids, and hydrolysable tannins (Pearson’s correlation coefficient MIC: r ≥ 963; MBC: r ≥ 963) which indicated that these structures highly contributed to antimicrobial activity [60]. Delgado et al. reported that antimicrobial activity of leaf extracts against E. coli is directly associated with total polyphenols (Pearson’s correlation coefficient r = 0.919) [61]. Moreover, individual phenolics, especially gallic acid, ellagic acid, and vanillic acid had strong negative relationship with microbial resistance (Pearson’s correlation coefficient MIC: r ≥ 834; MBC: r ≥ 834). However, S. aureus inhibition appeared to be highly influenced by hydroxybenzoic acid content (Pearson’s correlation coefficient MIC: r = − 918; MBC: r = − 918) but not the total polyphenol contents and the other individual phenolics. Biological activity of individual phenolics is closely related to their antimicrobial activity [62]. Therefore, the different correlations obtained in this study could be explained with the contents and structures (free hydroxyl group) of individual phenolics in the extracts [54]. Honey is rich in phenols and phenolic acids and exhibits strong antioxidant activity but not strong antimicrobial activity, demonstrating that specific structures such as hydrogen peroxide have more effective on the antimicrobial inhibition compared to total phenolics [63]. However, the correlations gave a lead to the conclusion that both total polyphenols and individual phenolics might be considered for the strong antimicrobial activity in this study.

Principal component analysis (PCA), a prominent group of factor analysis is a mathematical argument that aims to facilitate the interpretation of the large dataset by alleviating the dimensionality [64]. This statistical analysis converts the real dataset into new uncorrelated factors known as the principal component [65] and lets us to data evaluation for finding the relationship among all the variables obtained during research analysis [66]. PCs making up from the linear combination of the original data are usually placed on the axes depending on two-dimensional or three-dimensional. Each PC explains more the data variations than its predecessor i.e. PC1 permits us to more the data variation than PC2, PC2 permits us to more the data variation than PC3 and so on so forth [64].

PCA was employed on 25 different variables including TPC, TFC, HTC, antioxidant activities, antidiabetic activities, antimicrobial activities, and individual phenolics. The dimensionality of twenty-five partially correlated responses was decreased to two uncorrelated PC1 and PC2 with 1% of loss variation. PC1 and PC2 represented 78 and 21% of the total information, respectively (Fig. 3). So that, the first two PCs were responsible for 99% of the total variance. Many responses were associated with each other and some of them provided minor contributions. According to the scores of PC1 and PC2, the responses might be divided into two groups. Left part of the PC1 represented the first large group, consisted of antimicrobial and antidiabetic activities. The second large groups including individual phenolics and antioxidant activities were conglomerated on the right part of PC1. On the negative part of PC1 and positive part of PC2; antimicrobial and antidiabetic results were grouped. Positive part of PC1 and negative part of PC2 were correlated mainly phenolics and antioxidant results (ABTS and FRAP), pointing that phenolics had an incredible effect on antioxidant activities. Except for salicylic acid content and S. aureus inhibitions, other responses were uncorrelated exactly with PCs. Among all of the responses, only catechin, caffeic acid, p-coumaric acid, and hyroxybenzoic acid contents were correlated to the PC2 axis. The results could be assumed that this statistical analysis helps to evaluate for differences and similarities among responses by reducing the dimensionality.

Fig. 3
figure 3

Bi-plot from principal component analysis of analyzed 25 parameters of the pomegranate tissues for the first two principal components

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Conclusion

Considerable attention was given to studies on pomegranate as the reports indicate its positive effects on human health. Still, much work needs to be done to better elucidate phenolic contents, antioxidant, antidiabetic, and antimicrobial activities of distinct pomegranate tissues. Whereas PJ is widely consumed in our diet, a similar situation is not told for the other tissues: PP, PM, and PS. However, the results obtained in this study clearly showed that they could be a plausible alternative to the PJ. Different pomegranate tissues had a variety of phenolics and biological activities. PP possessed higher total content of phenolics (66.1 mg GAE/g), flavonoids (7 mg CE/g), hydrolyzable tannins (102 TAE/g) with beneficial effects on human health and biological activities compared to PJ (TPC: 12 mg GAE/g; TFC: 1.1 mg CE/g; HTC: 10.4 mg TAE/g). PM (TPC: 52.2 mg GAE/g; TFC: 5.6 mg CE/g; HTC: 72.3 mg TAE/g) also displayed a similar superiority. Ellagic acid was common individual phenolics in PP (1034.7 µg/g), PM (590.8 µg/g), PS (81.1 µg/g), and PJ (26.5 µg/g). The high antioxidant, antidiabetic, and antimicrobial activity of PP and PM could be associated with these bioactive compounds. In general, all samples were ranked as PP > PM > PJ > PS according to antioxidant and antimicrobial activities. The multivariate analyses also supported these phenomena. The biological activity results could be estimated indirectly by using polyphenol contents as a high correlation with those of these bioactive compounds was observed. Additionally, the data set was reduced to two components by using multivariate analysis (PCA), showing that it was useful for the discrimination of the results obtained. All these results recommended that pomegranate tissues could be considered as a natural functional ingredient that possesses valuable bioactive compounds instead of considering them as wastes. The consumption of PP, PM, and PS should be increased. For this purpose, further studies are essential for the incorporation of these materials and/or their extracts into food components such as dairy products. Additionally, different statistical analyses such as partial least squares algorithm could be applied to determine the correlation among other biological activities including the anti-inflammatory, anti-proliferative effects both in vitro and in vivo in further researches.