Insights into bioactivity guided chemical profiling of Ziziphus jujuba Mill. fruits wild-growing in Montenegro

Abstract

Jujube (Ziziphus jujuba Mill.) is a highly abundant wild-growing plant in Montenegro. It has been utilized since old times for various bioactive properties by the natives, however its detailed chemical characterization, antimicrobial, antioxidant and cytotoxic potential have not been extensively explored. Herein, we used crud methanol extract and three fractions (methylene chloride, n-butanol and aqueous) to asses bioactive features of fruits from this wild growing edible plant, after which we performed the targeted analysis of phenolic compounds of n-butanol fraction by (UHPLC-DAD-MS/MS). Our antioxidant assays showed the highest radical scavenging potential for n-butanol fraction using DPPH and ABTS methods. As for the antimicrobial activity of extract and three fractions, generally aqueous showed the least promising antibacterial and antifungal properties, whereas methylene chloride, methanol and n-butanol fractions showed quite promising antimicrobial potential. E. coli and S. aureus strains were the most susceptible to the compounds present in the methylene chloride and n-butanol fractions with MIC of 0.01–0.025 mg/mL, and MBC 0.025–0.05 mg/mL, along with MRSA strain, which was the most susceptible to the effects of methanol extract with MIC of 0.10 mg/mL and MBC 0.15 mg/mL. The results of antifungal activity showed lower potential to inhibit growth of pathogenic fungi than bacteria, whereas cytotoxicity assay showed extracts have no effects towards HaCaT skin cell line.The n-butanol fraction had the overall most promising activity, and therefore was subjected to more in depth chemical analysis.

Highlights

• •n-butanol extract had the highest share of phenolic and flavonoid compounds in tested samples.
• •Rutin was the most abundant compound in all tested samples.
• •The highest antioxidant potential was observed for n-butanol extracts.
• •All the tested extracts have strong antimicrobial activity towards tested pathogens.
• •All extracts from different localities showed IC₅₀ values greater than 400 μg/mL.

1. Introduction

Ziziphus jujuba Mill., also known as jujube or Chinese date, is a species of Ziziphus belonging to the buckthorn family (Rhamnaceae). Although it originated in southern Asia, it has been grown for more than 4000 years and is now found around the world, especially in the United States and the Mediterranean region. The fruits are tiny, one to two inches in diameter and of oblong, round, or oval shape. They resemble miniature plums or olives. Unripe fruits are green, after which they turn yellowish-green, and then reddish-brown. When jujube fruits are fresh, they taste sweet-tart like apples; when dried, they taste sweeter and more like dates. They're frequently consumed fresh, dried, or processed to make wines, sweets, teas, and syrups. Jujube fruits have a delicious flavor and are rich in vitamins, minerals, and antioxidants. In traditional medicine, jujubes are used for their several health benefits, including stress relief and immune system support [1].

Despite the global achievements in medicine, substantial health burden associated with bacterial and fungal infections is continuously rising. This can be partly associated related to the development of antimicrobial resistance in certain strains of microorganisms, which has recently increased exponentially. Consequently, alternative sources of compounds with antibacterial/antifungal properties have been explored. Plants (both edible and non-edible) emerged as an up-and-coming source of compounds with several noteworthy activities, including antimicrobial. Plant metabolites enable them to survive harsh environmental conditions, which includes protection from pathogenic microorganisms. This suggests that by utilizing plants as a source of antimicrobials, we use their natural defense mechanisms plants for our own needs [2]. Numerous studies have investigated the efficiency of medicinal plant extracts in combat against human and plant pathogens [[3], [4], [5], [6], [7], [8]]. Among them, Z. jujuba turned out to be a promising source of bioactive compounds (>400), mainly in group of flavonoids, phenolics, saponins, triterpenes, and alkaloids, which showed promising biological effects, including antioxidant, anti-inflammatory, antibacterial, antipyretic, antidiabetic, antidiarrheal, anticancer etc. [9]. The fruits of Z. jujuba contain flavonoids such as procyanidin B2, epicatechin, quercetin-3-O-rutinoside (Q-3-R), quercetin-3-O-galactoside (Q-3-G), kaempferol-glucosyl-rhamnoside (K-G-R). As the fruit ripens, the flavonoid content decreases, as does the antioxidant activity [10]. The jujube fruit has a high sugar content and high levels of vitamin C, A, and B complexes, phosphorus and calcium [11]. Phenolic compounds from Z. jujuba exert strong antioxidant effects against free radicals and reactive oxygen species (ROS) and exhibit skin-protective effects as well as potential therapeutic activity against liver injury [12]. Jujube also has neuroprotective properties, improves sleep quality and has a positive effect on learning and memory by reducing inflammation and oxidative stress, as well as through regulation of cholinergic transport and modulation of apoptosis [13].

Traditionally, fruits of Z. jujuba has been used worldwide (including Montenegro) for fever, nausea, liver problems, asthma, abdominal pain, vomiting, wounds, gout, hypertension, rheumatism, and diabetes [14,15]. However, the bioactive properties of the samples originating from the Adriatic coast have not yet been sufficiently investigated. The new results from an unexplored area (Montenegro) should be compared with previous studies on the same species, as the plant is considered commercially important. The obtained results can significantly increase the interest for jujube cultivation in the local communities of Montenegro. Therefore, it is very important to characterise the chemical composition and potential biological activity, as well as to promote the potential beneficial properties of jujube fruit. Additionally, using local products can help preserve traditions and cultural values while supporting local producers. Overall, the study of Z. jujuba is critical to conservation of natural resources, scientific progress, and sustainable development. Therefore, the aim of this study was to investigate (i) the total phenolic and total flavonoid content of phenols and flavonoids in n-butanol, methylene-chloride, methanol, and aqueous extracts of friut of Z. jujuba collected in Montenegro, (ii) assess their antioxidant properties using two assays such as DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)), (iii) evaluate their antimicrobial activity against a panel of pathogenic bacteria and fungi, and (iv) examine their cytotoxic potential using cell viability assay. Furthermore, this study aimed to (v) select the most prominent extracts based on their bioactivity for detailed chemical characterisation using advanced analytical techniques such as HPLC (High Performance Liquid Chromatography) and LC-MS (Liquid Chromatography-Mass Spectrometry). This comprehensive approach should provide new insights into the bioactive compounds in Z. jujuba from Montenegro, their potential health benefits, and their application in the pharmaceutical and food industries.

2. Material and methods

2.1. Standards and reagents and abbreviation list

Solvents for analyses (acetonitrile and formic acid) were LC-MS grade (Fisher Scientific, Loughborough, UK). Methanol and ethanol (HPLC grade) were purchased from AppliChem (Cheshire, CT, USA). The deionization (Millipore, Billerica, USA) system generated ultrapure water. Analytical standards of phenolic compounds (gallic acid, protocatechuic acid, gallocatechin, syringic acid, epigallocatechin, aesculetin, caffeic acid, isoorientin, rutin, vitexin, isoquercetin, ferulic acid, astragalin, apigetrin, luteolin, apigenin, naringenin, and hispidulin) as well as other chemicals were purchased from Sigma-Aldrich (Steinheim, Germany) unless stated otherwise.

The list of abbreviations: ABTS - 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid; BHA - Butylated hydroxyanisole; dH2O - Distilled water; DPPH - 2,2-diphenyl-1-picrylhydrazyl; FC - Folin-Ciocalteu reagents; GA - Gallic acid; GaE - Gallic acid equivalents; HaCaT Cell line - Human immortalized keratinocytes; HPLC - High Performance Liquid Chromatography; K2Cr2O7 - Potassium dichromate; LC-MS - Liquid Chromatography-Mass Spectrometry; MBC - Minimum bactericidal concentrations; MFC - Minimum fungicidal concentrations; MIC - Minimum inhibitory concentrations; MRSA - Methicillin-resistant Staphylococcus aureus; PG - Podgorica locality; QuE - Quercetin hydrate equivalents; TFC - Total flavonoid content; TPC - Total phenolic content; UHPLC-DAD- MS/MS - Ultra high-performance liquid chromatography-mass spectrometry; Ul - Ulcinj locality; ZIN-2018/1 - Z. jujuba fruits collected from Ulcinj; ZIN-2018/2 - Z. jujuba fruits colected from Šušanj; ZIN-2018/5 - Z. jujuba fruits colected from Podgorica; ŠU – Šušanj locality.

2.2. Material collection and preparation of the extract

Fully developed and maturated fruits of Z. jujuba were collected from individual tree in backyard from three localities: Podgorica (PG) ZIN-2018/5 (N43.63278; E19.64933), Ulcinj (Ul) ZIN-2018/1 (N44.16829; E22.10432) and Šušanj (ŠU) ZIN-2018/2 (N44.24138; E19.92472), Montenegro, in September 2018. The fruits of Z. jujuba were provided by Biotechnical Faculty, Centre for Subtropical Cultures, University of Montenegro, Montenegro. A total of 25 healthy fruits per location were separated from the pit and dried for five days at 25 °C for further analysis. Dry fruit flesh was pulverized into a powder (Waring 8010 ES) with methanol [16]. In brief, fruit flesh powder (10 g) was extracted with 100 ml of listed solvents for 24 h. The extract was filtered through Whatmann No. 4 paper and evaporated under decreased pressure (BÜCHI B-480) at the maximum temperature of 40 °C. Subsequently, crude methanol extracts (2 g) were suspended in distilled water dH₂O (100 mL) in a separating funnel and washed with dichloromethane (3 × 100 mL). The water layer was extracted with n-butanol (3 × 100 mL). The dichlormethane, n-butanol and dH₂O fractions concentrated under reduced pressure and obtained extracts stored at 4 °C till further analysis [17,18]. The extraction yields (v/v) are shown in Table 1. The following analyses used one crude extract (methanol) and three different fractions (dichloromethane, n-butanol and aqueous).

Table 1.

Extraction yields of Z. jujuba.

	Fractions			Crud extract
Localities of the plants collection	n-butanol Yield % (v/v)	Methylene-chloride Yield % (v/v)	Aqueous Yield % (v/v)	Methanol Yield % (v/v)
Šušanj	13	5	2	58
Ulcinj	15	9	5	59
Podgorica	13	5	2	48

Due to the high sugar content, methanol crude extract was fractionated with water and then sequentially partitioned with dichloromethane and n-butanol. We obtained the aqueous fraction, which presumably contained mainly sugars and some polar components. The dichloromethane fraction was used to extract less polar components such as lipophilic compounds. The n-butanol fraction served to dissolve the majority of phenolic compounds, for further detailed chemical analyses using UHPLC-DAD- MS/MS.

2.3. Total phenolic content (TPC) and total flavonoid content (TFC)

The TPC was quantified using the Folin-Ciocalteu (FC) reagent [19]. Briefly, 0.1 mL of the extract and fractions solution (2 mg/ml) was mixed with 0.5 mL of the 10 % FC reagent; after 6 min, 0.4 mL of 7.5 % sodium carbonate was added. After 2 h incubation, the absorbance at 740 nm was measured with JENWAY 7315 UV/Vis spectrophotometer versus a blank sample and TPC was calculated using gallic acid equivalents (GaE)calculated from a gallic acid (GA) calibration curve (10–100 mg/L).

TFC was determined as previously described by Mileski et al. [19]. A reagent mixture of 1 M potassium acetate, 10 % aluminium nitrate and 80 % ethanol (0.58 mL) was mixed with 0.6 mL tested plants solution (5 mg/ml). After 40 min of incubation, the absorbance was measured at 415 nm. TFC were calculated using quercetin hydrate equivalents (QuE) calculated from the calibration curve for quercetin hydrate (10–100 mg/L). The analysis was carried out in triplicates where TPC and TFC were expressed as mg of equivalents per g of dry extract.

2.4. Determination of ascorbic acid

Briefly, 10 g of depitted fruit samples were homogenized and pulverized using liquid nitrogen and extracted with 5 mL of 5 % metaphosphoric acid. The resulting mixture was centrifuged (15 min, 6000 rcf), and extract and fractions were stored no longer than 1 h in the dark. The analysis was carried out in triplicates, and the total amount of ascorbic acid was expressed as mg/100 g of dw [20].

2.5. Antioxidant activity of Z. jujuba extract and fractions

The antioxidant potential of Z. jujuba was evaluated via DPPH and ABTS assay.

DPPH solution (0.9 mL, 0.04 mg/mL) was added to extract stock solutions (0.1 mL, range of concentrations 2–5 mg/mL). After 30 min incubation in the dark, absorption was measured at 517 nm using JENWAY 7315 UV/Vis spectrophotometer. The results were obtained through three repetitions [20]. The decrease in DPPH absorption (presented in %) was calculated using equation Eq. (1): Percentage of absorption decrease = (Ac - As)/Ac x 100; (As) absorption of the sample, (Ac) absorption of the control.

As for the ABTS assay, radical cation content was estimated using the procedure previously described by Džamić et al. [16]. The reaction mixture consisiting 25 μL of the extract and fractions solution (2 mg/ml) and 1 mL ABTS was incubated for 30 min. Subsequently, absorption readings were measured at 734 nm. The ABTS inhibition percentage was calculated from the ascorbic acid (AaE) (Sigma, Burlington, MA, USA) calibration curve (0–2 mg/L) expressed as equivalents per g of dry extract. The measurements were performed in triplicates.

For the DPPH assay, results were expressed as IC₅₀ (mg/mL) values (concentration providing 50 % of antioxidant activity), while the ABTS assay, as vitamin C equivalents (mg of vitamin C per mL). Results were compared with the commercial standards BHA, α-tocopherol and ascorbic acid.

2.6. In vitro antimicrobial assays

The antibacterial and antifungal activity of the tested Z. jujuba fruits were evaluated using the microdilution method in 96-well microtiter plates. The minimum inhibitory concentrations (MIC) and minimum bactericidal/fungicidal concentrations (MBC/MFC) of the extract and fractions were determined [21,22]. The following isolates of Gram-positive Staphylococcus aureus (ATCC 11632), S. aureus (oral isolate), and Gram-negative Escherichia coli (ATCC 35210), Pseudomonas aeruginosa (ATCC 27853) bacteria were used. The following resistant strains were also evaluated: methicillin-resistant S. aureus (IBRS MRSA 011), E. coli (IBRS E003) and P. aeruginosa (IBRS P001). Their cultivation conditions were previously described in details by Kartsev et al. [23]. As for the microfungi, following strains were evaluated: Aspergillus flavus (ATCC 9170), A. niger (ATCC 6275), A. terreus (ATCC 16792), Penicillium ochrochloron (ATCC 9112), P. verrucosum var. cyclopium (food isolate) and Fusarium verticillioides (strawberry isolate). All tested microorganisms are deposited in the Mycological Laboratory, Department of Plant Physiology, Institute of Biological Research "Siniša Stankovic" - National Institute of the Republic of Serbia, University of Belgrade, Serbia.

Streptomycin, Ketoconazole and Previcur energy were used as positive controls. Ethanol solution (30 %) was used as a negative control. The experiments were repeated twice.

2.7. Cytotoxicity towards human keratinocytes (HaCaT cell line)

To evaluate the cytotoxic effect of the tested samples, a crystal violet assay was performed according to the protocol previously described by Stojkovic et al. [24] with modifications. The samples were dissolved in PBS to a final concentration of 8 mg/mL and the absorbance of the dye dissolved in methanol was measured at 570 nm (OD₅₇₀) in a plate reader. The results were expressed as IC₅₀ value indicating 50 % cell viability compared to the untreated control. Potassium dichromate (K₂Cr₂O₇) was used as a positive control and PBS as a negative control.

2.8. Ultra high-performance liquid chromatography-mass spectrometry (UHPLC-DAD- MS/MS) analysis of phenolic compounds of n-butanol fraction

The stock solutions of phenolics standards and ascorbic acid (1 mg/mL) were prepared in methanol and diluted to concentrations of 0.025–0.250 mg/mL. Separation, identification and quantification of the targeted compounds were performed using UHPLC with a tandem mass spectrometry system consisting of Dionex Ultimate 3000 (Thermo Fisher Scientific, Bremen, Germany) with a diode array detector (DAD) coupled to a triple-quadrupole (QqQ) mass spectrometer (MS) equipped with a heated electrospray ionisation source (HESI) (TSQ Quantum Access Max, Thermo Fisher Scientific, Basel, Switzerland). The crude n-butanol extracts were dissolved in methanol (20 mg/mL) and filtered through a 0.45 μm HPLC syringe filter (Lab Logistics Group GmbH, Meckenheim, Germany). Phenolics were identified and quantified according to the corresponding spectral characteristics of analytical standards: molecular ion, mass spectra, characteristic fragmentation, and characteristic retention time. The time-selected reaction monitoring (tSRM) experiment was used for quantitative analysis. Quantification was performed using two MS² fragments for each compound that were previously defined as dominant in the product ion scan (PIS) experiments. The procedure in details (chromatographic conditions, parameters of separation and mass spectrometric quantification) has already been described by Gašić et al. [25] and Mišić et al. [26]. The total amount of each phenolic compound is expressed as mg/kg of extracts DW.

2.9. StatisticalAnalysis

The experiments of the total phenolic and total flavonoid content of phenols and flavonoids, total amount of ascorbic acid and antioxidans properties were performed in three repetitions and the results were expressed as arithmetic mean value standard error of measurement. The results were analyzed using Tukey'sHSD test with p < 0.05. Statistical analyses were performed using SPSS Statistics software (IBMSPSS 148 Statistics for Windows, Version 22.0. Armonk, NY, USA:IBMCorp.).

3. Results

The extraction yields (% (v/v)) are presented in Table 1. Obtained results suggest that the methanol extracts from all three localities yielded a significantly higher amount dry weight then the n-butanol and methylene-chloride fractions (∼10 x and ∼100 x, respectively). As for the differences between localities, the highest yield was observed for the Ulcinj locality regardless to the solvent used.

3.1. Total phenolic and flavonoid content

Total phenolic and flavonoid content were estimated in n-butanol, methylene-chloride, and aqueous fractions as well as methanol extracts, prepared from fruits collected in localities Šušanj, Ulcinj and Podgorica. The obtained results in Table 2. Indicate that the n-butanol fraction had the highest share of phenolic and flavonoid compounds in the samples from all three localities. In the case of the n-butanol fraction, values were in the range of 99.34–155.98 mg GaE/g dw; for methylene-chloride fraction, the total phenolics were in the range of 48.09–76.12 mg GaE/g dw. Aquoeus fraction had a moderate phenolic content for the investigated localities, while methanol extract showed the lowest amount of the total phenolic content for all three localities, in the range of 37.14–41.20 mg GaE/g dw. Previously published results on 70 % (v/v) methanol extracts of jujube fruit showed that the content of phenolic compounds colud vary between the peel and pulp, being 5–6 times higher in the peel than in the pulp, possibily influencing antioxidant activity [27].

Table 2.

Total phenolic and flavonoid content of Z. jujuba extracts.

Assays	Total phenolic (mg GaE/g dw)			Total flavonoid (mg QuE/g dw)
Samples	ŠUa	ULa	PGa	ŠUa	ULa	PGa
n-butanol	155.98 ± 0.01	109.25 ± 0.01	99.34 ± 0.01	49.97 ± 0.00	50.67 ± 0.00	51.95 ± 0.00
Methylene-chloride	56.35 ± 0.01	48.09 ± 0.01	76.12 ± 0.01	10.69 ± 0.01	7.79 ± 0.01-	9.15 ± 0.00
Methanol	37.14 ± 0.01	39.27 ± 0.01	41.20 ± 0.01	41.69 ± 0.00	23.23 ± 0.00	20.18 ± 0.00
Aqueous	55.12 ± 0.01	52.01 ± 0.01	42.33 ± 0.01	7.15 ± 0.00	5.10 ± 0.00	6.62 ± 0.00

^aLocalities: (ŠU)- Šušanj (UL)-Ulcinj; (PG)-Podgorica.

As for the total flavonoid content, results indicate that the tested samples have lower amount of flavonoid than phenolic compounds. As was the case with phenolics, the highest share of flavonoids for all three samples was obtained for n-butanol fraction (49.97–51.95 mg GaE/g dw). The lowest flavonoid content was determined for aqueous fraction, in the range of 5.10–7.15 mg QuE/g dw). Methylene-chloride fraction showed a higher amount of phenolics than aqueous, while methanol was in range of 9.64–23.23 mg QuE/g dw.

The obtained results are in accordance with those published by Choi et al. [28], which suggest that Chinese jujube fruit is rich in phenolic compounds, with methanol extracts prepared from peel and pulp reaching value of 41.315 mg GaE/g dw. Furthermore, differences among the tested extracts made of different solvents for extraction and associated localities have been ascribed to fruit maturity, variety, geographic locations, soil and climatic conditions, with postharvest chemical changes not being detected [29]. Moreover, Wang et al. [30] demonstrated significant differences in the total phenolic content of Z. jujuba across four phenolic fractions (free, esterified, glycosidic, and insoluble-bound) at three edible maturity stages, with values ranging from 71.67 to 864.73 mg GAE/100g dry weight. Regarding flavonoid content in pulp, a wide range of 3.1–149.2 mg QuE/g dw was identified in methanol extracts [28]. According to Singh et al. [31], methanol extract of Z. spina-christi fruit had low-to-moderate amount of phenolics and flavonoids (16.44 mg GaE/g dw and 0.47 mg CeQ/g dw, respectively). Our detected values are somewhere in between, with n-butanol fraction having the highest amount 51.95 mg QuE/g dw, which may be ascribed to different climatic and geographic conditions as well as the solvent type used for the extraction. According to Bencheikh et al. [32], this amount may reach relatively high values: the aqueous extract of Z. lotus fruit had as much as 2242.89 mg QuE/g dw.

3.2. Antioxidant activity

The antioxidant properties of Z. jujuba extracts were investigated using DPPH and ABTS methods, and the results are shown in Table 3. The highest radical scavenging potential in the DPPH assay was observed for n-butanol fraction obtained from fruits collected in Podgorica and Šušanj (IC₅₀ = 3.36 mg/mL and IC₅₀ = 3.50 mg/mL, respectively). Same samples also showed the best antioxidant potential using ABTS method (1.44 mg Vit C/mL dw and 1.42 mg Vit C/mL dw, respectively).

Table 3.

Antioxidant activity of Z. jujuba extracts.

Assays	Antioxidant activity (IC50 values mg/mL)			Antioxidant activity (Vitamin C equivalents values mg/mL)
	DPPH			ABTS
Samples	ŠUa	ULa	PGa	ŠUa	ULa	PGa
n-butanol	3.50 ± 0.06	3.69 ± 0.08	3.36 ± 0.06	1.42 ± 0.03	1.17 ± 0.06	1.44 ± 0.21
Methylene-chloride	10.50 ± 0.01	23.13 ± 0.01	8.11 ± 0.06	0.19 ± 0.17	0.04 ± 0.02	0.31 ± 0.12
Methanol	9.64 ± 0.00	10.58 ± 0.00	9.84 ± 0.18	0.43 ± 0.14	0.44 ± 0.02	0.34 ± 0.06
Aqueous	16.55 ± 0.00	14.76 ± 0.00	17.53 ± 0.04	0.23 ± 0.09	0.23 ± 0.35	0.12 ± 0.08
BHA	0.13 ± 0.05			2.70 ± 0.00
α-tocopherol	0.15 ± 0.00
Ascorbic acid	0.07 ± 0.02			0.11 ± 0.01

^aLocalities: (ŠU)- Šušanj (UL)-Ulcinj; (PG)-Podgorica.

Previously published results regarding jujube antioxidant potential indicate a few times higher activity than we obtained for both (extracts and their fractions). According to Bencheikh et al. [32], the aqueous extract of Z. lotus fruit showed noteworthy antioxidant effect with IC₅₀ = 0.016 mg/mL in the DPPH method. In the case of a 50 % (v/v) ethanol extract of Z. joazeiro fruits, Brito et al. [33] also demonstrated a high free-radical scavenging potential, with an IC50 of 0.73 mg/mL. As for the previously published jujube ABTS results, data suggest that among seven Z. jujube cultivars, peel extracts of the 'Zhanhuadongzao' cultivar possessed the highest ABTS scavenging activity in the range of 37.59–53.74 mg Trolox/g dw [29].

Previously published data on jujube fruits antioxidant potential of several varieties (Z. jujuba, Z. lotus, Z. spina-christi and Z. joazeiro) proved that they possessed a high amount of phenolics and should be recommended as an essential dietary source of natural antioxidants [29,30,[33], [34], [35]].

3.3. Antimicrobial activity

The results of antibacterial activity are presented in Table 4. Overall, they indicate that all tested extracts and fractions have strong activity towards tested pathogens. Aqueous fraction was the least active among the tested samples, with non-determined bactericidal activity towards all tested S. aureus isolates. The best activity was observed for the methylene-chloride fraction from Podgorica locality towards oral isolate of S. aureus, and for the methanol extract from the locality Ulcinj towards E. coli with MIC 0.025 mg/mL, and MBC 0.05 mg/mL. Furthermore, methylene-chloride extract from the fruits collected in Šušanj, Ulcinj and Podgorica were also quite active with MIC 0.05 mg/mL and MBC 0.10 mg/mL towards two staphylococcal isolates (Table 4). Compared to the positive control Streptomycin, this antibiotic showed 10 fold or even higher antibacterial potential for selected strains. However, it colud not inhibit growth of IBRS MRSA 011 strain and S. aureus ATCC 11632, which seems quite important as some of our tested extracts showed activity towards this pathogen at 0.10 mg/mL and 0.15 mg/mL. Our previous publication analyzed results regarding the antibacterial activites of purified phytocompunds, rutin and ferulic acid against antibiotic resistant bacteria. Both compounds showed promising inhibitory potential, with MIC of 0.50–1.00 mg/mL [36].

Table 4.

Antibacterial activity of Z. jujuba extracts (mg mL⁻¹).

Compounds		S.aureus oral	S.aureus ATCC	MRSA	E.coli	E.coli rez	P.a.	P.a. rez
n-butanol ŠU∗	MIC	0.20	0.20	0.50	0.50	0.50	0.20	0.05
	MBC	0.50	0.50	0.75	0.75	0.75	0.50	0.10
n-butanol UL∗	MIC	0.10	0.20	0.20	0.20	0.20	0.10	0.20
	MBC	0.20	0.50	0.50	0.50	0.50	0.20	0.50
n-butanol PG∗	MIC	0.20	0.50	0.50	0.01	0.75	0.20	0.20
	MBC	0.50	0.75	0.75	0.025	1.50	0.50	0.50
Methylene-chloride ŠU∗	MIC	0.05	0.10	0.10	0.20	0.10	0.10	0.20
	MBC	0.10	0.20	0.20	0.50	0.20	0.20	0.50
Methylene-chloride UL∗	MIC	0.10	0.05	0.15	0.20	0.20	0.10	0.20
	MBC	0.20	0.10	0.20	0.50	0.50	0.20	0.50
Methylene-chloride PG∗	MIC	0.025	0.10	0.10	0.10	0.10	0.10	0.50
	MBC	0.05	0.15	0.15	0.20	0.50	0.20	0.75
Methanol ŠU∗	MIC	0.10	0.50	0.50	0.20	0.75	0.20	0.20
	MBC	0.50	0.75	0.75	0.50	1.50	0.50	0.50
Methanol UL∗	MIC	0.20	0.20	0.50	0.025	0.20	0.15	0.20
	MBC	0.50	0.50	0.75	0.05	0.50	0.20	0.50
Methanol PG∗	MIC	0.20	0.20	0.50	0.20	0.50	0.20	0.10
	MBC	0.50	0.50	0.75	0.50	0.75	0.50	0.20
Aqueous ŠU∗	MIC	0.50	0.50	0.50	0.50	0.50	0.15	0.10
	MBC	1.50	1.50	1.50	0.75	0.75	0.20	0.20
Aqueous UL∗	MIC	0.75	0.75	0.75	0.20	0.75	0.20	0.20
	MBC	1.50	1.50	1.50	0.50	1.50	0.50	0.50
Aqueous PG∗	MIC	1.50	n.d.	n.d.	0.20	0.75	1.00	0.10
	MBC	n.d.	n.d.	n.d.	0.50	1.50	1.50	0.20
Streptomycin	MIC	0.0012	0.01	n.d.	0.0025	0.005	0.005	0.005
	MBC	0.0025	n.d.	n.d.	0.005	0.01	0.01	0.01

∗n.d. – not detected.

∗Localities: (ŠU)- Šušanj (UL)-Ulcinj; (PG)-Podgorica.

Regarding antifungal activity, not all of the evaluated extracts and fractions exhibited significant antifungal potential. We demonstrated moderate antifungal potential towards P. ochrochloron for all the tested samples (MIC in the range of 0.50–1.00 mg/mL, MFC in the range of 1.00–2.00 mg/mL) and A. flavus with n-butanol fraction and methanol extract from Ulcinj (MIC in the range of 3.00–4.00 mg/mL, MFC in the range of 4.00–8.00 mg/mL). As for the rest of the tested pathogenic microfungi, the tested samples showed poor activity, or did not show it at all. Furthermore, tested aqueous fraction had no antifungal activity towards all the tested strains. The most abundant compounds in our fractions – rutin and ferulic acid were tested for antifungal activity (Table 5). Both compounds showed antifungal activities with MIC of 0.125–0.50 mg/ml and MFC of 0.0.25–1.00 mg/ml. Compared to the positive control Previcur, our extract, fractions and purified phytocompounds showed superior activity, though the other positive control ketoconazole had better antifungal potential than our ziziphus extracts. Since Previcur is a systemic fungicide that showed developmental toxicity and has a withholding period of a few days after the exposure of crops to its activity, our results which demonstrated antifungal effects for certain extracts are quite promising. Therefore, we provide utterly safe, natural and active extracts with solid antifungal potential towards several pathogenic microfungi.

Table 5.

Antifungal activity activity of Z. jujuba extracts (mg mL⁻¹).

Compounds		A.flavus	A.niger	P.ochrochloron	P.cyclopium	A.alternata	F. verticillioides
n-butanol ŠU	MIC	n.d.	0.75	1.00	2.00	2.00	12.00
	MFC	n.d.	1.00	2.00	4.00	4.00	16.00
n-butanol UL	MIC	4.00	0.75	1.00	2.00	1.00	16.00
	MFC	8.00	1.00	2.00	2.00	2.00	n.d.
n-butanol PG	MIC	n.d.	1.00	0.50	1.50	1.00	12.00
	MFC	n.d.	1.00	1.00	2.00	2.00	16.00
Methanol ŠU	MIC	n.d.	n.d.	1.00	2.00	n.d.	4.00
	MFC	n.d.	n.d.	2.00	4.00	n.d.	8.00
Methanol UL	MIC	3.00	n.d.	1.00	n.d.	0.50	n.d.
	MFC	4.00	n.d.	2.00	n.d.	1.00	n.d.
Methanol PG	MIC	n.d.	16.00	1.00	2.00	n.d.	3.00
	MFC	n.d.	n.d.	2.00	4.00	n.d.	4.00
Rutin	MIC	0.50	0.50	0.25	0.25	0.125	n.d.
	MFC	1.00	1.00	0.50	0.50	0.25	n.d.
Ferulic acid	MIC	0.50	0.50	0.50	0.50	0.125	n.d.
	MFC	1.00	1.00	1.00	1.00	0.25	n.d.
Previcur	MIC	50.0	75.0	25.0	50.0	50.0	50.0
	MFC	100.0	200.0	50.0	100.0	75.0	100.0
Ketoconazole	MIC	1.50	0.20	0.20	0.20	0.20	0.20
	MFC	2.00	0.50	0.50	0.30	0.30	0.50

∗n.d. – not determined.

∗Localities: (ŠU)- Šušanj (UL)-Ulcinj; (PG)-Podgorica.

3.4. Cytotoxicity

Table 6 presents the cytotoxicity results of extract and various fractions of Z. jujuba collected in Šušanj, Ulcinj, and Podgorica in Montenegro, together with a positive control, K₂Cr₂O₇. The IC₅₀ values, representing the concentration at which 50 % of cell viability is inhibited, were determined for methanol extract and n-butanol, methylene-chloride, and aqueous fractions. All samples from different localities showed IC₅₀ values greater than 400 μg/mL, indicating minimal cytotoxic effects under the tested conditions. In contrast, the positive control K₂Cr₂O₇ exhibited a significantly lower IC₅₀ value of 16.29 ± 1.42 μg/mL, confirming its strong cytotoxic activity as expected. Our previous publication analyzed results regarding the cytotoxicity of pure compounds rutin and ferulic acid towards HaCaT cells. Rutin has shown non-cytotoxic properties (IC₅₀ > 1 mg/mL), but on the other hand, the cytotoxic effect was found for gallic acid (IC₅₀ < 0.08 mg/mL) [36].

Table 6.

Cytotoxicity of Z. jujuba extracts.

Samples	IC50 (μg/mL)
n-butanol (ŠU, UL, PG)a	>400
Methylene-chloride (ŠU, UL, PG)a	>400
Methanol (ŠU, UL, PG)a	>400
Aqueous (ŠU, UL, PG)a	>400
K2Cr2O7	16.29 ± 1.42

^aLocalities: (ŠU)- Šušanj (UL)-Ulcinj; (PG)-Podgorica.

These results indicate that the tested Z. jujuba samples from Montenegro are not cytotoxic at the tested concentrations. This implies a potentially favorable safety profile for these extract and fractions, which increases their potential applications without adverse effects on cells. However, further studies are necessary to validate these results across different cell lines, and identify specific bioactive compounds responsible for the observed activities. Overall, this study provides valuable insights into the biological properties of Z. jujuba extracts and their potential applications in medicine and industry. Given their strong antibacterial activity, particularly against staphylococcal strains known to cause skin infections, these samples may have potential for the development of formulations used in topical treatments.

3.5. UHPLC-DAD MS/MS analysis of n-butanol fraction

The results of the chemical analysis are presented in Table 7. Our study showed the presence of 12 compounds in the n-butanol fraction: 11 belonging to phenolics and 1 cyclic polyol. Obtained results indicate presence of 12 compounds across all three samples, regardless of their locality. However, quercitrin was identified exclusively in the sample collected from the Ulcinj locality. Rutin was the most abundant compound in all analyzed samples: Ulcinj locality 620.18 mg/100 g dw, Podgorica locality 596.77 mg/100 g dw and Šušanj locality 551.39 mg/100 g dw. High abundance of rutin was followed by quinic, protocatechuic and syringic acid. The presence of these compounds varied depending on the locality, with quinic acid predominanting in the sample from Ulcinj locality (187.95 mg/100 g dw). In comparison protocatechuic and syringic acid were the most abundant phenolics in sample from in Šušanj (49.54 and 42.21 mg/100 g dw, respectively). These discrepancies among samples collected from different localities may be ascribed to other factors, including maturity stages and various sources of dates, which may affect the conversions of phenolics [35,37]. As reported by Wang et al. [30] rutin content differs depending on the maturity stage of fruits, being higher in the white rather than the red maturity phase (925.83 and 670.63 mg/kg dw respectively).

Table 7.

Chemical analysis of phenolic compounds (mg/kg dw) in n-butanol extract and vitamin C content in fruit flash (mg/100 g dw).

Compound	ŠU∗	UL∗	PG∗
Quinic acid	149.01	187.95	62.48
Protocatechuic acid	24.33	19.48	22.24
Syringic acid	49.54	30.91	32.10
Caffeic acid	10.94	8.45	5.19
Ferulic acid	42.21	34.22	24.06
Rutin	551.39	620.18	596.77
Quercitrin	n.i.∗	7.82	n.i.∗
Isoquercetin	24.40	25.13	27.49
Quercetin	28.20	18.30	11.84
Apigetrin	2.21	1.95	2.84
Aesculin	10.44	8.11	6.95
Hispidulin	n.i.∗	1.12	n.i.∗
Vitamin C	17.4 ± 1.4	9.8 ± 0.2	17.9 ± 1.2

∗n.i.: not identified.

∗Localities: (ŠU)- Šušanj (UL)-Ulcinj; (PG)-Podgorica.

Regarding previously published data on the chemical profile of Z. jujuba, phenolic compounds have generally been detected as the main compounds in various samples [38,39]. However, some results are in accordance to our results, but some show dissimilarities. For example, Pawlowska et al. [40] identified rutin as the most abundant flavonoid compound in methanol extracts of jujube fruits. Contrary to this, Wang et al. [30] and Xie et al. [29] identified gallic, p-coumaric, p-hydroxybenzoic, rosmarinic, chlorogenic, and caffeic acid in phenolic fractions and extracts of Z. jujuba fruits. These compounds were not identified in our samples originating from Montenegro.

3.6. UHPLC-DAD analysis of vitamin C

Since vitamin C shows photosensitivity and thermolability [41], analyses of the target compound were performed using freshly prepared extracts instead of dry ones. The Vitamin C content results are presented in Table 7, and they are accompanied by a figure which contains data from all three chromatograms (supplementary material 1). Results indicate a similar content of this compound in the samples from Šušanj and Podgorica (17.4 and 17.9 mg/100 g dw), but nealry half of this value in the sample from Ulcinj (9.8 mg/100 g dw). Obtained results are in accordance with those suggesting jujuba fruits are valuable source of vitamin C, A and B complex [1]. Even though our sample had a higher share of Vitamin C in comparison to the sample described by Benidir et al. [42] - 0.6 mg/100 g dw, the value we obtained was a few times lower than the value obtained by Zhang et al. [43] for the “Dongzao” - 534.94 mg/100 g dw. Observed differences in vitamin C content between jujube fruits may be affected by postharvest sorting, storage and drying processes as well as fruit maturity at the time of analysis [44,45].

4. Discussion

Jujube contains various nutrients and active substances, including polyphenols, polysaccharides, nucleotides, ascorbic acid, and triterpenoid acids. Recently, numerous in vitro and animal model studies have shown that bioactive compounds in jujube fruit have potential dietary and medicinal benefits for humans. They are especially rich in vitamin C, B-complex, phenols, flavonoids and triterpenic acids [1,46]. Triterpenic acids such as ceanothic, alphitolic, maslinic, oleanolic, oleanonic, corosolic, betulinic, betulonic, ursolic and ursonic acid have been associated with promising anticancer activities of the jujube extracts [47,48]. The alkaloids in jujube exert good antioxidant and antiviral activities [12]. As for the presence of sugars, the most abundant are glucose and fructose, while rhamnose, sorbitol, and sucrose are present to a lesser extent. Regarding mineral content, K, P, Ca, and Mn are highly abundant, while Fe, Na, Zn, and Cu are present in smaller quantities [11,49]. Dried pulp contains essential unsaturated fatty acids, various amino acids, and proteins. All these compounds contribute to various health-beneficial effects of jujube fruits, including hepatoprotective and immunoregulatory, with a significant impact on gut health and blood coagulation [50]. Furthermore, they exert antioxidant [[51], [52], [53]] and antitumor activity [46], affecting particularly melanoma cells [54]. Studies in vitro and on animals shows that essential oils from jujube seeds effectively suppress food-borne pathogens [55], have an anti-inflammatory effect on skin inflammation [56] and positively affect hair growth [57]. In a study by Daneshmand et al. [58] ethanolic extract of the jujube fruit exhibited a broad antimicrobial, antibacterial and antifungal activity. Thus, numerous studies on phytochemical composition and bioactivity rely on various extraction methods and solvent systems. Pu et al. [62] extracted dried jujube fruits subsequently with hexane and ethanol. In order to isolate bitter compounds, the ethanolic fraction was dispersed in deionized water and re-extracted with water-saturated butanol. A total of 35 compounds were identified by LC-MS/QTof analysis (7 nitrogen compounds, 14 flavonoids, 12 saponins, and 2 fatty oxides). Wang et al. [30] used a similar method for the preparation of crude jujube extracts (methanol) and fractionation of free and bound phenolic compounds (aqueous suspension of hydrochloric acid and ethylacetate). Obtained fractions of jujube extracts contained high phenolic content and exhibited good radical scavenging activity.

Aside from having potential for the pharmaceutical industry, Z. jujuba extracts have showed efficiency toward pathogenic microorganisms causing lossess in the food production chain. Given that each year, as much as 40 % of crops are affected by pathogenic microorganisms which reduce yield and qualityof products, there is an urgent need for efficient, yet natural and safe alternatives. The use of ziziphus extracts offers a sustainable approach to reducing these losses, posing no potential hazard to the environment and no detrimental effects on human health.

Our bioactivity guided exploration of active natural products from jujube fruits is a modern approach that enabled us targeted chemical characterization of only the most active extract. Our results revealed that n-butanol extract, which exhibited the highest overall activity with a notable emphasis on its antioxidant potential, is particularly rich in rutin and quinic acid. These compounds are likely responsible for the observed bioactive properties in all three samples collected from different localities. According to Benalli et al. [59], quinic acid indeed exerts potent antioxidant and antimicrobial properties, the latter being achieved by mechanisms that disrupt fluidity of membrane. As for the most abundant compound in our n-butanol extract – rutin, it has several prominent bioactive properties, with antimicrobial and antioxidant to name a few [60,61]. Rutin also plays a key role in reducing biofilm biomass by decreasing cell viability, exopolysaccharide production, and extracellular DNA levels [36]. Therefore, it should be isolated in larger quantities and further tested as a potential bioactive compound in the crude n-butanol extract.

5. Conclusion

Natural products play a pivotal role in the development of novel therapeutic drugs, a trend highlighted by ongoing research efforts. The rich traditional heritage of Mediterranean cultures provides a solid foundation for exploring the bioactive potential of plants such as Z. jujuba, ensuring a legacy of safety and efficacy. Our study contributes to this by highlighting the non-cytotoxic nature of Z. jujuba extracts from Montenegro, suggesting their safety and potential for various applications.

The results of this study underscore the potential of Z. jujuba extracts from Montenegro as a valuable source of bioactive compounds. The findings demonstrate a rich profile of phenolic and flavonoid compounds, with n-butanol fraction having the highest levels of both, particularly in the samples collected from the Ulcinj locality. These extracts showed promising antioxidant activity, especially in the DPPH and ABTS assays, which correlate with the substantial presence of phenolic compounds like rutin and quinic acid. The antimicrobial analysis also highlighted the efficacy of the methylene-chloride and methanol extracts, particularly against bacterial strains, suggesting Z. jujuba extracts could be useful for developing natural antimicrobial agents. The minimal cytotoxicity observed at concentrations greater than 400 μg/mL further supports the safety profile of these extracts, reinforcing their potential as functional ingredients in pharmaceutical and food applications.

This study provides valuable insights into the bioactive landscape of Z. jujuba from Montenegro, emphasizing its non-cytotoxic and bioactive properties, and highlighting the promising roles of rutin and quinic acid. These findings underscore the potential of Z. jujuba as a natural source for antioxidant and antimicrobial agents, paving the way for its integration into therapeutic applications and various other functional products. Further research into it's the molecular mechanisms and bioactivity in different formulations could facilitate the development of new health-promoting solutions.

Integration of various methods used in drug development, supported by fundamental scientific research and traditional knowledge, holds promise for developing therapies from bioactive compounds identified in Z. jujuba and other natural products as to meet changing needs of modern medicine.

CRediT authorship contribution statement

Tatjana Perović: Writing – original draft, Data curation, Conceptualization. Biljana Lazović: Writing – original draft, Resources, Investigation, Formal analysis. Mirjana Adakalić: Methodology, Formal analysis. Ana Džamić: Writing – original draft, Validation, Methodology, Formal analysis, Data curation. Lazar Žarković: Methodology, Formal analysis. Uroš Gašić: Formal analysis, Data curation. Marina Kostić: Investigation, Formal analysis, Data curation. Jovana Petrović: Writing – original draft, Validation, Investigation, Formal analysis. Dejan Stojković: Writing – review & editing, Supervision, Investigation, Conceptualization. Ana Ćirić: Writing – review & editing, Supervision, Investigation, Conceptualization.

Funding

This work has been supported by Ministry of Science, Technological Development and Innovations of the Republic of Serbia (451-03-66/2024-03/200007) and (451-03-65/2024-03/200178).

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Corresponding author Dejan Stojković serves as Associate Editor of the journal Heliyon. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the supplementary data to this article:

figs1.

figs2.