Active components and antioxidant activity of thirty-seven varieties of Chinese jujube fruits (Ziziphus jujuba Mill.)

ABSTRACT

Jujube has received more and more attention due to their nutritional value and pharmacological effects. This study evaluated the quality of different varieties of jujube by comparing the main active components and antioxidant activities of thirty-seven varieties of jujube through titration, UV spectrophotometry and High performance liquid chromatography (HPLC). The results revealed that there were differences in active components and antioxidant capacity among jujube cultivars. Among the thirty-seven varieties of jujube, Qingxuyuanzao (QXYZ) had the highest contents of total phenols and flavonoids, which were 16.33 mg GAE/g DW and 41.18 mg RE/g DW, respectively, and had the highest free radical scavenging (DPPH: 29.79 mg Vc/g DW; ABTS: 50.90 mg Trolox/g DW) and reducing abilities (62.13 mg Trolox/g DW). Correlation analysis indicated that the content of total flavonoids, total phenols and phenolic acid ((+)-catechin, caffeic acid, rutin) in jujube had high positive correlation with antioxidant capacity. According to principal component analysis (PCA), the comprehensive scores of different varieties of jujube were calculated. The top five jujube varieties with comprehensive scores are ‘QXYZ,’ Suyuanling (SYL), Xiangzao (XZ), Dunhuangdazao (DHDZ) and Meimizao (MMZ). In this paper, the main active components and antioxidant capacity of thirty-seven jujube varieties have been studied, which provides a theoretical basis for jujube breeding and the development of active nutrients for jujube.

KEYWORDS:

• Jujube cultivars
• active ingredients
• antioxidant activity
• PCA

Introduction

Jujube (Ziziphus jujuba Mill.) is a plant of the Rhamnaceae family, widely distributed in subtropical and tropical regions, especially in East Asia (China, Korea and India), the Middle East (Pakistan and Iran) and North African countries .^[1–3] Jujube originated in China and has a history of planting for more than 4,000 years .^[4] It is the world’s largest red date producer and only red date exporter, accounting for more than 90% of the world’s total red date output .^[5] In addition, it is also grown in Southeastern Europe (such as, Spain and Italy), Southwestern United States, Australia and Russia .^[6]

In the past decade, the interest and use of medicinal plant products have increased significantly .^[7] Therefore, more and more researchers begin to pay attention to a variety of bioactive substances in plants, fruits and vegetables and their importance to human health, such as Borago officinalis L. flower, ^[8] Galanthus transcaucasicus, ^[9] red cabbage, ^[10] Clematis orientalis and Clematis ispahanica .^[11] Jujube have always been regarded as an important food or traditional medicinal material. Other parts of jujube, such as seeds, fruits, leaves and flowers, are also used as medicinal preparations .^[2,12,13] Among them, the edible part (pulp and peel) of jujube fruit is the focus of research, which contains most of the active active substances. Some studies have indicated that jujube contains a large number of active substances, such as polyphenol, flavonoid, triterpene, polysaccharide, ascorbic acid, cyclic adenosine monophosphate (cAMP) .^[14,15] These active ingredients have antioxidant, antiinflammatory, antibacterial, liver protection, gastrointestinal protection, blood glucose reduction and anticancer effects .^[16–20] The nutritional value and beneficial effects of jujube on health, as well as people’s increasing attention to its antioxidant activity, indicate that it is an excellent health food with important research value. Therefore, it is necessary to select varieties with more relevant active ingredients according to the expected use of the fruit.

As far as we know, there are more than 700 varieties of jujube in China. However, the origin of its species is very confusing, and the nature of different varieties of jujube is still unclear. There are few studies on its active active ingredients and antioxidant activity. Therefore, in this study, the active components of thirty-seven Chinese jujube varieties were determined, and their antioxidant activities were quantitatively evaluated. In order to provide a theoretical basis for jujube breeding and the development of jujube active nutrients.

Materials and methods

Materials and chemicals

2, 2ʹ-diphenyl-1-picryhydrazyl (DPPH) from Shanghai Hualan Chemical Technology Co., Ltd (Shanghai, China). 2, 2ʹ-azinobis (3-ethylbenzothia zoline-6-sulfonic acid) diammonium salt (ABTS) from Hefei Bomei Biological Technology Co., Ltd (Anhui, China). Trifluoroacetic acid (TFA) from Shanghai McLean Biochemical Technology Co., Ltd (Shanghai, China) were purchased. Rutin, coumarin, caffeic acid, cinnamic acid, (+)-catechin, chlorogenic acid, and cAMP were obtained from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). All other reagents used are analytical grade unless otherwise specified.

Thirty-seven jujube varieties (Table 1) were collected from the red-ripe stage of Laoling city (Hundred jujube garden, latitude 37°81ʹ46, 70” N × longitude 117°31ʹ13, 16” E, 9 m above sea level), Shandong Province, China. The fruits of the similar sizes, without diseases, insect pests or mechanical damages, were randomly plucked from different tree species and brought to the laboratory on the same day and frozen at −20°C.

Table 1. Summary of tested cultivars of Ziziphus jujuba.

Sample ID	Cultivar	Sample ID	Cultivar	Sample ID	Cultivar
YL1H	Yuanling1hao	JZ1H	Jinzao1hao	DHDZ	Dunhuangdazao
DGZ	Daguazao	CTZ	Chengtuozao	SZDZ	Suizhoudazao
WDXZ	Wudixiaozao	MLZ	Malingzao	SYL	Suyuanling
LLJXZ	Laolingjixinzao	JS1H	Jinsi1hao	JSXZ	Jinsixiaozao
YL2H	Yuanling2hao	LYX	Liuyuexian	JDZ	Jidanzao
YTXZ	Yutianxiaozao	JZ	Jinzao	XZ	Xiangzao
TZ	Tanzao	JXZ	Jixinzao	DBL	Dabailing
JS4H	Jinsi4hao	QXYZ	Qingxuyuanzao	ZDZ	Zidanzao
BDXZ	Baodexiaozao	CYYZ	Chaoyangyuanzao	PZ	Pozao
LJZ	Lajiaozao	MPZ	Mopanzao	GTXZ	Gutouxiaozao
ZYMZ	Zhongyangmuzao	LXMZ	Lianxianmuzao	JIZ	Jianzao
DHZ	Dahuizao	JS3H	Jinsi3hao
HZ	Huizao	MMZ	Meimizao

Sample preparation

The extraction solution was prepared by using the optimized conditions of the extraction process of jujube polyphenols obtained in the laboratory. The specific conditions are as follows. The 1.0 g freeze-dried jujube powder was extracted with 20 mL of aqueous ethanol (67%, v/v) under ultrasound at 275 W for 30 min. The extraction was done twice. The extracts were used for the determination of total phenols, total flavones, phenolic acids and antioxidant capacity.

Determination of polysaccharides

Jujube powder (0.5 g) and 12 mL (80%) ethanol were added into a 50 mL centrifuge tube and shaken evenly. Ultrasonic extraction lasted for 30 min and centrifugation lasted for 10 min. The insoluble substance was transferred to 50 mL centrifuge tube and 25 mL water was added. Ultrasonic extraction was performed for 30 min and repeated for 2 times. The solution was filled to a constant volume into 100 mL volumetric flask. The total polysaccharide content was measured using phenol sulfuric acid colorimetric method^[21] and slight modification. Simply, 0.5 mL standard glucose solution (0.04–0.2 mg/mL) or sample solution was added with 1 mL phenol and 5 mL concentrated sulfuric acid and mixed well with water at 30°C for 30 min. The absorbance was measured at 490 nm.

Determination of ascorbic acid

Ascorbic acid content was determined by 2, 6-dichloro-indophenol titration method^[22] and modified appropriately. Homogenized sample (2.0 g) was blended with 40 mL of oxalic acid solution (20 g/L). The 5 mL filtered solution was diluted with 20 g/L oxalic acid to 10 mL, and titrated with 2, 6-dichloro-indophenol (0.69 mmol/L) to pink, and the color was kept fade-free for 15 seconds. The content was expressed as mg/100 g FW.

Determination of cAMP

The content of cAMP was determined by HPLC^[23] and slightly modified. Jujube powder (0.1 g) was blended with 50 mL distilled water. Ultrasonic assisted extraction was performed for 30 min. The extract solution was centrifuged for 15 min and the supernatant was filtered through a 0.45 µm polytetrafluoroethylene (PTFE) filter. HPLC conditions were as follows: Type LC-20A, equipped with PDA detector; Inertsil ODS-3 C18 column (250 mm × 4.6 mm, 5 µm); Mobile phase consisted of 0.05 mol/L KH₂PO₄-methanol (volume ratio = 80:20) at a flow rate of 0.6 mL/min; isometric elution; Column temperature was maintained at 30°C, injection volume 10 µL, detection wavelength 254 nm. The content of cAMP was computed employing the formula obtained from the regression equation. The concentration was represented as milligrams per 100 g of DW.

Determination of total phenolic

The total phenol content was measured by Folin-Ciocalteu method^[24] and some adjustments. Simply, 0.5 mL gallic acid standard solution (0.04–0.20 mg/mL) or polyphenol solution into 10 mL colorimetric tube was poured, 0.5 mL forinol-phenol reagent (0.5 N) was added, and shook well. Let stand for 5 min, added 2.5 mL Na₂CO₃ (10% w/v) solution, added deionized water and made the volume reach 10 mL. The solution was shaken well and placed still in the dark for 1 h. The detection wavelength was 760 nm. The results were expressed as milligrams of gallic acid equivalent (GAE)/g DW.

Determination of total flavonoids

The content of total flavonoids in jujube fruit was determined by colorimetric method .^[25] Simply, 1.0 mL rutin standard solution (0.10–0.50 mg/mL) or extract sample solution into 10 mL colorimetric tube was absorbed, 0.3 mL of 5% NaNO₂ and 0.3 mL of 10% Al(NO₃)₃ were added in turn, the solution was shaken well and then placed still for 5 min for settling. Then, 4.0 mL of 4% NaOH was added to the solution and diluted with 60% ethanol solution to scale. The solution was shaken well and placed still for 15 min. The detection wavelength was 510 nm. With rutin as the standard, the flavonoids content was expressed in rutin equivalent (RE.)/100 g DW.

Determination of triterpenic acid

Jujube powder (1.0 g) and 25 mL ethanol (70%) were ultrasonically extracted for 30 min at 60°C. The extract was extracted twice and the final volume was 100 mL. Sample solution (0.1 mL) or oleanolic acid standard solution (0.2–1.2 mg/mL) was poured into a test tube and evaporated till dryness in a water bath (T ≥ 85°C). Then, 0.2 mL of vanillin-acetic acid solution (5:95 w/v) and 0.8 mL HClO₄ were added and shaken well. The mixture was placed in a water bath (T = 60°C) for 15 min before it was cooled to room temperature. Glacial acetic acid (5.0 mL) was added to it and blended. The absorbance was performed at 547 nm. The result was expressed as mg oleanolic acid equivalent/g DW (mg OAE/g DW) .^[26]

Qualitative and quantitative analysis of phenolic substances

The extracting solution was concentrated by rotary evaporation at 40°C, and diluted to 25 mL with chromatography grade methanol. The extract was filtered through 0.45 µm organic membrane. HPLC conditions were as follows: Type LC-20A, equipped with PDA detector; C18 analytical column (250 × 4.6 mm i.d.); Column temperature was maintained at 30°C, injection volume 10 µL, detection wavelength 280 nm; Mobile phase consisted of (A) 5% methanol (0.1% TFA) and (B) methanol at a flow rate of 0.8 mL/min. Gradient elution was as follows: 0–30 min (20% B), 30–35 min (20–35% B), 35–60 min (35–60% B), 60–65 min (60–100% B), 65–75 min (100% B), 75–80 min (100–20% B), and 80–85 min (20% B). The phenolic acid was quantitatively analyzed by external standard method, and their contents were represented as mg/100 g DW.

Antioxidant capability

DPPH scavenging assay

The ability of jujube extract to scavenge DPPH free radicals was measured according to the previously reported method, ^[27] with appropriate modification. Extracting solution (0.1 mL) or the standard Vc solution (0–0.30 mg/mL) was blended with 5.0 mL ethanolic solution of DPPH (0.1 mmol/L). After 30 min, the absorbance value was measured at 517 nm. The DPPH clearance rate was calculated using the following Eq. (1)(1) $Y_1=\frac{\left(A_0-A_t\right)}{A_0}\times 100\mathrm{\%}$(1) . The results were expressed as mg Vc/g DW.

(1) $Y_1=\frac{\left(A_0-A_t\right)}{A_0}\times 100\mathrm{\%}$(1)

where A₀ and A_t are the absorbances of the control sample and the sample solution, severally.

ABTS scavenging assay

The scavenging capacity of ABTS cationic radical was measured by a reported method^[28] with some modifications. Mix 7.0 mmol/L ABTS solution and 5 mL 2.45 mmol/L K₂S₂O₈ solution in an equal proportion and leave for 12–16 h in the dark. On the day of testing, the absorbance of ABTS reserve solution diluted to 734 nm wavelength was 0.700 ± 0.050. The 0.2 mL extract or the standard solution (0–0.10 mg/mL of trolox) was blended with 5.0 mL ABTS solution. After 8 min, the absorbance was determined at 734 nm. The ABTS clearance rate was calculated using the following Eq. (2)(2) $Y_2=\frac{\left(A_0-A_t\right)}{A_0}\times 100\mathrm{\%}$(2) . The results were expressed as mg trolox/g DW.

(2) $Y_2=\frac{\left(A_0-A_t\right)}{A_0}\times 100\mathrm{\%}$(2)

where A₀ and A_t are the absorbances of the control sample and the sample solution, severally.

FRAP reducing power assay

Extracting solution (0.6 mL) or the standard trolox solution (0–0.50 mg/mL), 2.5 ml phosphate buffer solution (0.2 mol/L) and 2.5 mL K₃Fe(CN)₆ solution (1% w/v) were successively added into the colorimetric tube. Mixture was then immersed in a water bath (T = 45°C) for 20 min. After cooling, 2.5 mL trichloroacetic acid (TCA) (10% w/v) was added, shaken, and centrifuged. Supernatant (2.5 mL) was absorbed into another colorimeter tube and then 2.5 mL deionized water and 0.5 mL FeCl₃ (0.1% w/v) were added in turn .^[29] After 8 min, the absorbance was measured at 700 nm. The results were expressed as mg trolox/g DW.

Statistical analysis

The data in the tables were expressed as the mean ± standard deviation (SD). The significance of independent variables was compared using Duncan’s multiple range test and P < .05 indicated significant difference. The correlation of date was determined by Pearson correlation analysis. The data were also analyzed by Principal Component Analysis. Origin 2018 and SPSS 24 statistical software were used.

Results and discussion

Analysis of polysaccharides, ascorbic acid, and cAMP

Polysaccharide is an important biological component in jujube and has been shown to have antioxidant, anti-tumor, liver protection, blood glucose reduction, and other biological activities .^[20,30] In our study, the polysaccharides content in jujube fruits ranged from 1.64 g/100 g DW of ‘LJZ’ to 16.48 g/100 g DW of ‘GTXZ’ (Table 2). The polysaccharide content of ‘GTXZ’ is significantly higher than that of other jujube varieties, so it may be an ideal research object of jujube polysaccharide.

Table 2. Total phenols, total flavonoids, total triterpene, polysaccharides, Vc and cAMP of thirty-seven jujube cultivars

Cultivar	Total phenols (mg GAE/g DW)	Total flavonoids (mg RE /g DW)	Total triterpene (mg OAE/g DW)	Poly-sacchrides (g/100 g DW)	Vc (g/100 g FW)	cAMP (mg/100 g DW)
YL1H	12.25 ± 0.12^ij	13.51 ± 0.89^kl	66.65 ± 1.85 ^l-n	4.74 ± 0.09 ^g-j	359.22 ± 21.84 ^r	4.52 ± 0.14^d-f
DGZ	10.99 ± 0.11 ^f	12.24 ± 0.63^jk	70.49 ± 0.65^pq	3.41 ± 0.18^de	120.14 ± 6.01^a	20.34 ± 0.91^s
WDXZ	10.81 ± 0.23 ^f	9.97 ± 0.86 ^f-h	73.21 ± 0.65^rs	3.26 ± 0.14^d	296.06 ± 14.88^p	8.02 ± 0.43 ^j-l
LLJXZ	10.76 ± 0.12 ^f	9.28 ± 0.94^e-g	74.94 ± 1.18^st	3.68 ± 0.09^e	272.86 ± 10.07^no	7.32 ± 0.34^ij
YL2H	12.83 ± 0.34^k	13.01 ± 0.46^kl	68.49 ± 1.12 ^n-p	3.40 ± 0.04^de	265.70 ± 14.01^no	11.93 ± 0.22^q
YTXZ	9.85 ± 0.27^d	7.08 ± 0.90^ab	73.90 ± 1.52 ^r-t	5.54 ± 0.08^k	186.06 ± 6.43 ^cd	12.39 ± 0.49^q
TZ	11.68 ± 0.07^gh	13.44 ± 0.87^kl	58.57 ± 0.46^ij	2.32 ± 0.18^b	211.53 ± 9.08 ^f-i	10.07 ± 0.48^no
JS4H	8.45 ± 0.12^a	9.08 ± 0.60^d-f	53.81 ± 0.63^fg	4.79 ± 0.17 ^h-j	328.60 ± 9.42^q	10.79 ± 0.15°^p
BDXZ	11.65 ± 0.11^gh	10.02 ± 0.39 ^f-h	42.95 ± 1.53^e	9.00 ± 0.40°	208.71 ± 9.61 ^f-h	8.72 ± 0.46 ^lm
LJZ	12.28 ± 0.14 ^j	16.42 ± 0.57°	57.40 ± 0.85 ^h-j	1.64 ± 0.09^a	213.43 ± 10.55 ^f-i	5.07 ± 0.15^e-g
ZYMZ	11.41 ± 0.17 ^g	14.15 ± 0.51 ^lm	55.32 ± 0.88 ^gh	4.52 ± 0.08 ^gh	210.98 ± 9.74 ^f-i	7.86 ± 0.15^i-k
DHZ	9.47 ± 0.06 ^c	8.87 ± 0.30^d-f	65.71 ± 1.30 ^lm	2.81 ± 0.09 ^c	228.24 ± 10.07^il	5.68 ± 0.14 ^g
HZ	9.96 ± 0.13^de	10.85 ± 0.57^hi	64.98 ± 0.97 ^l	5.07 ± 0.04 ^j	234.16 ± 10.95 ^j-l	3.92 ± 0.15 ^cd
JZ1H	8.88 ± 0.07^b	8.40 ± 0.62^b-e	65.46 ± 1.80 ^lm	6.43 ± 0.36 ^m	182.60 ± 9.74 ^cd	5.23 ± 0.18^fg
CTZ	12.72 ± 0.12^k	11.41 ± 1.02^ij	59.46 ± 0.51^jk	1.95 ± 0.06^a	205.77 ± 7.77^e-g	9.92 ± 0.77 ^n
MLZ	12.29 ± 0.14^ij	10.52 ± 0.40 ^g-i	66.30 ± 1.13 ^lm	3.69 ± 0.13^e	225.17 ± 7.72 ^h-k	4.37 ± 0.24^de
JS1H	10.13 ± 0.23^de	7.07 ± 1.16^ab	52.05 ± 1.25 ^f	3.40 ± 0.11^de	242.45 ± 11.37^k-m	12.02 ± 0.38^q
LYX	10.01 ± 0.22^de	8.60 ± 0.55 ^c-e	72.33 ± 0.99^qr	3.43 ± 0.14^de	187.91 ± 10.23 ^c-f	41.56 ± 0.89 ^u
JZ	13.86 ± 0.13 ^l	13.12 ± 0.57^kl	73.12 ± 0.90^rs	4.09 ± 0.07 ^f	339.24 ± 9.81^q	8.56 ± 0.18^k-m
JXZ	11.62 ± 0.11^gh	11.25 ± 0.49 ^h-j	61.10 ± 1.58^k	3.19 ± 0.08^d	188.58 ± 8.78 ^c-f	7.14 ± 0.04^hi
QXYZ	16.33 ± 0.14°	41.18 ± 1.16^q	57.91 ± 1.34^ij	4.43 ± 0.06 ^g	274.22 ± 11.35^no	6.52 ± 0.20 ^h
CYYZ	12.48 ± 0.09^jk	10.90 ± 0.43^hi	56.62 ± 1.56^hi	6.44 ± 0.09 ^m	176.09 ± 7.56 ^c	27.20 ± 1.21 ^t
MPZ	12.47 ± 0.16^jk	14.99 ± 0.48^mn	69.30 ± 0.74°^p	9.09 ± 0.29°^p	136.68 ± 5.81^ab	3.54 ± 0.08 ^c
LXMZ	11.34 ± 0.09 ^g	13.17 ± 0.86^kl	75.61 ± 0.96 ^t	4.93 ± 0.09^ij	121.30 ± 6.26^a	8.13 ± 0.45 ^j-l
JS3H	11.98 ± 0.07 ^hi	11.10 ± 0.89 ^h-j	78.33 ± 1.99 ^u	4.69 ± 0.21 ^g-i	300.56 ± 7.77^p	8.03 ± 0.25 ^j-l
MMZ	15.77 ± 0.10 ^n	20.69 ± 0.75^p	64.75 ± 1.39 ^l	3.42 ± 0.13^de	336.35 ± 21.04^q	6.46 ± 0.20 ^h
DHDZ	15.42 ± 0.36 ^m	20.23 ± 0.91^p	71.89 ± 0.98q^r	9.35 ± 0.42^p	257.38 ± 6.35^mn	N.D.
SZDZ	10.98 ± 0.20 ^f	7.84 ± 0.50^b-d	24.97 ± 0.96^a	5.55 ± 0.26^k	199.36 ± 8.51^d-f	9.07 ± 0.55 ^m
SYL	16.05 ± 0.69^no	20.42 ± 0.47^p	67.50 ± 0.89 ^m-o	4.43 ± 0.10 ^g	277.21 ± 7.99°	7.68 ± 0.17^ij
JSXZ	10.12 ± 0.17^de	7.51 ± 0.51^bc	33.23 ± 0.67 ^c	9.16 ± 0.25°^p	197.78 ± 8.21^d-f	10.90 ± 0.60^p
JDZ	10.29 ± 0.23^e	6.00 ± 0.63^a	30.47 ± 2.26^b	5.76 ± 0.08^kl	244.23 ± 10.26 ^lm	9.87 ± 0.17 ^n
XZ	13.86 ± 0.13 ^l	19.93 ± 0.54^p	33.13 ± 1.11 ^c	3.22 ± 0.08^d	310.99 ± 10.47^p	4.06 ± 0.15 ^cd
DBL	12.74 ± 0.07^k	16.01 ± 1.08^no	26.58 ± 1.10^a	4.76 ± 0.08^g-j	198.81 ± 6.21^d-f	9.00 ± 0.47 ^m
ZDZ	9.49 ± 0.25 ^c	8.23 ± 0.42^b-e	33.28 ± 0.74 ^c	2.84 ± 0.13 ^c	211.42 ± 6.98 ^f-i	1.06 ± 0.07^a
PZ	9.37 ± 0.24 ^c	8.21 ± 0.51^b-e	39.29 ± 2.05^d	8.49 ± 0.27 ^n	146.78 ± 5.96^b	9.31 ± 0.42^mn
GTXZ	10.84 ± 0.14 ^f	8.27 ± 0.70^b-e	38.82 ± 0.94^d	16.48 ± 0.44^q	180.06 ± 9.32 ^cd	2.76 ± 0.12^b
JIZ	9.48 ± 0.32 ^c	8.45 ± 0.58 ^c-e	33.21 ± 1.16 ^c	6.01 ± 0.22 ^l	220.24 ± 8.21 ^g-j	19.02 ± 0.97 ^r

Values were presented as the means ± standard deviation (SD). Differences were compared by the Duncan’s multiple range test. Values in the same column with different letters (a-u) are significantly different (P < 0.05).

Ascorbic acid, as a natural antioxidant, has a good scavenging ability on reactive oxygen species (ROS), and plays a role in protecting cells from oxidative damage .^[31] Jujube is rich in Vc, which has the reputation of “natural vitamin pill.” An average of 20 grams of jujube can meet the daily Vc requirement of adults .^[32] The content of Vc varied greatly among different jujube varieties. The ascorbic acid content of thirty-seven jujube fruits ranged from 120.14 mg/100 g FW of ‘DGZ’ to 359.22 mg/100 g FW of ‘YL1H,’ which was basically consistent with the results measured by Kou et al., ^[33] who measured 1.67–4.24 mg/g FW. Jujube fruits were considered a good source of ascorbic acid in the diet.

cAMP is an important substance involved in the regulation of substance metabolism and biological function in cells, and is the second messenger of life information transmission .^[34,35] cAMP is an important active substance in jujube, which has the effects of anti-oxidation, improving immunity, nerve protection, and anticancer properties .^[36] Studies have shown that jujube has the highest cAMP content among more than 180 kinds of natural plants .^[37] In our study, the highest content of cAMP measured in ‘LYX’ was 41.56 mg/100 g DW; however, it wasn’t detected in ‘GHDZ.’ Therefore, ‘LYX’ was a promising source of cAMP.

Analysis of secondary metabolites in jujube fruit

Flavonoid is a kind of natural phenolic compound that have been shown to have a wide range of pharmacological effects, such as antiviral, anti-inflammatory and anti-obesity effects .^[38–40] The total flavonoids content of the jujube fruit measured ranged from 7.07 mg/g DW of ‘JS1H’ to 41.18 mg/g DW of ‘QXYZ,’ which was higher than the content of flavonoids determined by Rashwan et al., ^[2] which was 0.7–1.8 g/100 g DW. The total polyphenol content in jujube fruit ranged from 8.45 mg GAE/g DW of ‘JS4H’ to 16.33 mg GAE/g DW of ‘QXYZ,’ which was similar than 8.76–21.61 mg GAE/g determined by Ivanišová et al. .^[41] The difference of polyphenols content may be caused by the difference in jujube varieties and their origin .^[1]

Triterpenic acid has attracted much attention due to its anti-inflammatory, antibacterial, liver-protecting, and antioxidant effects .^[42] In our work, the total content of triterpenes in thirty-seven jujube varieties ranged from 24.97 mg/g DW of ‘SZDZ’ to 78.33 mg/g DW of ‘JS3H.’ In addition, the contents of triterpenoid acids in ‘LXMZ’ (75.61 mg OAE/g DW), ‘LLJXZ’ (74.94 mg OAE/g DW), ‘WDXZ’ (73.21 mg OAE/g DW) and ‘JZ’ (73.12 mg OAE/g DW) were also higher. Thus, ‘JS3H,’ ‘LXMZ,’ ‘LLJXZ,’ ‘WDXZ’ and ‘JZ’ are promising source of triterpenes. The growth conditions of all jujube varieties in this study are the same. The difference in composition between the varieties mentioned above may be caused by the genotype of jujube.

Composition and content of phenolic substances in different jujube fruit

Many studies have shown that polyphenols are a good source of natural antioxidants among the antioxidants consumed in the diet .^[43,44] Some studies compared the total phenols and antioxidant activities of 62 kinds of fruits and found that jujube is rich in phenols and has high antioxidant activity, making it an excellent natural antioxidant food .^[45] In this study, six phenolic substances in jujube were determined by high performance liquid chromatography (Figure 1 and Table 3). In general, the content of polyphenols in different red jujube varieties is very different. It can be seen from Table 3 that this kind of composition is found in the vast majority of date varieties, which is similar to the observations found by Zhao et al. .^[46] The contents of (+)-catechin, caffeic acid and rutin in jujube fruits of different varieties were relatively rich, which were higher than the other three phenolic compounds, and are obviously dominant. The contents of (+)-catechin, caffeic acid, and rutin in ‘QXYZ’ were the highest, which were 65.31 mg/100 g DW, 26.29 mg/100 g DW and 39.16 mg/100 g DW, respectively. A previous study^[33] reported the total phenolic content from fifteen jujube cultivars, but ignored the content of individual phenolic substances. As a matter of fact, the phenolic acid content measured in this work were higher than previously reported .^[24] The reason for this phenomenon may be due to differences in detection methods, varieties and regions .^[46]

Table 3. Contents of phenolic acids in extracts of thirty-seven jujube cultivars

Cultivar	(+)-Catechins (mg/100 g DW)	Chlorogenic acid (mg/100 g DW)	Caffeic acid (mg/100 g DW)	Coumarin (mg/100 g DW)	Rutin (mg/100 gDW)	Cinnamic acid (mg/100g DW)
YL1H	13.66 ± 0.62^k	2.11 ± 0.13 ^l	4.05 ± 0.16^k	0.30 ± 0.03 ^c	17.09 ± 0.48 ^l	0.10 ± 0.01^a-c
DGZ	13.21 ± 0.12^jk	0.96 ± 0.02 ^c-e	5.02 ± 0.02 ^l	0.81 ± 0.01^hi	14.10 ± 0.13 ^h-k	0.08 ± 0.01^a
WDXZ	12.73 ± 0.78 ^h-k	1.41 ± 0.13^i	2.94 ± 0.14^ij	0.98 ± 0.06 ^j-l	12.22 ± 0.34 ^c-f	0.12 ± 0.01 ^c-f
LLJXZ	10.92 ± 0.88 ^c-f	1.16 ± 0.04 ^f-i	2.67 ± 0.18 ^g-i	1.16 ± 0.02^p	11.27 ± 0.97^bc	0.09 ± 0.01^ab
YL2H	12.23 ± 0.64 ^f-k	2.15 ± 0.09 ^l	2.41 ± 0.10 ^f-i	0.11 ± 0.01^a	13.08 ± 0.15^e-h	0.23 ± 0.02 ^lm
YTXZ	12.30 ± 0.43 ^f-k	1.08 ± 0.04^d-g	2.57 ± 0.12 ^f-i	1.17 ± 0.03^p	11.99 ± 0.87 ^c-e	0.11 ± 0.01^b-e
TZ	18.87 ± 0.18^no	0.91 ± 0.03 ^c-e	5.25 ± 0.12 ^lm	0.59 ± 0.05 ^g	10.07 ± 0.23^a	0.17 ± 0.01 ^gh
JS4H	9.65 ± 0.10^bc	1.24 ± 0.13 ^f-i	2.11 ± 0.05^d-f	0.92 ± 0.02 ^j	13.28 ± 0.8^g-k	0.09 ± 0.01^a-c
BDXZ	11.02 ± 0.22 ^c-g	1.25 ± 0.17 ^f-i	1.81 ± 0.10 ^c-e	1.08 ± 0.07 ^m-o	10.33 ± 0.55^ab	0.11 ± 0.01^a-d
LJZ	19.49 ± 1.48°^p	1.85 ± 0.09^jk	5.71 ± 0.28 ^m	0.20 ± 0.02^b	22.99 ± 0.99^p	0.21 ± 0.01 ^j-l
ZYMZ	15.06 ± 0.46 ^l	0.58 ± 0.10^a	6.56 ± 0.15 ^n	0.47 ± 0.06^de	13.78 ± 1.05 ^g-j	0.19 ± 0.02 ^h-j
DHZ	11.37 ± 0.81^d-h	0.67 ± 0.06^ab	4.37 ± 0.06^k	0.40 ± 0.03^d	11.16 ± 0.43^bc	0.10 ± 0.01^a-c
HZ	13.68 ± 1.04^k	0.97 ± 0.13 ^c-e	5.70 ± 0.20 ^m	0.42 ± 0.04^d	13.41 ± 1.20 ^g-i	0.12 ± 0.01^d-f
JZ1H	18.63 ± 0.91^no	1.40 ± 0.13^i	2.12 ± 0.21^d-f	1.36 ± 0.08 ^r	13.05 ± 1.06^e-h	0.11 ± 0.01^a-d
CTZ	31.21 ± 1.02 ^t	3.61 ± 0.19°	5.13 ± 0.19 ^l	0.31 ± 0.03 ^c	19.24 ± 0.17^mn	0.14 ± 0.02 ^f
MLZ	11.61 ± 0.62^d-i	1.38 ± 0.11^hi	2.44 ± 0.09 ^f-i	1.11 ± 0.04 ^n-p	12.16 ± 0.42 ^c-f	0.10 ± 0.02^a-c
JS1H	10.20 ± 0.30 ^cd	1.96 ± 0.19^kl	1.54 ± 0.03^a-c	1.01 ± 0.04k^lm	12.02 ± 0.14 ^c-e	0.12 ± 0.02^d-f
LYX	12.40 ± 0.77 ^g-k	3.06 ± 0.28^a	1.04 ± 0.04^a	0.49 ± 0.02^e	14.34 ± 0.31^i-k	0.16 ± 0.01 ^g
JZ	21.92 ± 1.81^pq	2.01 ± 0.42^kl	2.74 ± 0.14^hi	1.26 ± 0.04^q	16.45 ± 1.41 ^l	0.10 ± 0.02^a-d
JXZ	12.78 ± 0.54 ^h-k	2.11 ± 0.11 ^l	3.39 ± 0.10 ^j	1.41 ± 0.04 ^r	21.33 ± 0.47°	0.11 ± 0.01 ^c-f
QXYZ	65.31 ± 0.87 ^u	2.46 ± 0.14 ^m	26.29 ± 0.26^s	0.62 ± 0.04 ^g	39.16 ± 0.31^s	0.33 ± 0.02°
CYYZ	16.76 ± 0.36 ^m	1.78 ± 0.12^jk	2.78 ± 0.02^i	1.72 ± 0.05^s	16.53 ± 0.19 ^l	0.22 ± 0.01^kl
MPZ	18.75 ± 0.20^no	2.96 ± 0.14 ^n	4.28 ± 0.11^k	0.47 ± 0.04^de	16.79 ± 0.33 ^l	0.08 ± 0.01^a
LXMZ	22.65 ± 1.42^q	1.99 ± 0.09^kl	6.94 ± 0.08^no	0.85 ± 0.02^i	20.00 ± 0.09 ^n	0.11 ± 0.01^b-e
JS3H	11.97 ± 0.41^e-j	1.71 ± 0.15 ^j	2.22 ± 0.11^e-h	1.14 ± 0.06°^p	14.67 ± 0.68 ^l-k	0.09 ± 0.01^ab
MMZ	30.63 ± 0.54^st	1.25 ± 0.09 ^f-i	18.45 ± 0.96^q	0.56 ± 0.02^fg	16.26 ± 0.65 ^l	0.08 ± 0.01^a
DHDZ	27.26 ± 1.80 ^r	1.14 ± 0.09^e-h	15.59 ± 1.00^p	0.40 ± 0.01^d	18.64 ± 0.47 ^m	0.20 ± 0.01^i-k
SZDZ	7.53 ± 0.43^a	0.80 ± 0.07^a-c	1.65 ± 0.07^b-d	0.52 ± 0.02^ef	15.17 ± 0.69^k	0.11 ± 0.01^a-d
SYL	29.73 ± 1.04^s	1.33 ± 0.09^hi	20.74 ± 0.77 ^r	N.D.	25.15 ± 0.82^q	0.14 ± 0.01^ef
JSXZ	10.67 ± 0.32 ^c-e	1.02 ± 0.07 ^c-f	1.35 ± 0.09^a-c	0.96 ± 0.01^jk	9.70 ± 0.09^a	0.10 ± 0.01^a-d
JDZ	12.73 ± 0.30 ^h-k	0.89 ± 0.07^b-d	2.68 ± 0.06 ^g-i	0.17 ± 0.05^ab	10.31 ± 0.25^ab	0.24 ± 0.02 ^m
XZ	20.98 ± 1.02^p	1.37 ± 0.05^hi	7.31 ± 0.07°	0.75 ± 0.07^h	28.01 ± 0.32 ^r	0.29 ± 0.02 ^n
DBL	18.45 ± 0.28^no	2.13 ± 0.10 ^l	5.24 ± 0.04 ^l-q	0.12 ± 0.01^a	21.81 ± 0.18°	0.16 ± 0.01^g
ZDZ	13.06 ± 0.22^i-k	1.70 ± 0.06 ^j	3.30 ± 0.04 ^j	0.62 ± 0.02 ^g	18.69 ± 0.65 ^m	0.19 ± 0.01^hi
PZ	17.79 ± 0.41^mn	0.60 ± 0.03^a	2.72 ± 0.04 ^g-i	0.31 ± 0.01 ^c	11.64 ± 0.32 ^cd	0.13 ± 0.01^ef
GTXZ	8.75 ± 1.72^ab	1.14 ± 0.04^e-h	1.15 ± 0.05^ab	0.95 ± 0.03^jk	12.67 ± 0.21^d-g	0.10 ± 0.01^a-c
JIZ	13.37 ± 0.19^jk	1.27 ± 0.03 ^g-i	2.18 ± 0.07^e-g	1.05 ± 0.01 ^l-n	17.27 ± 0.24 ^l	0.21 ± 0.01^kl

Values were presented as the means ± standard deviation (SD). Differences were compared by the Duncan’s multiple range test. Values in the same column with different letters (a-u) are significantly different (P < 0.05).

Figure 1. HPLC of the phenolic extracts from jujube (a: Standard; b: ‘QXYZ.’)

Note: 1. (+)-Catechin; 2. Chlorogenic acid; 3. Coffee acid; 4. Coumarin; 5. Rutin; 6. Cinnamic acid

At present, many studies have shown that these monomeric phenols can prevent and treat many related diseases caused by oxidative stress. For example, (+)-catechin and caffeic acid have a wide range of antibacterial properties, ^[47,48] antiviral activity, ^[49,50] etc. Rutin can be transformed into quercetin in the body, which inhibits adipocyte differentiation and plays a role in alleviating obesity .^[51] Therefore, jujube as a high quality source of polyphenols, has important research and development value.

Antioxidant capacity

Free radicals are constantly produced in the body due to continuous contact with the outside world, including respiration (oxidation reaction), external pollution, radiation exposure, and other factors .^[52] Scientific research has indicated that cancer, aging or other diseases are mostly linked to the production of excess free radicals .^[53] The mechanism of antioxidant action is to directly act on free radicals or indirectly consume substances that are easy to generate free radicals to prevent further reactions. Therefore, in recent years, the natural antioxidants contained in food and their effects have become the focus of extensive research .^[54]

In this work, the antioxidant abilities of DPPH, ABTS and FRAP extracts from different jujube fruits were measured, and the results were shown in Figure 2. The results of DPPH measurement showed that ‘QXYZ’ has high antioxidant activity with a value of 29.79 mg Vc/g DW, followed by ‘DHDZ.’ The ‘QXYZ’ was shown to have the highest antioxidant activity based on ABTS and FRAP assays, with values of 50.90 mg Trolox/g DW and 62.13 mg Trolox/g, followed by ‘SYL’ and ‘MMZ.’ The ABTS scavenging capacities of thirty-seven jujube species ranged from 17.50 mg Trolox/g DW of ‘JS1H’ to 50.90 mg Trolox/g DW of ‘QXYZ,’ and FRAP values ranged from 26.87 mg Trolox/g DW of ‘JS4H’ to 62.13 mg Trolox/g DW of ‘QXYZ,’ which were lower than the measured results (ABTS: 59.67–118.56 mg Trolox/g DW; FRAP: 44.20–87.15 mg Trolox/g DW) by Wojdyło et al. .^[55] This phenomenon may be caused by the variety and regional differences of jujube. Studies have shown that showed that the antioxidant capacity of jujube fruit decreased with the increase of maturity .^[5,56,57] Nonetheless, the ABTS free radical scavenging ability of jujube fruit was 2–3 times of that of common fruits, such as lemon, litchi, apple, avocado, cantaloupe, banana, caramel, durian, longan, papaya, etc .^[39] Thus, jujube fruit was a good source of natural antioxidants.

Figure 2. (a) DPPH radical scavenging capacity in mg Vc/g DW. (b) ABTS cation radical scavenging capacity in mg Trolox/g DW. (c) FRAP reduction capability in mg Trolox/g DW

Correlation analysis

In order to investigate the effect of the main active ingredients on antioxidant capacity of jujube fruit, the correlation coefficients were calculated (Table 4). Our results showed that both total phenols and total flavonoids were significantly correlated with antioxidant capacity, which is similar with earlier report (DPPH: R = 0.565; FRAP: R = 0.871*) (DPPH: R = 0.654*; FRAP: R = 0.836**) .^[5] In addition, (+)-catechin, caffeic acid, rutin, and ascorbic acid were found to have a positive correlation with antioxidant capacity. However, the contents of cAMP and coumarin were also negatively correlated with the three types of antioxidant capacities. The content of total polysaccharides was negatively correlated with DPPH free radical scavenging capacity (DPPH, R = −0.052). Kou et al.^[33] reported similar result (DPPH, R = −0.041). The mechanism behind this outcome was unknown, as polysaccharides had been certified to have antioxidant capacity .^[58,59]

Table 4. Correlation coefficients (R) between each antioxidant capacity method and the active ingredients of jujube fruits

	DPPH	ABTS	FRAP
Total polyphenol	0.888**	0.788**	0.838**
Total flavonoids	0.891**	0.875**	0.754**
Ascorbic acid	0.272**	0.305**	0.165
Total triterpene	0.244**	0.053	0.117
Polysacchride	−0.052	−0.204*	−0.087
cAMP	−0.252**	−0.130	−0.135
(+)-Catechins	0.839**	0.811**	0.618**
Chlorogenic	0.312**	0.193*	0.206*
Caffeic acid	0.876**	0.841**	0.704**
Coumarin	−0.253**	−0.302**	−0.218*
Rutin	0.751**	0.768**	0.656**
Cinnamic acid	0.335**	0.433**	0.379**

Correlations between the data obtained were run using a standard Pearson correlation. *P < 0.05. **P < 0.01

Principal component analysis and hierarchical cluster analysis

It is distinctly that the active components and antioxidant activities of different varieties of jujube fruits are different. All the results were analyzed by Principal component analysis (PCA) and hierarchical clustering analysis (HCA). The results are shown in Figs. 3, 4 and Table 5. All the variables were divided into four principal components, accounting for 76.31% of the total variance. The first principal component explained 47.80% of the total variance, including total phenols, total flavonoids, Vc, (+)-catechin, caffeic acid, rutin, DPPH, ABTS, FRAP. The second principle component explains 10.77% of total variance with triterpene acid and chlorogenic acid. The third principal component covers cAMP and cinnamic acid can explain 9.82% of total variance, while the forth principal component possesses polysaccharide and coumarin with 8.54% of total variance.

Table 5. Eigenvalues, cumulative contribution and eigenvectors associated with four principal components for the jujube fruits

	Components
	PC1	PC2	PC3	PC4
Eigenvalues	7.170	1.616	1.473	1.187
Cumulative variance contribution rate (%)	47.80%	58.57%	68.39%	76.31%
Fruit trait	Eigenvectors
Total phenol	0.331	0.036	−0.151	0.078
Total flavonoids	0.358	−0.027	−0.008	0.048
Vc	0.122	0.081	−0.380	−0.534
Triterpene acid	0.055	0.597	−0.368	0.120
Polysaccharide	−0.075	−0.423	−0.104	0.627
cAMP	−0.082	0.371	0.504	0.084
(+)-Catechins	0.336	0.023	0.070	0.114
Chlorogenic acid	0.119	0.461	0.309	0.131
Caffeic acid	0.340	−0.079	−0.129	0.028
Coumarin	−0.125	0.236	−0.101	0.400
Rutin	0.319	−0.011	0.218	0.027
Cinnamic acid	0.190	−0.204	0.493	−0.229
DPPH	0.349	0.025	−0.118	0.164
ABTS	0.343	−0.038	0.017	−0.029
FRAP	0.311	−0.021	−0.014	0.140

Figure 3. PCA of thirty-seven different jujube cultivars. (a) Loading plot of PCA, (b) scores scatter plot of PCA, and (c) Scores histogram of PCA

Figure 4. Cluster analysis results of thirty-seven different cultivars of red fresh jujubes

Thirty-seven kinds of jujube fruits were scored, as shown in Figure 3(c). The top five jujube varieties with comprehensive score were ‘QXYZ,’ ‘SYL,’ ‘MMZ,’ ‘XZ’ and ‘DHDZ.’ The results of our study indicated that ‘QXYZ’ had high total phenols, total flavonoids, FRAP, DPPH and ABTS scavenging activities, and was suitable for food production. This study provides important information for further research on jujube fruits and provides ideas for investigating the potential health benefits of jujube fruits.

HCA was used to analyze the genetic relationship between different jujube species (Figure 4). The clusters obtained from principal component analysis can be confirmed by the clusters obtained from HCA. From all the results, ‘QXYZ’ forms an independent cluster, which contains the highest active ingredients and antioxidant capacity. The main varieties in the second cluster are ‘SYL,’ ‘MMZ,’ ‘XZ,’ ‘DHDZ,’ and their active ingredients and antioxidant activities are relatively high. Sixteen jujube cultivars, such as ‘JS4H,’ ‘BDXZ,’ ‘DHZ’ and ‘SZDZ,’ formed a cluster, and the content of active ingredients and antioxidant capacity of them were the lowest. Therefore, according to its active ingredient content and antioxidant capacity, thirty-seven jujube varieties were distinguished.

Conclusion

In summary, this study conducted on thirty-seven red jujube varieties, including total phenols, total flavonoids, Vc, polysaccharides, triterpene acids, cAMP, six phenolic acids and antioxidant activity. The results exhibited that there were differences in active components and antioxidant capacity among jujube cultivars. Among them, the contents of total phenols (16.33 mg GAE/g DW), total flavones (41.18 mg RE/g DW), (+)-catechin (65.31 mg/100 g DW), caffeic acid (26.29 mg/100 g DW), rutin (39.16 mg/100 g DW) and cinnamic acid (0.33 mg/100 g DW) in ‘QXYZ’ were the highest. The highest contents of total triterpenes, polysaccharides, ascorbic acid, cAMP, chlorogenic acid, and coumarin were ‘JS3H,’ ‘GTXZ,’ ‘YL1H,’ ‘LYX,’ ‘CTZ,’ and ‘CYYZ,’ respectively. ‘QXYZ’ has the highest free radical scavenging ability and reducing ability among thirty-seven kinds of jujube. Among them, total phenols, total flavonoids, (+)-catechins, caffeic acid and rutin have a high positive correlation with antioxidant capacity. The results of principal component analysis showed that ‘QXYZ,’ ‘SYL,’ ‘MMZ,’ ‘XZ’ and ‘DHDZ’ were the top five jujubes, which contained more active components. Our research results can provide references for the selection of varieties in the planting and production of jujube and the development of active foods.

Acknowledgments

The authors declare that there is no conflict of interest.

Additional information

Funding

This work was supported by the Shandong Province Key Research and Development Plan (2016GNC113015; 2019GNC106061) and Shandong Province Major Application of Agricultural Technological Innovation Project and Shandong Land Rural Revitalization Group Co. Ltd & Shandong Agricultural University co-Building Project of ‘Research Institute of Shandong Ningyang Jujube Industry.’