Fruit quality and antioxidant potential of Prunus humilis Bunge accessions

Hongbo Fu; Xiaopeng Mu; Pengfei Wang; Jiancheng Zhang; Baochun Fu; Junjie Du

doi:10.1371/journal.pone.0244445

. 2020 Dec 30;15(12):e0244445. doi: 10.1371/journal.pone.0244445

Fruit quality and antioxidant potential of Prunus humilis Bunge accessions

Hongbo Fu ^1,^2,³, Xiaopeng Mu ¹, Pengfei Wang ¹, Jiancheng Zhang ¹, Baochun Fu ³, Junjie Du ^1,^*

Editor: Branislav T Šiler⁴

PMCID: PMC7773198 PMID: 33378359

Abstract

In this study, we aimed to evaluate the fruit quality of Prunus humilis and identify cultivars that could provide superior human health benefits. We measured the basic characteristics, bioactive compounds, and antioxidant capacities of 137 P. humilis accessions. Flavonoid and phenol content were determined via colorimetry and ultrahigh performance liquid chromatography. Single fruit and stone weights varied widely and were genetically diverse among accessions. The variation in soluble solid content was comparatively narrow. Total flavonoid content (TFC) ranged from 3.90 to 28.37 mg/g FW, with an average of 10.58 mg/g FW in 2019. Significant differences between accessions in terms of TFC, total phenol content, and antioxidant capacity were found. TFC in the accessions was normally distributed and predominantly in the medium range (9.57–15.23 mg/g FW). Red was the predominant peel color over all other phenotypes (i.e., dark red, red, light red, red-orange, and yellow). There was no obvious correlation between peel color and TFC. Catechin was the major flavonoid component in the fruit. Principal component analysis showed that TFC, ABTS, single fruit weight, and vertical and horizontal diameter contributed to the first two principal components for each accession. Accessions 10–02, 3-17-2, 3-17-4, and JD1-6-7-37 were characterized by high TFC, ABTS, and large fruit. We believe that our results will aid in the breeding and functional food processing of Prunus humilis.

Introduction

Prunus humilis Bunge (Cerasus humilis (Bunge) S.Ya.Sokolov) (Rosaceae) is a small deciduous shrub [1]. Like peach, plum, and apricot, it is an ancient tree species in China with a cultivation history dates back to around 3000 years [2]. In the north of China, it is recorded from about 13 provinces, including Shanxi, Hebei, and Liaoning [3]. The root of P. humilis is well developed and has strong ecological adaptability [4]. Prunus humilis can grow on barren land, withstand drought, and conserve soil water. It has been used by the Forestry Bureau in the Sand Control Project around Beijing and Tianjin areas and in the Three-North Shelter Forest Program [5]. The fruits of P. humilis are known as ‘calcium fruit’ in China due to the high calcium content [6]. The fruits are also rich in amino acids, vitamins, organic acids, and mineral elements and contain relatively high levels of anthocyanins, flavanols, flavonols, tannins, and other substances [7]. They can be consumed fresh or used in wine, juice, jam, and several other products. This plant has both ecological and economic value. It is highly nutritious, resistant to various abiotic and biotic stressors, and has human health benefits. It has great research, development, and utilization potential [3].

As human standards of living continue to improve, the importance of fruit quality among both producers and consumers has increased. Fruit quality parameters include color, flavor, shape, size, taste, and texture. These criteria are used to assess fruit commodity value and substantially influence consumer preference [8,9]. The acid and sugar content and proportion affect fruit taste [10,11] and determine fruit quality. Flavonoids are polyphenols and important secondary metabolites in plants. They possess strong biological activity and affect fruit color and flavor [12]. Additionally, flavonoids have antioxidant, antiaging, antiviral, and antitumor properties. They inhibit mutagenesis and enhance microcirculation [13–15]. Free radicals are associated with many human diseases [16]. An increasing incidence of non-communicable diseases, all closely associated with oxidative stress, is motivating scientists to look for natural disease prevention method. Fruits are an important component of traditional food, and are also essential items in a healthy diet for the modern urban population [17]. Moreover, in this view fruits such as Prunus humilis, represents potent sources of bioactive compounds, with strong health-promoting and disease-preventing activities [18]. These compounds have a strong antioxidant capacity and also can scavenge free radicals. Foods derived from plant materials with high antioxidant activity are currently receiving much attention and constitute a new trend in plant resource utilization [19].

Fruit trees are major cash crops that help to repurpose or reclaim barren agricultural lands and remediate soil and forest ecosystems. Breeding is vital to the development of the fruit tree industry. Accessions are original sources for breeding new fruit tree varieties and serve as the basis for studying the origin and evolution of tree species. Fruit tree accessions harbor numerous valuable genes [20]. In recent years, many countries have focused on the collection and preservation of plant genetic resources. Accession evaluation is of great practical significance in the maintenance of species diversity and is now a topic of global concern.

In previous studies, researchers have measured and analyzed the fruit quality of P. humilis accessions. Total flavonoid content and radical scavenging activity of 16 P. humilis genotypes have been determined [3]. The polyphenol compounds of 28 and 13 different genotypes of P. humilis in Liaoning province [21] and Beijing city [2] have been systematically characterized, respectively. Seven varieties of P. humilis have been evaluated [22]. Although the fruit quality of P. humilis has been evaluated, the number of accessions was small. To the best of our knowledge, this is the first study to assess the fruits derived from P. humilis accessions on a large scale. We selected 137 P. humilis accessions and analyzed the essential characteristics, bioactive compounds, and antioxidant capacities of the fruit to evaluate the fruit quality in P. humilis and identify cultivars that could provide superior human health benefits. Such investigations may help in promoting the use of P. humilis in functional foods and as an ingredient in pharmaceutical and nutraceutical products.

Materials and methods

Plant materials and chemicals

The experimental materials were acquired from the horticultural station, the accession nursery, and the Juxin experimental orchard of Shanxi Agricultural University, Jinzhong, China (37°23′N, 112°29′E). Field management included conventional irrigation and fertilization methods. During the growth period, the plants were fertilized twice a year, once before germination in spring and once after winter. The amount was 40 kg of nitrogen fertilizer per 667m², mainly by hole application. From May to August, the plants were watered once or twice a month, and, in early November the plants were irrigated with enough water for wintering. Between June and October, in 2018 and 2019, 137 fully ripe P. humilis accessions (S1 Table) were harvested. Three healthy plants were selected per accession. Thirty pest- and disease-free fruits were sampled from the top, middle, and bottom of each of the three sampled plants and stored at -40°C before analysis.

Methanol and acetonitrile used in ultrahigh performance liquid chromatography (UHPLC) were purchased from OmniGene LLC (Morrisville, NC, USA). Analytical flavonoid standards, i.e., catechin, epicatechin, rutin, liquiritigenin, cyanidin-3-O-glucoside, gallic acid, Trolox, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), quercetin-7-O-β-D-glucopyranoside, and 2,4,6-tri-(2-pyridyl)-1,3,5-triazine (TPTZ) were purchased from Solarbio Technology Co. Ltd. (Beijing, China).

Single fruit and stone weights and fruit diameter

Fruit and stone weights were measured using an electronic balance (JJ224BC Changshu Shuangjie Instrument Co. Ltd., Changshu, China) with an accuracy of 0.0001 g. The vertical and horizontal diameters of the fruit were measured with an electronic digital caliper (Guilin Guanglu Digital Dynamometer Co. Ltd., Guilin, China).

Fruit color

Peel color was sensorially evaluated by a team of experienced students and teachers. Thereafter, peel color parameters were measured with a spectrocolorimeter (YS3060 Shenzhen San’enshi technology Co. Ltd., Shenzhen, China) using five representative fruits. Color parameters were measured at the intersection of the fruit equator and suture line, at a single point per 90° rotation along the equator. Therefore, the color parameters were measured at four points per fruit, the average was calculated, and the means of the five fruits were averaged. The following color indices were used to determine fruit color.

Soluble solid content (SSC)

The SSC was measured with a handheld refractometer (LH-T32 Hangzhou Luheng Technologies Co. Ltd., Hangzhou, China).

Total flavonoid and phenol content

The extracts were prepared according to the methods of Bai [23] and Guo et al. [24]. All the sampled fruits were pulverized in liquid nitrogen and extracted with 40% (v/v) acidified methanol. The ratio of material to solution was 1:10. The suspension was mixed by whirlpool oscillation, extracted by ultrasound at 40 kHz for 30 min, and centrifuged at 10 000 × g for 15 min at 4°C. The extraction was repeated thrice, and the filtrates were pooled.

Total flavonoid content (TFC) was determined by NaNO₂-Al(NO₃)₃ colorimetry [24]. First, 0.8 mL of the extracted solution was transferred to a 10 mL volumetric flask containing 0.3 mL of 5% (w/v) NaNO₂. The mixture was shaken well and kept in the dark for 6 min. Thereafter, 0.3 mL of 10% (w/v) Al(NO₃)₃ was added to the mixture, shaken well, and kept in the dark for another 6 min. Then, 4 mL of 4% (w/v) NaOH was added to the mixture and shaken well. The volume was adjusted to 10 mL using 40% (v/v) methanol, and the mixture was shaken well and kept in the dark for 10 min. Finally, the absorbance was read at 510 nm in a spectrophotometer (UV-5200 Shanghai Yuanxi Instrument Co. Ltd., Shanghai, China) calibrated with a rutin standard curve.

Total phenol content (TPC) was determined by Folin-Ciocalteu colorimetry [25]. First, 0.2 mL of the extracted solution was added to a 10 mL volumetric flask containing 0.2 mL Folin-Ciocalteu reagent. The mixture was shaken well and left to stand for 4 min. Then, 1 mL of 10% (w/v) Na₂CO₃ was added to the mixture, and the volume was adjusted to 8 mL with ddH₂O. The mixture was shaken well and incubated in a water bath at 35°C for 1 h. The absorbance was read at 760 nm in a spectrophotometer (UV-5200 Shanghai Yuanxi Instrument Co. Ltd., Shanghai, China) calibrated using a gallic acid standard curve.

Antioxidant capacity

DPPH assay

Following the method of Zhang [26], we added 2.8 mL of 0.1 mM DPPH to 0.2 mL of extracted solution. The mixture was shaken well and kept in the dark for 30 min at 25°C. For the blank, the extracted solution was replaced with 40% (v/v) methanol. The absorbance was read at 517 nm in a spectrophotometer (UV-5200 Shanghai Yuanxi Instrument Co. Ltd., Shanghai, China).

ABTS assay

Following the method of Zhang [26], we added 3.9 mL of ABTS⁺ solution (7 mL ABTS plus 140 mM K₂(SO₄)) to 0.1 mL of extracted solution. The ABTS⁺ solution was prepared in the dark at 25°C over 12–16 h. The sample mixture was shaken well and incubated in the dark for 10 min at room temperature. For the blank, the extracted solution was replaced with 40% (v/v) methanol. The absorbance was read at 734 nm in a spectrophotometer (UV-5200 Shanghai Yuanxi Instrument Co. Ltd., Shanghai, China).

Ferric reducing antioxidant power (FRAP) assay

Following the method of Zhang [26], we added 4.9 mL of FRAP solution (0.1 M CH₃COONa [pH = 3.6], 10 mM TPTZ, and 20 mM FeCl₃ in a 10:1:1 volumetric ratio) to 0.1 mL of extracted solution. The sample mixture was shaken well and incubated in the dark for 10 min at room temperature. For the blank, the extracted solution was replaced with 40% (v/v) methanol. The absorbance was read at 593 nm in a spectrophotometer (UV-5200 Shanghai Yuanxi Instrument Co. Ltd., Shanghai, China).

Trolox evaluation

The standard antioxidant Trolox was used to plot a standard curve and evaluate antioxidant activity [27]. The results of DPPH, FRAP and ABTS were expressed as mg of Trolox equivalents (TE) per g of fruit (on a fresh weight basis)

Flavonoid components

The content of six flavonoid components was determined by UHPLC in an Agela Venusil ABS C18 column (4.6 mm × 250 mm; 5 μm) (Agela Technologies, Wilmington, DE, USA). In particular, 1 mL of flavonoid extract was passed through a 0.22 μm Millipore membrane filter (EMD Millipore, Burlington, MA, USA) and placed in a liquid sample bottle. The solvent system consisted of 0.5% (v/v) formic acid water (solvent A) and acetonitrile (solvent B). The flow rate was set to 0.8 mL min^-1, and the run time was 69 min. The sample injection volume was 20 μL. The gradient program was as follows: 10% B at 0 min; 13% B for 0–5 min; 16% B for 5–25 min; 21% B for 25–30 min; 22% B for 30–45 min; 25% B for 45–50 min; 25% B for 50–65 min; and 10% B for 65–69 min. The detector was set to 280 nm (for detecting catechin, epicatechin, and liquiritigenin), 360 nm (rutin and quercetin-7-O-β-D-glucopyranoside), and 520 nm (cyanidin-3-O-glucoside) for the simultaneous monitoring of the various flavonoid components.

Statistical analysis

Data were analyzed in Microsoft Excel v. 2007 (Microsoft Corporation, Redmond, WA, USA). Cluster analysis, principal component analysis and differences among the mean values were evaluated using ANOM (analysis of means), and statistical significance was set at P <0.05 using the Statistical Analysis System v. 9.2 (SAS Institute, Cary, NC, USA). The coefficient of variation (CV) was calculated as CV (%) = S/F × 100 (SAS Institute, Cary, NC, USA), where S and F are the standard deviation and average, respectively. The abbreviations are detailed in S2 Table.

Results

Changes in flavonoid content over two years

The flavonoid content and the antioxidant capacities could be greatly affected by climatic conditions, we collected two-year data for analysis. In 2018 and 2019, the average contents of flavonoid were 11.11 mg/g FW and 10.58 mg/g FW, respectively (Table 1), with a difference between two years was of 0.53 mg/g. The average variation coefficient of flavonoid contents in the same P. humilis accession was 12.43%, less than 20%. The results showed that the change in the flavonoid content of the same P. humilis accession was small and the flavonoid content was relatively stable between the two years.

Table 1. Flavonoid content and variation analysis of Prunus humilis fruits in 2018 and 2019.

	The average of total flavonoid content (mg/g FW)
2018	11.11
2019	10.58
Coefficient of variation (%)	12.43

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Fruit character

The average single fruit and stone weights for 137 P. humilis accessions were 6.13 g and 0.452 g, respectively, with a CV of 45.98% and 38.02%, respectively (Table 2). As both had a CV >20%, the fruit and stone weights showed large variation and rich genetic diversity. The average vertical and horizontal diameters were 19.62 mm and 21.88 mm, respectively, with a CV of 15.52% and 17.19%, respectively. As both had a CV <20%, the vertical and horizontal diameters showed small variation, and their inheritance was relatively simple. The average vertical diameter was smaller than the average horizontal diameter, which is consistent with the oblate shape found in most P. humilis fruits. The average SSC was 14.15%, and the CV was 17.71%. Hence, the variation in SSC was small, and the inheritance relatively simple.

Table 2. Fruit character variation in 137 Prunus humilis accessions.

Fruit trait	Average	Maximum	Minimum	Standard deviation	Coefficient of variation (%)
Single fruit weight (g)	6.13	15.61	1.20	2.82	45.98
Stone weight (g)	0.452	0.954	0.174	0.17	38.02
Vertical diameter (mm)	19.62	26.41	12.13	3.04	15.52
Horizontal diameter (mm)	21.88	31.32	12.84	3.76	17.19
Soluble solid content (%)	14.15	20.64	7.04	2.51	17.71

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The average TFC and TPC were 10.58 mg/g FW and 3.93 mg/g FW, respectively, with a CV of 35.78% and 34.06%, respectively (Table 3). Therefore, both had a CV >20%, and as we found significant differences among the accessions (P < 0.01), this indicated a large variation in the TFC and TPC of the P. humilis accessions and a rich genetic diversity. The highest average ABTS scavenging ability was 12.38 mg (TE)/g FW. The average of FRAP scavenging ability was 8.03 mg (TE)/g FW. The lowest average DPPH scavenging ability was 4.42 mg (TE)/g FW. The CV for these antioxidant indices was >20%, and we found significant differences among accessions (P < 0.01). Thus, the antioxidant capacities of the P. humilis accessions varied greatly and had rich genetic diversity.

Table 3. Fruit bioactive compounds and antioxidant capacities in 137 Prunus humilis accessions.

Fruit trait	Average	Maximum	Minimum	Standard deviation	Coefficient of variation (%)	F-value
TFC (mg/g FW)	10.58	28.37	3.90	3.78	35.78	94.08**
TPC (mg/g FW)	3.93	9.02	1.44	1.34	34.06	99.80**
DPPH (mg TE/g FW)	4.42	8.19	2.11	1.14	25.86	58.11**
FRAP (mg TE/g FW)	8.03	16.82	3.25	2.56	31.88	69.50**
ABTS (mg TE/g FW)	12.38	24.23	4.68	3.88	31.32	35.29**

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Note: F-value and **indicates significance at P < 0.01. TFC, total flavonoid content; TPC, total phenol content; DPPH, 2,2-diphenyl-1-picrylhydrazyl free radical scavenging capacity; FRAP, ferric reducing antioxidant power; ABTS, 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging capacity.

Cluster analysis of fruit flavonoid content

In the cluster analysis, the accessions were divided according to flavonoid content into four major types and six subgroups (S1 Fig; Table 4). One accession with ultrahigh flavonoid content (28.37 mg/g FW) was assigned to subgroup one. Ten accessions with high flavonoid content (range 15.96–21.99 mg/g FW; average 18.12 mg/g FW) were assigned to subgroup two. Twenty-two accessions with medium-high flavonoid content (range 12.91–15.23 mg/g FW; average 14.07 mg/g FW) were assigned to subgroup three. Forty-two accessions with medium flavonoid content (range 9.57–12.84 mg/g FW; average 11.06 mg/g FW) were assigned to subgroup four. Forty accessions with medium-low flavonoid content (range 7.21–9.40 mg/g FW; average 8.32 mg/g FW) were assigned to subgroup five. Twenty-two accessions with low flavonoid content (range 3.90–6.92 mg/g FW; average 6.03 mg/g FW) were assigned to subgroup six. Overall, we found that the flavonoid content of the 137 accessions was normally distributed (Fig 1) and mainly occurred at medium concentrations in the fruit (Table 4).

Table 4. Flavonoid content levels in Prunus humilis accessions.

Flavonoid content types	Subgroup	Number/proportion of accessions (%)	No.	Flavonoid content range (mg/g FW)	Average flavonoid content (mg/g FW)
Ultrahigh		1/0.73	1	28.37	28.37
High		10/7.30	2–11	15.96–21.99	18.12
Medium	Medium-high	22/16.06	12–31, 129–130	12.91–15.23	14.07
Medium	Medium	42/30.66	32–65, 126–128, 131–135	9.57–12.84	11.06
Low	Medium-low	40/29.20	66–105	7.21–9.40	8.32
Low	Low	22/16.06	106–125, 136, 137	3.90–6.92	6.03

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Flavonoid content in different-colored fruit peels

We divided the 137 accessions into different peel colored phenotypes, i.e., dark red, red, light red, red-orange, and yellow (Fig 2). We then classified the flavonoid content for each phenotype into subgroups through cluster analysis (Table 5). The flavonoid content in the dark red accession was concentrated in the medium and medium-low subgroups (both with 40%). The flavonoid content in the red accession was distributed across all six subgroups but mainly occurred in the medium- and medium-low subgroups (28.40% and 27.16%, respectively). The flavonoid content in the light red accession was distributed across the high, medium, medium-low, and low subgroups. However, the highest proportion occurred in the medium-low subgroup (42.86%). The flavonoid content of the red-orange and yellow accession varied widely across the subgroups, except in the ultrahigh subgroup. The highest flavonoid content in the red-orange and yellow accession occurred in the medium (41.67%) and medium-low (40%) subgroups, respectively.

Table 5. Flavonoid content in different-colored fruit peels of Prunus humilis accessions.

Peel color	Flavonoid content
Peel color	Ultrahigh (%)	High (%)	Medium-high (%)	Medium (%)	Medium-low (%)	Low (%)
Dark red				40.00	40.00	20.00
Red	1.23	6.17	18.52	28.40	27.16	18.52
Light red		14.29		28.57	42.86	14.29
Red-orange		8.33	25.00	41.67	20.83	4.17
Yellow		10.00	5.00	25.00	40.00	20.00

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Bioactive compounds and antioxidant capacity

Based on the flavonoid cluster analysis and phenotype classification, we selected 62 of the 137 accessions for further analysis. The TFC and TPC of 62 accessions were 5.07–28.37 mg/g FW and 2.02–9.02 mg/g FW, respectively, and comprised all six subgroups (Fig 3). The flavonoid content was higher than the TPC in each accession.

Fig 4 shows the antioxidant capacities of 62 accessions. They most strongly scavenged ABTS (6.55–24.23 mg (TE)/g FW) followed by FRAP (3.82–16.82 mg (TE)/g FW) and DPPH (2.47–8.19 mg (TE)/g FW).

Flavonoid components

The content of six flavonoid components were determined using analytical flavonoid standards (Fig 5). There were significant differences in the content of the flavonoid components among the 62 accessions (Table 6). The content of the flavonoid components differed significantly among the five different phenotypes (P < 0.01). Catechin was the only flavonoid component detected in all 62 accessions (15.76–120.81 mg/100 g FW). Epicatechin was detected in all accessions, except 628, DS-1, S-D-1, and 19–05 (1.98–20.47 mg/100 g FW). Liquiritigenin was detected in 54 accessions (1.05–1.95 mg/100 g FW). Its content and variation were the lowest among all six flavonoid components. Cyanidin-3-O-glucoside was detected in 39 accessions (15.24–231.18 mg/100 g FW). Cyanidin-3-O-glucoside was not detected in 23 accessions include all the yellow accessions, 80% of the light red accessions and 84.62% of the red-orange accessions, but it was detected in all red and dark red accessions. Overall, the color index increased with cyanidin-3-O-glucoside content. Thus, there appeared to be a correlation between cyanidin-3-O-glucoside and red peel color. Rutin was detected in all accessions, except Y13-09, Ft-1, T17-1, and 03–35 (1.57–3.25 mg/100 g FW). Quercetin-7-O-β-D-glucopyranoside was detected in all accessions, except Ft-1 and T17-1 (2.39–4.65 mg/100 g FW).

Table 6. Flavonoid components in different-colored fruit peels of 62 Prunus humilis accessions.

Accession name	Peel color	Color index	CC (mg/100 g FW)	EC (mg/100 g FW)	LG (mg/100 g FW)	C3G (mg/100 g FW)	RT (mg/100 g FW)	Q3G (mg/100 g FW)
3-17-4	Red	188.71	81.12±2.51	9.11±1.11	1.52±0.01	23.47±0.15	1.95±0.02	2.63±0.04
JD1-6-7-37	Red	75.01	120.81±5.41	15.32±1.32	1.28±0.02	31.38±0.71	1.95±0.01	3.43±0.05
10–02	Red-orange	24.13	53.64±2.29	3.03±0.26	1.61±0.08	-	2.13±0.08	2.90±0.04
3-17-2	Red	93.40	116.43±3.82	11.73±0.91	1.60±0.07	24.02±0.45	2.55±0.09	3.45±0.08
3-60-2-8	Light red	38.96	64.47±1.21	10.12±0.54	1.24±0.04	-	2.89±0.11	4.65±0.15
TWS	Red	112.08	84.87±1.98	8.53±1.52	1.67±0.03	47.73±0.60	2.14±0.03	3.08±0.01
K1	Red	79.88	119.89±4.96	20.47±3.46	1.59±0.02	17.36±0.13	2.26±0.03	3.26±0.01
Y09-15	Red	54.01	81.37±1.55	9.02±0.98	1.28±0.02	36.20±0.74	2.30±0.04	3.62±0.02
3-17-5	Red-orange	15.69	54.68±4.44	4.06±0.87	1.12±0.03	-	2.20±0.08	2.85±0.03
99–02	Red-orange	12.04	68.67±3.23	5.15±0.05	1.68±0.04	-	2.22±0.01	3.66±0.10
DG-41	Red	60.71	83.82±2.24	3.48±0.42	1.41±0.02	27.75±0.94	2.09±0.02	3.17±0.04
19–09	Red-orange	28.17	60.12±0.86	14.17±1.01	1.22±0.02	-	1.89±0.01	2.80±0.03
Y04-27	Yellow	5.15	65.30±3.26	2.99±0.10	1.16±0.01	-	2.43±0.05	4.29±0.13
08–16	Yellow	1.88	68.00±2.14	4.41±0.21	1.29±0.01	-	2.05±0.05	2.79±0.02
XZ-2	Light red	74.25	52.55±2.21	2.69±0.01	1.21±0.03	-	1.97±0.02	2.81±0.01
02–17	Light red	11.66	39.64±2.15	2.96±0.28	1.25±0.02	-	1.91±0.03	2.94±0.15
Y14-26	Red	90.02	49.92±0.57	4.49±0.34	1.25±0.02	21.18±0.63	1.60±0.01	2.45±0.01
JO-1	Dark red	454.33	45.61±2.00	5.97±0.26	1.29±0.02	139.78±1.91	2.10±0.02	3.46±0.07
19–04	Red	67.29	52.26±2.42	7.46±0.78	1.28±0.03	43.5±2.50	1.92±0.06	3.93±0.13
JO-2	Dark red	576.91	57.46±4.57	9.17±1.13	1.33±0.03	143.11±4.51	2.41±0.06	3.52±0.15
Y07-14	Red-orange	7.03	45.83±1.16	2.75±0.07	1.49±0.01	15.24±0.01	1.95±0.08	2.97±0.04
JO2H	Red	124.17	51.56±4.48	3.40±0.30	1.37±0.04	68.81±2.07	2.27±0.02	3.55±0.05
19–06	Red	97.38	38.85±1.60	2.12±0.07	1.19±0.01	38.51±1.63	1.87±0.02	2.58±0.02
01–01	Red-orange	15.68	52.36±2.20	5.33±0.32	1.63±0.03	-	2.00±0.01	3.31±0.04
19–03	Red-orange	16.43	63.88±5.67	3.37±0.08	1.95±0.04	-	1.94±0.05	3.44±0.17
Y05-17	Red-orange	25.75	59.85±1.96	6.26±0.63	1.13±0.01	-	1.73±0.05	2.71±0.01
X17-01	Red	36.79	39.81±4.63	3.25±0.12	1.58±0.04	20.63±2.94	2.08±0.03	2.89±0.03
03–38	Red-orange	8.84	33.66±0.57	2.94±0.16	1.39±0.02	-	1.71±0.04	2.54±0.06
03–25	Red	256.74	60.25±0.64	5.32±0.25	1.28±0.01	58.67±1.81	2.09±0.05	3.26±0.10
19–07	Red-orange	11.73	37.96±3.91	3.66±0.82	1.05±0.02	23.82±0.89	1.88±0.02	3.99±0.04
15–02	Red	53.33	31.57±2.31	3.64±0.28	1.17±0.01	20.89±1.79	1.92±0.01	3.01±0.04
09–19	Yellow	3.24	46.44±2.00	3.70±0.08	1.51±0.04	-	1.85±0.03	3.21±0.03
11-20M	Red	83.14	35.85±2.76	4.99±0.43	1.38±0.03	15.9±1.27	3.25±0.02	3.05±0.12
JHY	Red	69.14	31.84±3.07	3.44±0.21	1.19±0.01	23.05±0.22	1.84±0.05	2.76±0.01
10–33	Red	108.92	29.61±0.87	3.25±0.31	1.10±0.03	42.45±3.37	1.89±0.06	2.65±0.05
Y08-22	Red	59.99	34.03±2.73	2.05±0.11	1.09±0.03	34.87±1.88	1.63±0.04	2.66±0.04
Y03-10	Red	72.73	31.01±1.61	3.51±0.07	1.11±0.01	45.51±1.09	2.17±0.03	3.10±0.08
10–06	Yellow	5.92	28.05±2.47	2.08±0.10	1.46±0.69	-	1.58±0.01	2.92±0.02
DG-7	Red	66.92	36.75±1.94	3.01±0.38	1.36±0.01	17.93±0.04	2.22±0.01	3.12±0.06
JO1H	Red	90.12	42.69±3.95	2.93±0.02	1.35±0.03	28.93±1.02	2.08±0.03	2.81±0.03
F3-1	Red-orange	47.75	39.46±1.01	2.84±0.08	1.19±0.02	-	1.83±0.04	2.70±0.01
J-2	Yellow	5.58	38.43±1.78	2.26±0.01	1.29±0.03	-	2.00±0.06	3.98±0.09
15–01	Red-orange	22.34	36.34±1.07	2.82±0.07	1.54±0.04	-	1.99±0.03	2.86±0.04
11–07	Dark red	2802.48	18.69±1.38	4.11±0.60	1.43±0.03	231.18±8.98	1.70±0.01	3.92±0.09
M19-4	Red	42.75	25.29±0.08	1.98±0.05	1.56±0.03	18.94±0.21	1.59±0.01	2.70±0.18
GS-2	Red	92.73	27.43±0.63	3.72±0.12	1.21±0.01	38.69±1.37	1.75±0.03	2.62±0.05
3-17-1	Red	73.81	31.61±1.32	2.97±0.21	1.46±0.06	29.33±1.34	2.54±0.07	3.03±0.08
DS-1	Light red	31.73	21.43±0.48	-	1.37±0.08	18.33±0.20	1.84±0.01	2.39±0.06
03–35	Red	109.86	28.37±1.23	3.23±0.05	-	33.55±1.12	-	2.67±0.03
13–05	Red-orange	36.04	32.70±0.97	2.55±0.03	1.22±0.02	-	1.57±0.04	2.63±0.05
XZ-1	Red	163.45	28.97±1.40	3.56±0.93	1.55±0.01	17.87±0.90	1.67±0.04	3.32±0.03
16-10M	Light red	45.82	28.56±2.85	2.10±0.04	1.63±0.09	-	1.75±0.10	2.68±0.06
19–05	Yellow	3.12	28.51±1.44	-	-	-	2.07±0.04	3.59±0.15
Ft-1	Red	49.98	24.63±0.93	2.55±0.03	-	18.77±0.06	-	-
10–03	Red	106.06	24.86±1.60	2.72±0.12	-	32.68±1.57	1.77±0.02	3.30±0.12
Y13-09	Yellow	7.04	18.74±2.01	2.24±0.08	-	-	-	2.62±0.04
S-D-1	Red	81.85	24.18±0.76	-	-	22.94±0.58	1.86±0.02	2.86±0.04
15–51	Red	104.05	29.10±1.57	2.78±0.55	1.08±0.01	69.1±3.81	1.60±0.02	2.87±0.06
628	Dark red	2601.90	15.76±0.30	-	1.45±0.04	149.16±3.56	1.67±0.01	3.42±0.05
08–24	Red	98.57	17.49±0.04	2.16±0.04	-	16.03±0.08	1.60±0.01	2.50±0.01
HB-6	Yellow	1.12	29.06±0.74	2.76±0.04	1.23±0.04	-	1.80±0.01	2.51±0.01
T17-1	Red	69.61	26.83±0.64	2.61±0.5	-	20.42±0.24	-	-
F-value			281.63**	83.57**	11.38**	1277.93**	167.39**	130.14**

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Note: F-value and ** indicate significance at P < 0.01. Data are means ± SD. CC, catechin; EC, epicatechin; LR, liquiritigenin; RT, rutin; Q3G, quercetin-7-O-β-D-glucopyranoside; C3G, cyanidin-3-O-glucoside.

Correlations among fruit color parameters, bioactive compound content, flavonoid components, and antioxidant capacities

A correlation analysis between the color parameters, bioactive compound content, flavonoid components, and antioxidant capacities of 62 P. humilis fruit accessions revealed that the color index was significantly positively correlated with cyanidin-3-O-glucoside content (correlation coefficient = 0.85; P < 0.01) (Fig 6). Catechin, epicatechin, and rutin content was significantly positively correlated with TFC (P < 0.01). The strongest correlation was found between catechin and TFC (correlation coefficient = 0.80). TFC and TPC were significant positively correlated with the antioxidant indices (P < 0.01). Correlation coefficients between TFC and ABTS and between TPC and FRAP were 0.92 and 0.94, respectively.

Fig 6 — Note: * and ** indicate significance at P <0.05 and P <0.01, respectively. CC, catechin; EC, epicatechin; LR, liquiritigenin; RT, rutin; Q3G, quercetin-7-O-β-D-glucopyranoside; C3G, cyanidin-3-O-glucoside.

Principal component analysis

The PCA results identified two components that explained 86.12% of the total variation in fruit quality among the 62 P. humilis accessions (Table 7). The first principal component (Prin1) contributed 52.56% of the total variation, and large positive values were associated with total flavonoid content and ABTS, suggesting that these two indices contributed significantly to Prin1. The second principal component (Prin2) contributed 33.56% of the total variation; large positive values were associated with single fruit weight,vertical and horizontal diameter, suggesting that these three indices greatly contributed to Prin2.

Table 7. Principal component analysis of fruit traits in 62 Prunus humilis accessions.

Traits	Eigenvectors
Traits	Prin1	Prin2
Single fruit weight (g)	-0.26	0.45
Stone weight (g)	-0.01	0.03
Vertical diameter (mm)	-0.20	0.48
Horizontal diameter (mm)	-0.30	0.60
Soluble solid content (%)	0.10	-0.05
Total flavonoid content (mg/g FW)	0.57	0.31
Total phenol content (mg/g FW)	0.19	0.08
DPPH (mg TE/g FW)	0.13	0.08
FRAP (mg TE/g FW)	0.35	0.14
ABTS (mg TE/g FW)	0.54	0.27
Eigenvalue	45.40	28.99
Proportion (%)	52.56	33.56
Cumulative (%)	52.56	86.12

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Note: DPPH, 2,2-diphenyl-1-picrylhydrazyl free radical scavenging capacity; FRAP, ferric reducing antioxidant power; ABTS, 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging capacity.

Scatterplots from the PCA based on the fruit quality showed that 10–02, 3-17-2, 3-17-4, and JD1-6-7-37 belonged in the first group (Fig 7), characterized by high TFC, ABTS and large fruit. The other four groups had different characteristics. These data suggest that crossing within groups maybe more efficient for directed breeding.

Discussion

In this study, we selected 137 P. humilis accessions and analyzed the basic characteristics, bioactive compounds, and antioxidant capacities of their fruit to elucidate flavonoid biosynthesis and identify cultivars that could provide superior human health benefits. Typically, consumers first notice fruit size and appearance. These morphological traits are also important commercial indicators of fruit quality and have become the focus of plant breeders. The range of single fruit weights was wide (1.2–15.61 g; average: 6.13 g). Prunus humilis fruits in Hebei province were measured, and it was found that the largest weight was 10.11 g and the smallest weight was 3.67 g [22]; however, we found that our P. humilis weight varied more widely. It is well known that fruit weight varies between areas, but under similar cultivation conditions in the same area, P. humilis also produces fruit of different weights. This finding means that fruit weight depends on accessions and not geographic areas. Moreover, the genetic diversity in our study was rich. Hence, these accessions furnish abundant material for the selection and breeding of fruits of the required size. SSC directly determines fruit flavor; usually, the higher the soluble solid content, the better the flavor and quality. Thus, the selection of P. humilis with high SSC is an important breeding target. In this study, the SSC in P. humilis fruit was 7.04%–20.64%. Compared with the seven P. humilis fruits in Hebei province (7.43%-15.90%), our fruits have higher soluble solid content. Therefore, accessions with relatively higher SSC should be used in future breeding programs.

Flavonoids occur widely in plants and perform various physiological functions. Flavonoids are some of the most abundant polyphenols in the human diet [28] and are vital functional substances. Liu et al. [29] determined the TFC of five mulberry fruit varieties and found that the highest value was 1.46 mg/g and the lowest value was 0.86 mg/g. Wang et al. [30] measured the TFC for 13 strawberry fruit cultivars and reported a range of 0.26–0.54 mg/g. Xia et al. [31] evaluated the TFC of 13 Prunus salicina fruits and indicated a range of 0.14–1.44 mg/g and an average of 0.85 mg/g. Chen et al. [32] assessed the TFC of six apple fruit lines and found that the highest value was 4.40 mg/g, while the lowest value was 0.86 mg/g. Cao [33] determined the TFC of 186 pear accessions and showed that the values ranged between 0.19 mg/g and 6.77 mg/g and their average was 0.83 mg/g. Wu et al. [34] evaluated the TFC of five blueberry cultivars and demonstrated that the highest value was 3.06 mg/g and the lowest value was 0.52 mg/g. Jiang [35] measured the TFC of 50 grape materials including ten species (46 strains) of East Asian wild grapes; the range for the four control materials was 0.005–0.04 mg/g. In the present study, the TFC of 137 P. humilis accessions ranged from 3.90 mg/g FW to 28.37 mg/g FW, with an average of 10.58 mg/g. The polyphenol content of 28 different genotypes in Liaoning Province was determined, and a total of 31 polyphenols was detected, including 17 flavonols and 1 flavone; among them, the Qu3Ara (quercetin 3-O-arabinoside) was the dominant component, and content ranging from 7.44 to 37.89 mg/100g DW [21]. Catechin was the only flavonoid component detected in all 62 accessions analyzed. Its content ranged from 15.76 to 120.81 mg/100 g FW, and it was significantly and positively correlated with TFC (P < 0.01; correlation coefficient = 0.80). Thus, catechin was the most important flavonoid component of all six components analyzed in this study. Catechin is a natural flavonoid in the flavanol family. It has a strong antioxidant capacity and scavenges free radicals in the human body. It can protect the heart and kidneys, normalize blood pressure, and prevent and cure cancer and inflammation [36]. Cyanidin-3-O-glucoside is a member of the anthocyanin family, which comprises the largest group of pigments in reddish fruits such as grapes, cherries, blueberries, blackberries, plums, and apples [37–39]. Cyanidin-3-O-glucoside is also the major anthocyanin in P. humilis. In the present study, cyanidin-3-O-glucoside was detected in all red and dark red P. humilis accessions and was absent in 23 other accessions, including in all the yellow, 80% of the light red, and 84.62% of the red-orange peels. Moreover, the color index increased with cyanidin-3-O-glucoside content, and the two parameters were significantly positively correlated (P < 0.01; correlation coefficient = 0.85). As we know red fruits and mainly dark red fruits have large quantities of these anthocyanidins. In our early research, the metabonomics of 19–04 (red) were determined, and 20 components of anthocyanins were detected. After absolute quantitative analysis by UHPLC, the content of cyanidin 3-O-glucoside accounted for more than 63% of the total content. Therefore, cyanidin-3-O-glucoside is a vital component of red peel color formation in P. humilis fruit.

Vitamins, organic acids, amino acids, phenols, flavonoids, superoxide dismutase, and other active substances in fruits play important roles in human antioxidant responses. They scavenge free radicals and participate in anti-aging, anti-radiation, and anticancer mechanisms [40]. These active substances also have strong free radical scavenging and antioxidant capacities in plant cells. Free radicals are unstable and therefore have a short life span. It is an important method to evaluate the antioxidant activity of antioxidants by studying their scavenging ability. Due to the difference of chemical property and reaction environment of different free radicals, it is important to select suitable free radicals for evaluating the biological activity and structure-activity relationship of free radical scavengers [1]. In the present study, DPPH, FRAP, and ABTS were selected as antioxidant indices. Regarding sweet cherry, the fruits were characterized by higher antioxidant activity, DPPH radical scavenging activity was about 10 mmol TE/100g DW [41], and that was strongly and positively correlated with fruit phenolic content [18]. The antioxidation test on P. humilis in Liaoning Province found that the FRAP free radical scavenging capacity (9.52–29.44mg/g DW) was the highest and the ABTS free radical scavenging capacity was the lowest (3.40–12.88mg/g DW) [21]. However, in our study, ABTS free radical scavenging capacity (4.68–24.23mg/g FW)was the highest; the difference may be due to fresh and dry samples, and the flavonoids may have changed during drying. This effect could cause a difference in free radical scavenging ability. Our results showed a significant positive correlation between flavonoid content and antioxidant capacity (P < 0.01). Although the P. humilis fruit is abundant in flavonoids, which act as potent antioxidants, the fruit might contain relatively more components that scavenge ABTS and fewer components that scavenge DPPH. We found that ABTS was the most scavenged, while DPPH was the least scavenged, and that the correlation coefficient for ABTS scavenging was the highest, while that for DPPH scavenging was the lowest. These findings are consistent with those reported for Actinidia spp. (kiwifruit) [42], Lonicera caerulea (honeyberry) [43], and Rosa roxburghii (chestnut rose) [44]. More than 9000 flavonoid components occur widely in plants. According to their structure, these flavonoids are classified as flavones, flavanones, flavanols, isoflavones, flavonols, anthocyanidin, and flavanonols. Each component has its own function, and at the same time, the difference in flavonoid content causes differences in antioxidant activity. So different species differ in antioxidant capacity. In particular, P. humilis has a stronger ability of scavenging ABTS free radicals probably because contain a higher number of different antioxidant metabolites.

Cluster analysis divided the 137 P. humilis accessions by flavonoid content into four major types and six subgroups. The TFC in P. humilis accessions showed a normal distribution and was predominantly in the medium range (9.57–15.23 mg/g FW). These quantitative genetic traits are controlled by multiple genes. The accessions were also divided into five different peel color phenotypes. Fruits with red peel had the highest flavonoid content. When we integrated the flavonoid content phenotypes into the clustering results, we found that P. humilis accessions with different peel colors were distributed across all flavonoid concentrations. Hence, there was no obvious correlation between peel color and TFC.

The principal component analysis results identified two components, including TFC, ABTS free radical scavenging activity, single fruit weight,vertical and horizontal diameter. Based on these five indices, we grouped different accessions into five categories with different characteristics; 10–02, 3-17-2, 3-17-4 and JD1-6-7-37 are rich in TFC and strong antioxidant activity that could be more widely used in the general population and the food industry as a source of bioactives to improve human health.

Conclusions

We explored the basic traits, bioactive compounds, and antioxidant capacities of P. humilis fruits. The single fruit and stone weights varied greatly and presented abundant genetic diversity. In contrast, the variation in SSC was small. Therefore, inheritance is relatively simple in this crop. The fruit shape was found to be basically oblate. We found significant differences among all accessions (P < 0.01) in terms of their TFC, TPC, and antioxidant capacity. These findings confirm that there is wide variation and rich genetic diversity among accessions. The TFC for most P. humilis accessions were in the medium range and were normally distributed. This quantitative genetic trait is controlled by multiple genes. Relatively more accessions had a red peel color than peels of other colors; however, we found no obvious correlation between peel color and TFC. The catechin content was high in the accessions and most strongly correlated with TFC. Therefore, catechin appears to be a vital flavonoid component in P. humilis fruit. Additionally, we found that cyanidin-3-O-glucoside was essential for peel color formation in red P. humilis fruits. We selected four accessions (10–02, 3-17-2, 3-17-4, JD1-6-7-37) with high TFC, ABTS free radical scavenging capacity, and large fruit.

Supporting information

S1 Fig. Cluster analysis of fruit flavonoid content in 137 Prunus humilis accessions.

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Click here for additional data file.^{(16KB, doc)}

S1 Table. The background, maturing period and peel color of Prunus humilis accessions.

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Click here for additional data file.^{(198KB, doc)}

S2 Table. A list of abbreviations.

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Click here for additional data file.^{(21KB, doc)}

S3 Table

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Click here for additional data file.^{(495.8KB, doc)}

Acknowledgments

We are very grateful to the Wiley Editing for the assistance with language editing.We also thank the editors and reviewers for their helpful comments regarding this manuscript.

Data Availability

All relevant data are in the manuscript and its Supporting Information files.

Funding Statement

This work was supported by the Key Research and Development Projects of Shanxi Province (Grant No. 201703D221028-4), the Key Projects of Key Research and Development of Shanxi Province (Grant No. 201703D211001-04-04), and the Applied Basic Research Project of Shanxi Province (Grant No. 201801D121251).

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39.Qin LN, Zhang JL, Qin ML. Protective effect of cyanidin 3-O-glucoside on beta-amyloid peptide-induced cognitive impairment in rats. Neurosci Lett. 2013;534: 285–288. 10.1016/j.neulet.2012.12.023 . [DOI] [PubMed] [Google Scholar]
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41.Celli GB, Pereira-Netto AB, Beta T. Comparative analysis of total phenolic content, antioxidant activity, and flavonoids profile of fruits from two varieties of Brazilian cherry (Eugenia uniflora L.) throughout the fruit developmental stages. Food Research International. 2011;44(8): 2442–2451. 10.1016/j.foodres.2010.12.036 [DOI] [Google Scholar]
42.Huang CH, Liang GM, Xie M, Tao JJ, Qu XM, Chen L, et al. Dynamic changes in total phenols, flavonoids and antioxidant capacity during fruit development of different kiwifruit cultivars. J Fruit Sci. 2019;36(02): 174–184. [Google Scholar]
43.Sun J. Comparison of bioactive component contents and antioxidant activities in three varieties of Lonicera caerulea fruits. Non-Wood Forest Res. 2019;37(03): 95–100. [Google Scholar]
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PLoS One. doi: 10.1371/journal.pone.0244445.r001

Decision Letter 0

Branislav T Šiler

9 Oct 2020

PONE-D-20-31344

Fruit quality and antioxidant potential of Chinese dwarf cherry (Cerasus humilis) germplasms

PLOS ONE

Dear Dr. Fu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The manuscript preparation did not follow the Submission Guidelines for PLOS ONE. The authors are advised to meticulously chech and adapt the text according to the Submission Guidelines, since the manuscript cannot be sent to reviewers in this form.

==============================

Please submit your revised manuscript by Nov 23 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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2. Please include a copy of Table 4 which you refer to in your text on page 12. (Table 3 2x)

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Reviewers' comments:

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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PLoS One. 2020 Dec 30;15(12):e0244445. doi: 10.1371/journal.pone.0244445.r002

Author response to Decision Letter 0

22 Oct 2020

Dear editor:

All relevant data are within the manuscript and its Supporting Information files.And all available database are present as tables and figures.

Thanks!

PLoS One. doi: 10.1371/journal.pone.0244445.r003

Decision Letter 1

Branislav T Šiler

3 Nov 2020

PONE-D-20-31344R1

Fruit quality and antioxidant potential of Chinese dwarf cherry (Cerasus humilis) germplasms

PLOS ONE

Dear Dr. Fu,

==============================

The authors show lack of familiarity with the study species since they did not perform literature survey in order to compare the results they have obtained with the ones already published, e.g.:

Shuang, C. H. E. N. G. (2007). Free Radical Scavenging Activities of Polyphenols from Prunus humilis Bge Fruit [J]. Food Science, 9.

Liu, S., Li, X., Guo, Z., Zhang, X., & Chang, X. (2018). Polyphenol content, physicochemical properties, enzymatic activity, anthocyanin profiles, and antioxidant capacity of Cerasus humilis (Bge.) Sok. Genotypes. Journal of Food Quality, 2018.

Wu, Q., Yuan, R. Y., Feng, C. Y., Li, S. S., & Wang, L. S. (2019). Analysis of polyphenols composition and antioxidant activity assessment of Chinese dwarf cherry (Cerasus humilis (bge.) Sok.). Natural Product Communications, 14(6), 1934578X19856509.

and other relevant articles.

Cerasus humilis (Bunge) S.Ya.Sokolov is a synonym of Prunus humilis Bunge (http://www.plantsoftheworldonline.org/taxon/urn:lsid:ipni.org:names:721943-1; http://www.theplantlist.org/tpl1.1/record/rjp-33381)

and should be treated this way throughout the manuscript.

Main title: "accessions", not "germplasms" (and further in the text). Germplasm is a collective noun and cannot read in plural.

Many data are not provided in the M&M section, such as info on the accessions studied. Inconsistencies in measuring process are also notified by the reviewers, while the data visualization can be improved.

The whole text has to be thoroughly reorganized in order to keep the flow and improve readability. Some parts (particularly Introduction and Discussion sections) need to be fully rewritten as suggested in the reviewers' comments.

==============================

Please submit your revised manuscript by Dec 18 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this study authors analysed in 137 Cerasus humilis germplasms the fruit features, polyphenol and flavonoid contents, and the antioxidant activity of fruit extracts with the aim to identify cultivars with increased nutritional and nutraceutical properties.

The aim of the study is interesting, but the quality of the manuscript is poor.

The introduction is superficial, and more background is needed in order to understand the overall purpose of this study: authors have not considered the presence in literature of many papers dealing with antioxidant metabolites and antioxidant properties in cherry fruits (some of them in the links below).

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912798/

https://www.sciencedirect.com/science/article/abs/pii/S096399691100007X

https://www.sciencedirect.com/science/article/abs/pii/S0308814617313158

Material and Methods

The methodology is missing important details rendering the reproducibility of this study impossible.

Authors deal with 137 C. humilis germplasms; however, they do not include any detail of the species or accessions in the Material and Methods section. A more detailed explanation of plant material must be included in this section, and a list of the 137 plant accessions should be added as supplementary table.

“Field management included conventional irrigation and fertilization methods”. This is an international journal and the conventional specifications for cultivation in China may be different from those of other Nations. Therefore, everything must be written in detail or referred to another publication in which the cultivation method has been already explained in detail.

“Total phenol content (TPC) was determined by Folin-Ciocalteu colorimetry [17]. A certain volume of extracted solution”: what do you mean with “a certain volume”? This is a scientific article, everything must be correctly defined.

“Following the method of Zhang [18], we added 2.8 mL of 0.1 mM DPPH to 0.2 mL of sample diluent.”: What do authors mean with “sample diluent”? Methanolic extract?

About the FRAP assay: “For the blank, the diluent was replaced with 40% (v/v) methanol.”: Do authors mean “40% (v/v) acidified methanol”?

The Trolox assay must be better specified.

About the cluster analysis, I do not understand why the heat map analysis has been performed using only one parameter and not including all the analyses parameters. An analysis like heat map or PCA represents a useful tool for the analysis of multivariate data that could offer an overview of the results and visualize the real differences between different accessions.

The discussion is too general and not very focused on the data obtained. Probably the fact that the entire set of data was not subjected to a multivariate analysis (heay map or PCA) did not allow the authors to fully understand the differences between the accessions analysed and the potential of their own study.

I think at the moment the manuscript requires an extensive revision, including missing details, a better data analysis and a deep re-writing of the introduction and discussion. Therefore, I reject the manuscript encouraging the authors to resubmit it after addressing all my concerns.

Reviewer #2: The paper entitled “Fruit quality and antioxidant potential of Chinese dwarf cherry (Cerasus humillis) germplasms¨ aims to analyse flavonoid, total phenol content and antioxidant potential of 137 dwarf cherry germplasms.

Article satisfy all PLOS ONE publication criteria.

The presented studies were well planned, and methodology is appropriate for the aim of study. Authors decided to use various methods. In order to determine antioxidant activity against free radicals the following methods used: DPPH, ABTS and FRAP. Additionally, statistical analysis of the studies was performed.

In my opinion the manuscript is well prepared and obtained results are interesting and novelty. The results are well analysed, and form of their presentation is good. References are adequate. The obtained results are interesting and can be interpreted as good direction for future studies.

Authors point out that they aimed to elucidate flavonoid biosynthesis and I do not agree with them. In my opinion they have studied the fruit quality, mainly related to flavonoids, but not their biosynthesis.

A list of abbreviations will be very helpful.

Define the abbreviations at Table 6 legend.

Have you studied other anthocyanidins apart from Cyanidin-3-gluscoside? Red fruits and mainly dark red fruits should have large quantities of these compounds.

Discussion is comprehensive and readable but can be improved. A deeper discussion relating data is needed.

May be a Principal component analysis of data should be interesting.

In my opinion the paper can be published in PLOS ONE after some revisions.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. 2020 Dec 30;15(12):e0244445. doi: 10.1371/journal.pone.0244445.r004

Author response to Decision Letter 1

6 Nov 2020

Dear editor:

1.We have revised the information about the author (Baochun Fu and Jinming Guo).

2.We have upload the figures(1-8) as the Item type 'Figure' files.

Thanks!

Attachment

Submitted filename: response to reviewers.doc

Click here for additional data file.^{(44KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0244445.r005

Decision Letter 2

Branislav T Šiler

10 Nov 2020

PONE-D-20-31344R2

Fruit quality and antioxidant potential of Chinese dwarf cherry (Cerasus humilis ) accessions

PLOS ONE

Dear Dr. Fu,

==============================

The authors rushly returned the revised manuscript failing to address all the concerns raised in the editor's and reviewers' reports. Moreover, in their response to reviewers, for the comments which need a thorough discussion, it is not sufficient to reply with one universal answer such as: "modification have been made as required...". The authors should express their opinion about each and every issue noted by the reviewer while pointing out to the exact place in the text (line numbers) where the changes were made. I strongly encourage the authors to meticulously check the previous editor's and reviewers' reports in order to avoid potential future flaws in the manusript preparation process.

==============================

Please submit your revised manuscript by Dec 25 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

PLoS One. 2020 Dec 30;15(12):e0244445. doi: 10.1371/journal.pone.0244445.r006

Author response to Decision Letter 2

12 Nov 2020

Dear editor:

We have upload the files about our manuscript.

Thanks!

Attachment

Submitted filename: response to reviewers.doc

Click here for additional data file.^{(82.5KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0244445.r007

Decision Letter 3

Branislav T Šiler

27 Nov 2020

PONE-D-20-31344R3

Fruit quality and antioxidant potential of Chinese dwarf cherry (Cerasus humilis ) accessions

PLOS ONE

Dear Dr. Fu,

==============================

The language is still one of the major concerns. Some of them are stated in the reviewers' reports but I strongly recommend revising the manuscript by a professional editing agency.

Another major point: in the Revision #2, I advised:

"Cerasus humilis (Bunge) S.Ya.Sokolov is a synonym of Prunus humilis Bunge (http://www.plantsoftheworldonline.org/taxon/urn:lsid:ipni.org:names:721943-1; http://www.theplantlist.org/tpl1.1/record/rjp-33381)

and should be treated this way throughout the manuscript."

The authors did not respond to this important issue through two rounds of the review process. To make myself more clear: Prunus humilis Bunge is the internationally accepted botanical name of the study species and Cerasus humilis (Bunge) S.Ya.Sokolov is a synonym. Therefore, P. humilis should be used through the text, while its synonym can be written in parenthesis first time it is mentioned in the Introduction section.

Moreover, the authors did not respond to the Editor's comment:

"The authors show lack of familiarity with the study species since they did not perform literature survey in order to compare the results they have obtained with the ones already published, e.g.:

Shuang, C. H. E. N. G. (2007). Free Radical Scavenging Activities of Polyphenols from Prunus humilis Bge Fruit [J]. Food Science, 9.

Wu, Q., Yuan, R. Y., Feng, C. Y., Li, S. S., & Wang, L. S. (2019). Analysis of polyphenols composition and antioxidant activity assessment of Chinese dwarf cherry (Cerasus humilis (bge.) Sok.). Natural Product Communications, 14(6), 1934578X19856509.

and other relevant articles."

I find the adequate approach to the proper taxonomic position of the studied species highly important. In addition, meticulous literature survey which would NOT exclude the most important articles in the field while comparing obtained results reported therein is essential towards increasing the manuscript quality.

==============================

Please submit your revised manuscript by Jan 11 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: No

**********

6. Review Comments to the Author

Reviewer #1: I have previously reviewed this manuscript raising several concerns. The authors have addressed all the points raised; however, the English of the new included parts is very poor. There are too many grammatical flaws. In particular, there are errors in grammar and sentence structure and the paper sometimes is difficult to read. I have corrected some of them, but the paper needs a careful revision and a professional English editing.

Line 40: substitute “out” with “our”

Lines 46- 49: change “ It is like peach, plum and apricot, which is an ancient tree species in China with a cultivation history dated back to around 3000 years [2]. It is grown in the north of China recorded about 13 provinces, i.e., Shanxi, Hebei, Liaoning provinces, and so on [3].” with “ Like peach, plum and apricot, it is an ancient tree species in China with a cultivation history that dates back to around 3000 years [2]. Its growth in the north of China is recorded in about 13 provinces, among which Shanxi, Hebei and Liaoning [3].”

Lines 53-54: “C. humilis plants are known as ‘calcium fruit’ in China due to their high calcium content [6].”

Lines 73-74: “[17], including Cerasus humilis, represent” change with ““[17]. In this view, Cerasus humilis represents”

Line 87: “In the early research,” change with “Previously”

Line 93: change “assessions” with “accessions”

Lines 105-106: “The growth period was fertilized twice a year,” change with “During the growth period, plants were fertilized twice a year”

Lines 107-108: “From May to August watering once or twice a month, in the early November enough water was irrigated for overwintering.” Change with “From May to August the plants were watered once or twice a month; in early November the plants were irrigated with enough water for wintering.”

Lines 186: Trolox evaluation

Lines 394-397: “It is well known that fruit weight in different area, but under similar cultivation conditions in the same area also produce differernt weights, it mean that it has a close relationship between the fruit weight and accessions.” change with “It is well known that fruit weight in different areas but similar cultivation conditions produce fruits with different weights; it means that fruit weight depends on accessions and not geographic areas”.

Line 400: the better

Line 403: “our fruits are with high soluble solid content.” change with “our fruits have higher soluble solid content.”

Line 422:” a total of 31 polyphenols were detected” change with “a total of 31 polyphenols was detected” or “31 polyphenols were detected”

Line 424: ranging

Line 426: significantly and positively

Line451: About the research of sweet cherry plants,

Lines 453-454: “it is strong positive correlations between fruit phenolic content [18]” change with “that was strongly and positively correlated with fruit phenolic content [18].”

Line 457: “However, in our study” instead of “But in our study”

Lines 472-476: “So there are differences in the ability on antioxidant indices in different species. It may be that there are more components in C.humilis fruit which have the ability of scavenging ABTS free radicals, so that the C.humilis fruit has a stronger ability of scavenging ABTS free radicals.” Change with “So different species differ in antioxidant capacity. In particular, C.humilis has a stronger ability of scavenging ABTS free radicals probably because contain a higher number of different antioxidant metabolites.”. Moreover, the name of species must be italicized.

The results and discussion now appear reliable and also interesting, but the paper needs a careful revision and a professional English editing.

Reviewer #2: The article has been corrected following reviewers’ indications. Now it is acceptable for its publication after minor revision.

New paragraphs have been added to the manuscript. Please review English in these new parts of the article mainly the part that has been added to the discussion.

For example: Line 396 ..., it mean must be it means

Line 400 ...content ,the beter..... replace by: content, the better...

Line 400…quality. thus.. replace by …Thus..

Line 424 and content tanging… replace by ranging

Lines 475 and 476 C. humilis must be in italics.

These are just examples, please review in detail and correct some expressions and sentences, because it is not always easy to understand.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Petronia Carillo

Reviewer #2: No

PLoS One. 2020 Dec 30;15(12):e0244445. doi: 10.1371/journal.pone.0244445.r008

Author response to Decision Letter 3

2 Dec 2020

Dear edit:we have upload the file incliding cover letter, manuscript, revised manuscript with track changes, and response to reviewer, and figures and supplements.

Attachment

Submitted filename: Response to reviewer.docx

Click here for additional data file.^{(20.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0244445.r009

Decision Letter 4

Branislav T Šiler

4 Dec 2020

PONE-D-20-31344R4

Fruit quality and antioxidant potential of Chinese dwarf cherry (Prunus humilis) accessions

PLOS ONE

Dear Dr. Fu,

==============================

As I commented in the previous review round, full scientific name of the studied species must stand. Hence, "Prunus humilis Bunge" must stand in the main title (without quotation marks), and in the first mention in the Introduction section. There (in the Introduction section), its synonym "Cerasus humilis (Bunge) S.Ya.Sokolov" should be provided in parenthesis (in brackets) of the actual scientific name.

One more most important point: Figure 1, Figure 2 and Figure 6 are of very poor quality.

Labels in Fig.1 and Fig.6 are not visible. Resolution of Fig.2 is too low.

==============================

Please submit your revised manuscript by Jan 18 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

PLoS One. 2020 Dec 30;15(12):e0244445. doi: 10.1371/journal.pone.0244445.r010

Author response to Decision Letter 4

4 Dec 2020

Dear editor:

Thanks for your understand my mood about my degree, and we have revised my manuscript according your comments, and we have update and upload the figures.

Wish you have a good life!

Sincerely!

Attachment

Submitted filename: response to reviewers.docx

Click here for additional data file.^{(13.6KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0244445.r011

Decision Letter 5

Branislav T Šiler

7 Dec 2020

PONE-D-20-31344R5

Fruit quality and antioxidant potential of Prunus humilis Bunge accessions

PLOS ONE

Dear Dr. Fu,

==============================

Authors did not make visible labels in Figure 2. Since the information presented there is hardly readable, I suggest moving the Figure 2 into the Supplementary material. Figures 3 - 6 must be renamed consecutively and their references in the text should be updated too.

==============================

Please submit your revised manuscript by Jan 21 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

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PLoS One. 2020 Dec 30;15(12):e0244445. doi: 10.1371/journal.pone.0244445.r012

Author response to Decision Letter 5

7 Dec 2020

Dear editor: we have moFved Figure 2 into supplement and changed figures 3-8 to 2-7.

Thanks for your comments!

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PLoS One. doi: 10.1371/journal.pone.0244445.r013

Decision Letter 6

Branislav T Šiler

9 Dec 2020

PONE-D-20-31344R6

Fruit quality and antioxidant potential of Prunus humilis Bunge accessions

PLOS ONE

Dear Dr. Fu,

==============================

Dear authors, I owe you a big apology. In the previous review round I asked you to move Figure 2 into the supplementary material, but I really meant Figure 1. Can you please put back Figure 2 and move Figure 1 to supplementary? Once again, I deeply regret for asking you for another revision.

==============================

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PLoS One. 2020 Dec 30;15(12):e0244445. doi: 10.1371/journal.pone.0244445.r014

Author response to Decision Letter 6

9 Dec 2020

Dear editor:

we have revised the figure 1 to the Fig S1, and we update the figures 1-7.

Thank you for your support!

PLoS One. doi: 10.1371/journal.pone.0244445.r015

Decision Letter 7

Branislav T Šiler

10 Dec 2020

Fruit quality and antioxidant potential of Prunus humilis Bunge accessions

PONE-D-20-31344R7

Dear Dr. Fu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0244445.r016

Acceptance letter

Branislav T Šiler

14 Dec 2020

PONE-D-20-31344R7

Fruit quality and antioxidant potential of Prunus humilis Bunge accessions

Dear Dr. Fu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Cluster analysis of fruit flavonoid content in 137 Prunus humilis accessions.

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S1 Table. The background, maturing period and peel color of Prunus humilis accessions.

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S2 Table. A list of abbreviations.

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S3 Table

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Data Availability Statement

All relevant data are in the manuscript and its Supporting Information files.

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PERMALINK

Fruit quality and antioxidant potential of Prunus humilis Bunge accessions

Hongbo Fu

Xiaopeng Mu

Pengfei Wang

Jiancheng Zhang

Baochun Fu

Junjie Du

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Abstract

Introduction

Materials and methods

Plant materials and chemicals

Single fruit and stone weights and fruit diameter

Fruit color

Soluble solid content (SSC)

Total flavonoid and phenol content

Antioxidant capacity

DPPH assay

ABTS assay

Ferric reducing antioxidant power (FRAP) assay

Trolox evaluation

Flavonoid components

Statistical analysis

Results

Changes in flavonoid content over two years

Table 1. Flavonoid content and variation analysis of Prunus humilis fruits in 2018 and 2019.

Fruit character

Table 2. Fruit character variation in 137 Prunus humilis accessions.

Table 3. Fruit bioactive compounds and antioxidant capacities in 137 Prunus humilis accessions.

Cluster analysis of fruit flavonoid content

Table 4. Flavonoid content levels in Prunus humilis accessions.

Fig 1. Normal distribution of total flavonoid content in 137 Prunus humilis accessions.

Flavonoid content in different-colored fruit peels

Fig 2. Prunus humilis accession phenotypes.

Table 5. Flavonoid content in different-colored fruit peels of Prunus humilis accessions.

Bioactive compounds and antioxidant capacity

Fig 3. Flavonoid and total phenol content in 62 Prunus humilis accessions.

Fig 4. Antioxidant capacities of 62 Prunus humilis accessions.

Flavonoid components

Fig 5. Flavonoid standards used to detect flavonoid content in 62 Prunus humilis accessions by ultrahigh performance liquid chromatography (UHPLC).

Table 6. Flavonoid components in different-colored fruit peels of 62 Prunus humilis accessions.

Correlations among fruit color parameters, bioactive compound content, flavonoid components, and antioxidant capacities

Fig 6. Correlations among fruit color parameters, bioactive compound content, flavonoid components, and antioxidant capacities in 62 Prunus humilis accessions.

Principal component analysis

Table 7. Principal component analysis of fruit traits in 62 Prunus humilis accessions.

Fig 7. Scatterplot of principal component analysis based on fruit quality of 62 Prunus humilis accessions.

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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