Nutritional composition and antioxidant properties of the fruit of Berberis heteropoda Schrenk.

OBJECTIVE: This study assessed the major nutrients and antioxidant properties of Berberis heteropoda Schrenk fruits collected from the Nanshan Mountain area of Urumqi City, Xinjiang Uygur Autonomous Region, China. METHODS AND MATERIALS: We assessed the basic nutrients, including amino acids, minerals, and fatty acids, and determined the total phenol, flavonoid, and anthocyanin contents of the extracts.
RESULTS: The analytical results revealed the average water (75.22 g/100 g), total fat (0.506 g/100 g), total protein (2.55 g/100 g), ash (1.31 g/100 g), and carbohydrate (17.72 g/100 g) contents in fresh B. heteropoda fruit, with total phenol, flavonoid, and anthocyanin contents of B. heteropoda fruits at 68.55 mg gallic acid equivalents/g, 108.42 mg quercetin equivalents/g, and 19.83 mg cyanidin-3-glucoside equivalent/g, respectively. Additionally, UPLC-Q-TOF-MSE analysis of polyphenols in B. heteropoda fruit revealed 32 compounds.
CONCLUSION: B. heteropoda fruits may have potential nutraceutical value and represent a potential source of nutrition and antioxidant phytochemicals in the human diet.

Introduction

Berberis heteropoda Schrenk is a shrub of the family Berberidaceae, which is distributed in the Altai, Tianshan, and Baluke mountains of the Xinjiang Uygur Autonomous Region, China, as well as in Mongolia and Kazakhstan [1]. The roots, bark, stems, and fruits of B. heteropoda are traditionally used as an herbal medicine, and the fruits in particular have historically been consumed as a tea [2, 3]. In modern times, studies have confirmed that this fruit can be used to treat dysentery, enteritis, pharyngitis, stomatitis, eczema, and hypertension [4, 5]. Because the nutritional and antioxidant properties of B. heteropoda fruit are related to its molecular and secondary metabolite content, there are potential benefits to its consumption.

In addition to proteins, fats, dietary fiber, minerals, and other nutrients, plants also contain numerous phenolic components that can play an important role in human health [6]. Polyphenols are secondary metabolites produced by plants and often observed in vegetables, fruits, and forages [7]. Phenolic compounds are effective at preventing oxidation at the cellular and physiological levels, with their antioxidant capacity determined based on the arrangement of hydroxyl and carbonyl groups in their structures, as well as the gain and loss of electrons from hydrogen atoms to reduce free radicals and form stable phenoxy groups [8, 9]. Flavonoids are major components of plant polyphenols and play important roles in antioxidant effects, including in reduction reactions as a hydrogen donor for singlet oxygen quenching and metal chelation. Thus, evaluating the polyphenol, flavonoid, and antioxidant contents of B. heteropoda Schrenk fruit and evaluating its medicinal and nutritional value are important.

A previous study focused on the anthocyanin composition of B. heteropoda fruit [3]; however, the nutritional and phenolic composition of B. heteropoda remains unclear. Therefore, in the present study, we assessed the major nutrient content and antioxidant properties of B. heteropoda and investigated the active components of the plant, as well as how this information can guide its nutritional use.

Materials and methods

Plant material

A total of 3 kg of ripe B. heteropoda fruit was collected from dozens of shrubs in a ravine in the Nanshan Mountain area of Urumqi City, Xinjiang Uygur Autonomous Region, China (latitude 89°29′36″E, longitude 43°27′32″N), in September 2019. The specimens were identified by expert Lude Xin from Xinjiang Medical University, and a voucher specimen (WR2101079001) was deposited in the Institute of Clinical Nutrition, People’s Hospital of Xinjiang Autonomous Region. The fruits were then transported to the laboratory for a pre-cooling treatment (−20 °C) 2 h after harvest. The fruits for study were selected after being combed, and we ensured that all of the selected fruits were even and full, with uniform size and maturity. Fruits without mechanical damage, rot, or other miscellaneous defects were selected for further analysis. Subsequently, the stem and seeds were removed, and fruits were placed in dark storage at −20 °C until further use.

Standards and reagents

The reagents 1,1-diphenyl-2-picryl-hydrazl (DPPH) and 2,2-azinobis-(3-ethylbenzthiazoline-6- sulfonic acid) (ABTS) were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Gallic acid and rutin standards were purchased from Chengdu Munster Biotechnology Co., Ltd. (Chengdu, China). Folin–Ciocalteu’s phenol reagent was purchased from Tianjin Kaitong Chemical Reagent Co., Ltd. (Tianjin, China). Anhydrous methanol, anhydrous ethanol, concentrated hydrochloric acid, sodium nitrite, sodium hydroxide, sodium carbonate, and ferrous sulfate were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shenyang, China).

Nutritional composition

Determination of general nutrients

Crude protein content was determined using the Kjeldahl method according to Chinese National Standard (CNS) GB/T5009.5–2016 “Determination of protein in food.” Ash content was measured using the muffle furnace burning method according to CNS GB 5009.4–2016 “Determination of ash in food.” Crude fat was determined using the Soxhlet extraction method according to CNS GB 5009.6–20163 “Determination of fat in food.” Moisture was measured using the direct drying method according to CNS GB5009.3–2016 “Determination of moisture in food.” Carbohydrate content was determined based on CNS NY/T 2332–2013. The total energy of each sample was calculated as follows: Total Energy (kJ) = 17 × (g crude protein + g total carbohydrate) + 37 (g crude fat) [10].

Mineral composition

Mineral and element contents were determined according to CNS GB5009.268–2016 “Determination of multi-elements in food” using inductively-coupled plasma (ICP)-mass spectrometry [MS; 5110 ICP optical emission spectrometer (OES); Agilent Technologies, Santa Clara, CA, USA]. Briefly, a 1.0-g slurry sample was digested in 2 mL of concentrated HNO3 in a microwave oven and then diluted with distilled water to 25 mL. The solution was filtered before storage, and a blank digest was performed in a similar manner. The blank solution and the test solution were each injected into the ICP OES to determine the contents of K, Ca, Na, Mg, Fe, Cu, Zn, and P.

Amino acid analysis

Amino acid contents were measured by an automatic amino acid analyzer (L-8900; Hitachi, Tokyo, Japan) according to CNS GB 5009.124–2016 “Determination of amino acid in food.” Continuous flash evaporation at reduced pressure was used to remove excess acid, and the sample was dissolved in citrate buffer (pH 2.2) [11].

Fatty acids

Fatty acid composition and content was determined by gas chromatography–MS (7890B/7000D; Agilent Technologies) according to CNS GB 5009.168–2016. Triglyceride undecarbonate was used as an internal reference, 37 different fatty acid methylester standard solutions were used as external references. The fatty acid content was quantitatively measured using chromatographic peaks.

Extraction and quantification of Total Phenol Content (TPC), Total Flavonoid Content (TFC), and Total Anthocyanin Content (TAC)

Extraction

Sample extraction was performed using a previously reported method [12], with slight modification. Briefly, 1.0 g of B. heteropoda fruit was added to 30 mL of 70% acidified ethanol (0.1% HCl, v/v), and the solution was extracted three times under ultrasonic conditions (40 kHz, 100 W) for 30 min at 25 °C. The mixture was then centrifuged at 1000 r/min for 15 min, and the supernatant was collected. The residue was subsequently extracted twice, all of the collected supernatant was mixed together and concentrated under vacuum, and the extraction was preserved at −20 °C until further analysis. The solvents used for fruit extraction included methanol, acetone, and ethanol. The final extract was used for the quantification of TPC, TFC, TAC, and antioxidant activity.

TPC Determination

The TPC was measured using Folin–Ciocalteu’s phenol reagent with the colorimetric method [13]. Briefly, 0.5 mL of reagent and 1.5 mL of sodium carbonate solution (10%, w/v) were added to 1 mL of B. heteropoda fruit extract, followed by immediate addition of 8 mL of distilled water and incubation for 10 min in a water bath at 75 °C. The absorbance was the measured using an ultraviolet–visible (UV–vis) spectrophotometer (New Century T6; Persee Analytics, Beijing, China) at 760 nm. We generated a standard curve of the absorbance value of gallic acid solution, and then TPC was determined as milligram of gallic acid equivalent per gram of fresh fruit mass.

TFC Determination

The TFC was measured using rutin as a reference standard with the aluminum nitrate method [14]. Briefly, 0.5 mL of B. heteropoda fruit extract was added to 1 mL of sodium nitrite and incubated for 6 min, followed by mixture with 1 mL of 10% aluminum nitrate and then incubation for another 6 min. We then added 10 mL of 1.0 M sodium hydroxide, adjusted the volume of water to 20 mL, and incubated the solution for 15 min. UV–vis spectrometry was then used to detect the absorbance at 510 nm and generate a standard curve. TFC was denoted as milligram of rutin equivalent per gram of weight of fresh fruit mass.

TAC Determination

The TAC was determined by the pH differential method [15]. Briefly, 2 mL of fruit extract was added to a centrifuge tube for centrifugation at 1000 r/min for 5 min, after which 0.5 mL of supernatant was added into two 10-mL volumetric flasks: one with a buffer at pH 1.0 and the other with a buffer at pH 4.5. The absorbance at 517 nm and 700 nm was measured after a 15-min incubation, and data were expressed as milligram of cyanidin-3-glycoside equivalents per gram of fresh fruit mass. The TAC was calculated according to the following formula: where MW represents the molecular weight of centrinin-3-glycoside [449.2 g/mol; according to the centrothrin-3-glycoside molar extinction coefficient (26900 L·cm−1·mol−1)], DF represents diluted multiples, L denotes absorption pool thickness (1 cm), V represents extraction volume (mL), and M denotes the weight of peel powder.

Measurements of antioxidant capacity

DPPH free radical assay

The DPPH free radical-scavenging assay was performed according to the method described by Vlase et al. [16]. Briefly, B. heteropoda fruit extract was dissolved in 70% ethanol at different concentrations and mixed with 2 mL of a freshly prepared ethanol solution of DPPH free radicals (100 μM). The solution was mixed vigorously and stored in darkness at room temperature for 30 min, followed by UV–vis spectrometry detection of the absorbance at 517 nm. The positive control group was measured using vitamin C (VC). The results were expressed as half maximal inhibitory concentration (IC50), which was used to indicate the corresponding concentration of the extract when the anti-oxidation free radical-scavenging capacity was 50%: where AC denotes the absorbance value of the control, A0 represents the absorbance value of the blank, and AS is the absorbance value of the sample.

ABTS free radical assay

The ABTS free radical-scavenging assay was performed according to the method described by Lyu et al. [17]. Briefly, 2 mL of 10 mM potassium persulfate solution and 2 mL of 10 mM ABTS free radical solution were mixed and then stored in the dark for 12 h. Ethanol was then added to the mixed solution until its UV–vis absorbance value reached 0.700 ± 0.020 at 736 nm. Subsequently, 2 mL of B. heteropoda fruit extract or ascorbic acid solution was mixed vigorously with 2 mL of ABTS working solution and stored in the dark at room temperature for 10 min. The IC50 values of the sample extract were calculated based on the concentration and capacity designated by the free radical-scavenging curves: where AC represents the absorbance value of the control, and AS denotes the absorbance value of the sample.

Hydroxyl free radical assay

The hydroxyl free radical assay was performed according to the method described by Liang et al. [18]. Briefly, 0.5 mL of 7.5 mM ferrous sulfate heptahydrate, 0.5 mL of 7.5 mM salicylic acid, 1 mL of B. heteropoda fruit extract, and 0.2 mL of 30% hydrogen peroxide were mixed and incubated for 30 min in a water bath at 37 °C. After cooling, the absorbances of the hydroxyl radical sample, blank, and control groups were determined at 510 nm on the UV–vis spectrometer, and hydroxyl radical scavenging activity (HRSA) was determine as follows: where AC denotes the absorbance value of the control, A0 represents the absorbance value of the blank, and AS represents the absorbance value of the sample.

Superoxide anion free radical assay

The superoxide anion free radical assay was performed according to the method described by Liu et al. [15]. Briefly, 4.5 mL of 50 mM Tris–hydrochloric acid and 1 mL of B. heteropoda fruit extract were mixed and incubated 15 min in a water bath at 25 °C, followed by the addition of 0.4 mL of 5 mM pyrogallic acid and incubation for 5 min in a water bath at 25 °C. Subsequently, 0.1 mL of 8 M hydrochloric acid was added to terminate the reaction, and the absorbance values of the sample, blank, and control were measured at 325 nm on the UV–vis spectrometer to determine the following rate: where AC denotes the absorbance value of the control, and AS represents the absorbance value of the sample.

Chromatography and mass spectrometry

Chromatographic conditions

Chromatographic separations were performed using an ultra-high performance liquid chromatography (UPLC) 1290 system with a Waters UPLC BEH C18 column (1.7 μm 2.1 × 100 mm; Agilent Technologies). The flow rate was set to 0.4 mL/min, and the sample-injection volume was set to 5 μL. The mobile phases comprised 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The multi-step linear elution gradient program was as follows: 0 to 3.5 min, 95% to 85% A; 3.5 to 6 min, 85% to 70% A; 6 to 6.5 min, 70% to 70% A; 6.5 to 12 min, 70% to 30% A; 12 to 12.5 min, 30% to 30% A; 12.5 to 18 min, 30% to 0% A; 18 to 25 min, 0% to 0% A; 25 to 26 min, 0% to 95% A; and 26 to 30 min, 95% A.

MS conditions

We used a Q Exactive Focus mass spectrometer coupled with Xcalibur software (Thermo Fisher Scientific, Waltham, MA, USA) was employed to obtain MS and MS/MS data in independent data acquisition mode. During each acquisition cycle, the mass range was set to a range of 100 to 1500, the top three data points in every cycle were screened, and the corresponding MS/MS data were further acquired. The following parameters were used: sheath gas-flow rate, 45 Arb; auxiliary gas-flow rate, 15 Arb; capillary temperature, 400 °C, full MS resolution, 70,000; MS/MS resolution, 17,500; collision energy, 15/30/45 in normalized collision energy mode; and spray voltage, 4.0 kV (positive) or −3.6 kV (negative).

Statistical analysis

All experimental data were collected in triplicate, and data were expressed as the mean ± standard deviation. Statistical analyses were performed using GraphPad Prism (v.7.0; GraphPad Software, La Jolla, CA, USA) and SPSS (v.23.0; IBM Corp. Armonk, NY, USA).

Results

Nutritional composition of B. heteropoda fruit

Proximate composition of B. heteropoda fruit

The major nutrients of B. heteropoda fruit are summarized in Table 1 and S1 File. The major components were identified as water, crude fiber, and total protein, with values of 75.22±1.75 g/100 g, 17.72±0.52 g/100 g, and 2.55±0.03 g/100 g, respectively Ash content was 1.31±0.04 g/100 g, indicating that the fruit is rich in minerals. The total sugar and total fat contents were 0.05±0.00 g/100 g and 0.51±0.02 g/100 g, respectively, and the energy content per 100 g of fruit was 363.52 kJ.

Table 1

Proximate nutritional composition of fresh Berberis heteropoda fruit.

Composition (Unit)
Water (g/100 g)	75.22±1.75
Total fat (g/100 g)	0.506±0.02
Total Protein (g/100 g)	2.55±0.03
Ash (g/100 g)	1.31±0.04
Total sugars (g/100 g)	0.05±0.00
Carbohydrates (g/100 g)	17.72±0.52
Total Energy (kJ)	363.52±7.51

Minerals

We detected a total of eight minerals in B. heteropoda fruit (Table 2 and S1 File). We found that K (582.67±8.02 mg/100 g) was the most abundant element [19], with Ca (78.5±1.62 mg/100 g), P (73.24±1.72 mg/100 g), and Mg (30.61±0.56 mg/100 g) also abundant.

Table 2

Nutritional composition (minerals and amino acids) of Berberis heteropoda fruit.

Minerals	Composition (mg/100 g FW)	Total minerals (%)
Na	1.38±0.03	0.18
K	582.67±8.02	75.73
Ca	78.5±1.62	10.20
Cu	0.27±0.01	0.04
Zn	0.59±0.01	0.08
Fe	2.31±0.05	0.30
Mg	30.61±0.56	3.97
P	73.24±1.72	9.51
Total minerals	769.843
Amino acids	Composition (g/100 g FW)	Total amino acids (%)
Phenylalanine	0.12±0.01	4.24
Alanine	0.17±0.01	6.01
Methionine	0.015±0.00	0.53
Proline	0.2±0.02	7.07
Glycine	0.21±0.01	7.43
Glutamic acid	0.53±0.01	18.74
Arginine	0.22±0.01	7.78
Lysine	0.21±0.00	7.43
Tyrosine	0.11±0.00	3.89
Leucine	0.18±0.01	6.36
Serine	0.13±0.01	4.60
Threonine	0.13±0.01	4.60
Aspartic acid	0.27±0.01	9.55
Valine	0.15±0.01	5.30
Histidine	0.073±0.00	2.58
Isoleucine	0.11±0.01	3.89
Total amino acids	2.828

FW, fruit weight.

Amino acids

The 16 amino acids identified in B. heteropoda fruit are shown in Table 2 and S1 File. Glutamic acid was the most abundant amino acid, followed by aspartic acid, arginine, lysine, and glycine. The fruit contained six types of essential amino acids (EAAs) to a value of 0.9 g/100 g fruit weight and accounting for 31.8% of the total amino acids, with the content of the remaining 10 non-EAAs (NEAAs) at 1.93 g/100 g fruit weight.

The fatty acid content in B. heteropoda fruit is presented in Table 3 and S1 File. We identified a total of 10 different fatty acids, including saturated and unsaturated varieties. Tetrahexanoic acid (C24:0) was the dominant fatty acid, followed by octadecentrienoic acid (C18:3) and octadecadienoic acid (C18:2). The unsaturated fatty acid (UFA) content was slightly higher than that of the saturated fatty acid (SFA) content (51.51% vs. 48.48%).

Table 3

Fatty acid content in Berberis heteropoda fruit.

Fatty acids	Formula	Composition (g/100 g fatty acid)	Proportion (%)
Myristic acid (C14:0)	C14H28O2	0.0039	1.41
2-methyl-heptanoic acid (C8:0)	C8H16O2	0.0019	0.71
Hexadecanoic acid (C16:0)	C16H32O2	0.0285	10.35
Stearyl acid (C18:0)	C18H36O2	0.0041	1.50
Octadecenoic acid (C18:1)*	C18H32O2	0.0263	9.55
Octadecadienoic acid (C18:2)*	C18H32O2	0.0526	19.11
Octadecentrienoic acid (C18:3)*	C18H30O2	0.0630	22.86
Arachidic acid (C20:0)	C20H40O2	0.0038	1.38
Docosanoic acid (C22:0)	CH3(CH2)20COOH	0.0112	4.06
Tetrahexanoic acid (C24:0)	CH3(CH2)22COOH	0.0801	29.08
Subtotal		0.2754	100.00

* UFAs

TPC, TFC, and TAC

The TPC, TFC, and TAC values for B. heteropoda fruit are shown in Fig 1 and S2 File. The methods used to determine flavonoid and polyphenol contents showed a good linear relationship within the measurement range (r2 = 0.995 and 0.999, respectively), with the following regression equations used: y = 0.0109x + 0.0157 and y = 0.067x − 0.0173, respectively. The extraction effect of each solvent (high to low) was methanol > acetone > ethanol for total phenol, total flavonoids, and total anthocyanins. Using methanol as the extraction solvent yielded TFC, TPC, and TAC values of 108.42 mg/g, 68.55 mg/g, and 19.83 mg/g fresh fruit weight, respectively. These results suggested that methanol as the extraction solvent obtained higher total flavonoid and total phenol values.

Fig 1

Quantification of TPC (A), TFC (B), and TAC (C) in Berberis heteropoda shrub extract.

Comparison of the extraction effect of methanol, acetone, and ethanol. **P < 0.05.

Antioxidant activity of B. heteropoda fruit extract

The antioxidant activity of the B. heteropoda fruit extracts was evaluated using VC as the control, with the IC50 values for the DPPH free radical-, ABTS radical-, •OH-, O2•-scavenging abilities at 20.27±0.26 μg/mL, 13.89±0.13 mg/mL, 5.81±0.13 mg/mL, and 0.57±0.02 mg/mL, respectively (Fig 2 and S2 File). We observed that methanol extract had the best antioxidant activity, with IC50 values for DPPH radical-, hydroxyl radical-, ABTS radical-, and superoxide anion radical-scavenging abilities at 20.13 μg/mL, 5.44 mg/mL, 8.79 μg/mL, and 1.35 mg/ mL, respectively. The IC50 values for methanol extraction were higher than those of VC but lower than those of ethanol and acetone extraction. The ranking from high to low according to free radical-scavenging ability was methanol > acetone > ethanol and suggested that B. heteropoda fruit extracts showed good antioxidant activity based on effective free radical scavenging.

Fig 2

IC50 values (mg/mL) of different extracts on free radicals.

Comparison of IC50 values using methanol, ethanol, acetone, and Vc for extraction and scavenging of (A) DPPH radical, (B) ABTS radical, (C) hydroxyl radical, and (D) superoxide anion radical. **P < 0.05.

Identification of phenols in B. heteropoda fruit extract using chromatography and MS

The UPLC-quadrupole time-of-flight (Q-TOF)-MS spectra indicated that the compounds in the extract of B. heteropoda fruit were primarily identified within 2 min to 10 min and when the mobile phase was at 15% to 70% ethyl alcohol solution, indicating that the polyphenols of B. heteropoda fruit belonged to polar compounds (Table 4, S3 File and S1 Fig) [10, 12, 20–38].

Table 4

Characterization of phenolic compounds of Berberis heteropoda fruit by UPLC-Q-TOF-MSE.

Compound	tR/min	Ionization mode	Identification	Molecular formula	MS(m/z)	MS2(m/z)
1	1.52	[M-H]−	Corilagin	C27H22O18	633.0787	261.667; 181.051
2	2.96	[M-H]−	Petunidin-3-O-beta-glucopyranoside	C22H23O12	477.1030	299.013; 314.043
3	3.67	[M-H]−	Cianidanol	C15H14O6	289.0719	245.0827
4	4.53	[M-H]−	Gossypetin-8-C-glucoside	C21H20O13	479.0835	316.0244; 271.216
5	5.03	[M-H]−	Syringetin-3-O-glucoside	C23H24O13	507.1144	301.067; 345.0604
6	5.50	[M-H]−	Myricetin-3-O-galactoside	C21H20O13	479.0834	115.0551; 133.014
7	5.80	[M-H]−	kaempferol 7-O-glucoside	C21H20O11	447.0926	285.0365
8	5.84	[M-H]−	Syringetin-3-O-galactoside	C23H24O13	507.1143	344.053; 273.032
9	5.85	[M-H]−	Flavanomarein	C21H22O11	449.1095	287.0572; 150.0037
10	5.89	[M-H]−	Luteolin	C15H10O6	285.0393	151.0022; 133.0302
11	5.91	[M-H]−	Spiraeoside	C21H20O12	463.089	301.034; 179.0188
12	6.02	[M-H]−	Myricetin	C15H10O8	317.0300	137.0248; 151.00568
13	6.14	[M-H]−	Luteolin-4’-O-glucoside	C21H20O11	447.0927	285.0388
14	6.20	[M-H]−	Dihydromyricetin	C15H12O8	319.0458	150.999; 107.0111
15	6.73	[M-H]−	Morin	C15H10O7	301.0356	165.02
16	6.97	[M-H]−	Quercetin	C15H10O7	301.0357	121.0272; 151.0038; 178.9974
17	7.97	[M-H]−	Kaempferol	C15H10O6	285.0408
18	8.04	[M-H]−	Kaempferide	C16H12O6	299.0556	284.0329; 256.036
19	8.19	[M-H]−	Isorhamnetin	C16H12O7	315.0507	300.029
20	9.56	[M-H]−	Galangin	C15H10O5	269.0455	225.0558
21	4.52	[M+H]+	Genistein	C15H10O5	271.0588	121.028
22	4.75	[M+H]+	Flavokawain B	C17H16O4	285.1122	249.1829; 267.141
23	4.85	[M+H]+	Epicatechin	C15H14O6	291.0858	123.0446; 139.039
24	5.05	[M+H]+	Herbacetin	C15H10O7	303.0478	257.042
25	5.08	[M+H]+	Dihydro-Quer	C15H12O7	305.0650	289.631; 290.365
26	5.55	[M+H]+	Flavonol base + 4O, 1MeO	C16H12O8	333.0602	58.065; 318.036
27	7.25	[M+H]+	Naringenin-7-O-glucoside	C21H22O10	435.1279	153.0385; 273.0744
28	7.29	[M+H]+	Phlorizin	C21H24O10	437.1445	107.045; 275.0905
29	7.75	[M+H]+	Hyperoside	C21H20O12	465.1028	61.0285; 85.0285
30	8.22	[M+H]+	Aurantio-obtusin beta-D-glucoside	C23H24O12	493.1329	331.0826
31	9.92	[M+H]+	Kaempferol 3-glucorhamnoside	C27H30O15	595.1650	85.0305; 287.0686
32	9.92	[M+H]+	Vicenin 2	C27H30O15	595.1658	325.071; 317.0645

Discussion

A previous study on the anthocyanin composition of B. heteropoda fruit considered it as a potential anthocyanin pigment source [3] and focused on chemical characterization of B. heteropoda fruit; however, there has been a comprehensive investigation of the overall nutritional composition of the fruit. The present study systematically evaluated the major nutrients and antioxidant properties of B. heteropoda fruits and found them to be rich in various nutrients, thereby providing evidence for their potential health-related or nutritional use. Moreover, we identified a total of 32 polyphenols in B. heteropoda fruit extract.

The results revealed that B. heteropoda fruit exhibits nutritional properties suggesting potential nutraceutical value. The major nutrients of B. heteropoda fruit were comparable to those of wolfberry (Lycium ruthenicum Murr.), which is a wild plant and widely observed in Xinjiang [39]. Additionally, the protein content of B. heteropoda fruit was higher than that in black mulberry (1.17±0.06%) [19], and the contents of fat and sugar in B. heteropoda fruit were low, suggesting a low risk for causing obesity and consideration for use as a functional food or medicine rather than an edible fresh fruit due to its poor taste.

We found that B. heteropoda fruit contains numerous minerals, including Na, K, Ca, Cu, Zn, Fe, Mg, and P. Previous studies demonstrate that these minerals play important roles in the physiological function of human tissues, maintaining cellular osmotic pressure, supporting the pH balance of the body, and regulating specific physiological functions as cofactors [40, 41]. Additionally, we observed that the Na:K ratio in B. heteropoda fruit was 0.002, which could promote the prevention of hypertension [42]. These findings suggest that B. heteropoda fruit might be considered helpful for controlling blood pressure.

The EAA:NEAA ratio was 0.47, which does not meet the ideal protein condition proposed by the Food and Agriculture Organization of the United Nations and the World Health Organization [43]. Therefore, this fruit is not recommended as a high-quality protein food. The percentages of glutamic acid, glycine, and aspartic acid were 18.72%, 7.42%, and 9.54%, respectively, and accounting for >33% of the total amino acids in B. heteropoda fruit. Moreover, the UFA:SFA ratio in B. heteropoda fruit was 1.06, suggesting that it should not be recommended as a food rich in fatty acids.

Phenolic and flavonoid compounds are as important phytonutrients in plants [44, 45]. Flavonoids are secondary metabolites and abundant [46], and phenols are important plant compounds that mimic the biological effects of vitamin E [47]. A previous study reported that anthocyanins are rich in many plants and responsible for red, yellow, purple, black, and other colorful pigments [48]. The basic structural unit of anthocyanins is 2-phenylbenzopyran, which comprises a C6–C3–C6 backbone [49]. Its unique structure enables it to exert anti-oxidation, anti-inflammation, and antitumor functions [50], as well as those related to the prevention of cardiovascular disease and enhancement of vision [51]. In the present study, we found that the anthocyanin content of B. heteropoda fruit was higher than that of wolfberry [39] but lower than that of Passiflora foetida [10]. Moreover, the TPC of B. heteropoda fruit was lower than barberry (Berberis vulgaris L.), and calafate (Berberis microphylla) fruits and other native berries, suggesting that the antioxidant activity of B. heteropoda is likely lower than that of several other Berberis fruits [52, 53]. A possible reason for this could be that the TPC is significantly related to geographical, climate, and soil conditions. Furthermore, we found the anthocyanin content in B. heteropoda fruit was inconsistent with that of a previous study [3], which reported a TAC of 20.37 mg/g fresh weight of B. heteropoda. A possible explanation could be that the samples in the present study were obtained from the Nanshan Mountain area of Urumqi City (latitude 89°29′36″E, longitude 43°27′32″N), whereas those in the previous study were from Daxigou (latitude 44°26′ N, longitude 80°46′ E).

A previous study reported the free radical-scavenging activity of flavonoids and polyphenols from Stachys affinis [54]. The IC50 value is typically used to evaluate antioxidant activity, with smaller IC50 values indicating stronger antioxidant capacity. The present results indicated that B. heteropoda fruit extract showed strong scavenging effects on DPPH•, •OH, O2−•, and ABTS+•, suggesting that B. heteropoda fruit could be considered an excellent source of natural antioxidants.

This study has some limitations. First, we did not address the functional monomers of B. heteropoda fruits, nor did we assess the structures of specific phenolic compounds and their antioxidant effects. Furthermore, the mechanism of action for the phenolic compounds needs further evaluation, and the potential effect of B. heteropoda fruits on general human health needs further assessment.

Conclusions

This study analyzed the major nutrients, mineral elements, fatty acids, and amino acids in B. heteropoda fruits and identified a wide array of important nutrient components. We found that B. heteropoda fruits had high TPC, TFC, and TAC values, as well as potentially excellent antioxidant properties. These findings suggest that B. heteropoda fruit could potentially be used as a health-promoting food for resisting oxidative damage; however, further studies are necessary to assess the biological activities of B. heteropoda fruit.

Representative total ion chromatogram of an extract sample obtained from Berberis heteropoda fruit in positive ion mode (A) and negative ion mode (B) by UPLC-Q-TOF-MSE.

(TIF)

Click here for additional data file.

Nutritional composition of B. heteropoda fruit.

(XLSX)

Click here for additional data file.

Antioxidant Activity of B. heteropoda fruit extract.

(XLSX)

Click here for additional data file.

Identification of Phenols in B. heteropoda fruit extract using chromatography and MS.

(XLSX)

Click here for additional data file.

11 Nov 2021

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**********

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Reviewer #1: Manuscript No. PONE-D-21-29715

Manuscript Title: Nutritional Composition and Antioxidant Properties of the Fruit of Berberis heteropoda

Schrenk

Comments: The study discussed in this manuscript explains the major nutrients and antioxidant properties of Berberis heteropoda Schrenk fruits. Berberis plants are reported to possess antimicrobial, antiemetic, antipyretic, antioxidant, anti-inflammatory, anti-arrhythmic, sedative, anti-cholinergic, cholagogic, anti-leishmaniasis, and anti-malaria, and thus are important plants to provide bioactive secondary metabolites.

The present findings revealed that plant material under study is rich in phenolics which are considered as strong antioxidant, and can be part of nutraceuticals or functional foods.

In ABSTRACT, RESULTS: ……. in Berberis heteropoda Schrenk fruits were 75.22, 0.506, 2.55, 1.31, and 17.72 g100 g fresh fruit, probably it is 16.72g/100 g…?????

Similarly, …. The total phenol, flavonoid, and anthocyanin 34

contents of Berberis heteropoda Schrenk fruits were 68.55 mg gallic acid equivalentsg, 108.42 mg quercetin equivalentsg, and 19.83 mg cyanidin-3-glucoside equivalentg, should it be equivalent/g ?????

…The UPLC-Q-TOF-MSE analysis of phenols revealed 32 compounds……, what is mean by analysis of phenols???

Introduction: …..shrub of the family Berberidaceae and is distributed in… it should be …which is distributed in Altai, ……

Don’t use full name of the plant every time, for example …..and fruits of Berberis heteropoda Schrenk…. Can be written as and fruits of B. heteropoda…….

In introduction, language composition is not good, some parts are deleted, some are under lined please address this issue carefully. Several notes are not clear due to poor language

Introduction Line 66 states about ..recent study…, the reference quoted at 67 is of 2014, so it is not recent….

Provide voucher specimen No.????

Extraction: Line 125….. The samples were extracted using a modification method…extracted using reported method with modification….

Rewrite extraction part: it is again language problem

Determination of TPC: Line 136….Pure water means?? Distilled or deionized???

Nutritional Composition of the Berberis heteropoda Schrenk Fruit: Lines 237 and 238; Water content was the highest (75.22±1.75 g/100 g),…… Remove the word highest, just provide value.

Table 1: column composition per unit, ….remove repeated word “fresh fruit” and add in table caption.

Line 290 and onward… The details of 32 kinds of compounds are listed as follows….. Since all these compounds are listed in Table 4, it is just repetition in the text, so remove it, and better to add comments on percentage of components and their possible role in bioactivities.

Chromatograms and mass spectra of 32 compounds should be provided as supplementary material.

There are serious grammatical errors, language should be improved, repetition should be avoided

Reviewer #2: while going through the manuscript, the manuscript did not have much interesting finding and lacking novelty as far as my knowledge is concerned. The manuscript should include some biological activities like antimicrobial activity, anticancer etc.

Reviewer #3: PONE-D-21-29715. Nutritional Composition and Antioxidant Properties of the Fruit of Berberis heteropoda Schrenk

The subject of this manuscript falls within the general scope of the journal, and the study of Nutritional Composition and Antioxidant Properties of the Fruit of Berberis heteropoda Schrenk is relevant.

Keywords: the words Berberis heteropoda Schrenk; nutritional composition and antioxidant properties are in the title too. These keywords must be replaced by other ones.

Abstract: the precise units for the total content of phenol, flavonoid, and anthocyanin are not found this section, i.e. “…68.55 mg gallic acid equivalentsg…”. g of fresh fruit weight or dry fruit weight?

Introduction: this section contains the information that justifies this work. The antecedents on B. heteropoda Schrenk fruits are described, although briefly. The objective of the manuscript is correctly stated.

Line 47: “…The roots, bark, stems, and fruits of Berberis heteropoda Schrenk…”. Delete the term Schrenk in this sentence and in the following ones.

Materials and Methods: even though this section includes details about the methods employed, several topics remain unclear.

Line 74: “…Mature Berberis heteropoda Schrenk fruits were collected…”. The correct term is ripe rather than mature.

Line 76: “…Fig. 1 shows its distribution in Xinjiang…”. Show the site where the fruits were collected. Site 1, 2 or 3 or all? This Figure must show the geographical position.

Line 79: “…High-quality Berberis heteropoda Schrenk fruit were selected…” The term “High quality” must be more precise. Nothing is said about the sample size and the number of shrubs selected for the fruit harvest.

Line 125-132. The different solvents used for the fruit extraction is not described in this section, i.e. methanol, acetone and ethanol. It is necessary to add this information here.

Line 140: “…TPC was denoted as milligram of gallic acid equivalent per gram of plant mass…”. Plant mass must be replaced by fruit mass. Fresh or dry fruit weight?

Line 149-150: The same comment for Line 140.

Results: this section is presented in 2 Figures and 3 Tables. Legends of Figures 2 and 3 do not content sufficient information about the statistical analysis of the results, i.e. the meaning of the asterisks above the bars.

Line 266-267: “…The regression equations used were y = 0.0109x + 0.0157 and y = 0.067x - 0.0173…”. Rewrite as “…The regression equations used were y = 0.0109x + 0.0157 and y = 0.067x - 0.0173, respectively…”.

Line 270: “…the TFC, TPC, and TAC values were 108.42, 68.55, and 19.83 mg/g fruit, 271 respectively…” Please add the term of expression referred, i.e. mg/g fresh fruit weight.

Discussion:

Lines 317-318: this sentence is more suitable for the Introduction section.

Lines 327-328: the term “this study” means the study of reference 3 or the study of this manuscript? Please, be clearer.

Lines 362-364: “…The anthocyanin content of Berberis heteropoda Schrenk fruit found in this study was slightly inconsistent with a prior study, possibly because the previous samples were obtained from Yili, Xinjiang [3]…”. The meaning of this sentence must be clearer.

As a general comment of the Discussion section, the authors do not discuss the obtained results with other Berberis fruits species, in particular with respect to TPC, TFC and TAC.

Conclusions: the conclusions are well stated.

References: the inclusion of references of other Berberis fruit species is suggested to be discussed.

Final comment: this manuscript needs to incorporate the corrections suggested before its publication in PLOS ONE.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Submitted filename: PONE-D-21-29715_reviewer.pdf

Click here for additional data file.

6 Dec 2021

Point-By-Point Response

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Response: Thanks for this suggestion, and the Figure 1 have already removed in the revised manuscript.

Response to reviewer #1

General comments: The study discussed in this manuscript explains the major nutrients and antioxidant properties of Berberis heteropoda Schrenk fruits. Berberis plants are reported to possess antimicrobial, antiemetic, antipyretic, antioxidant, anti-inflammatory, anti-arrhythmic, sedative, anti-cholinergic, cholagogic, anti-leishmaniasis, and anti-malaria, and thus are important plants to provide bioactive secondary metabolites.

The present findings revealed that plant material under study is rich in phenolics which are considered as strong antioxidant, and can be part of nutraceuticals or functional foods.

Response: As behalf of all co-authors, I would like to appreciate this referee due to thoughtful comments proposed by the peer review. After we revised the manuscript, those significant issues could be changed.

Question 1: In ABSTRACT, RESULTS: ……. in Berberis heteropoda Schrenk fruits were 75.22, 0.506, 2.55, 1.31, and 17.72 g100 g fresh fruit, probably it is 16.72g/100 g…?????Similarly, …. The total phenol, flavonoid, and anthocyanin 34 contents of Berberis heteropoda Schrenk fruits were 68.55 mg gallic acid equivalentsg, 108.42 mg quercetin equivalentsg, and 19.83 mg cyanidin-3-glucoside equivalentg, should it be equivalent/g ?????

Response: Thanks for this suggestion, and these sentences have already changed in the revised manuscript.

Question 2: The UPLC-Q-TOF-MSE analysis of phenols revealed 32 compounds……, what is mean by analysis of phenols???

Response: Thanks for this suggestion, and this sentence have already changed into: “The UPLC-Q-TOF-MSE analysis of polyphenols of Berberis heteropoda Schrenk fruit revealed 32 compounds”

Question 3: …Introduction: …..shrub of the family Berberidaceae and is distributed in… it should be …which is distributed in Altai, ……

Response: Thanks for this suggestion, and the sentence have already changed in the revised manuscript.

Question 4: Don’t use full name of the plant every time, for example …..and fruits of Berberis heteropoda Schrenk…. Can be written as and fruits of B. heteropoda…….

Response: Thanks for this suggestion, and the name of the plant have already changed in the revised manuscript.

Question 5: In introduction, language composition is not good, some parts are deleted, some are under lined please address this issue carefully. Several notes are not clear due to poor language

Response: Thanks for this suggestion, and the English revision have already performed in the revised manuscript by Editage Company.

Question 6: Introduction Line 66 states about ..recent study…, the reference quoted at 67 is of 2014, so it is not recent….

Response: Thanks for this suggestion, and this sentence have already changed in the revised manuscript.

Question 7: Provide voucher specimen No.????

Response: Thanks for this suggestion, and voucher specimen No have already added in the revised manuscript.

Question 8: Extraction: Line 125….. The samples were extracted using a modification method…extracted using reported method with modification….Rewrite extraction part: it is again language problem

Response: Thanks for this suggestion, and this sentence have already changed in the revised manuscript. Moreover, the language of whole manuscript have already revised by Editage Company.

Question 9: Determination of TPC: Line 136….Pure water means?? Distilled or deionized???

Response: Thanks for this suggestion, and pure water have already changed into distilled water.

Question 10: Nutritional Composition of the Berberis heteropoda Schrenk Fruit: Lines 237 and 238; Water content was the highest (75.22±1.75 g/100 g),…… Remove the word highest, just provide value.

Response: Thanks for this suggestion, and this sentence have already changed into: “Water content was 75.22±1.75 g/100 g, crude fiber was 17.72±0.52 g/100 g and total protein was 2.55±0.03 g/100 g.”

Question 11: Table 1: column composition per unit, ….remove repeated word “fresh fruit” and add in table caption.

Response: Thanks for this suggestion, and this change have already performed in the revised Table 1.

Question 12: Line 290 and onward… The details of 32 kinds of compounds are listed as follows….. Since all these compounds are listed in Table 4, it is just repetition in the text, so remove it, and better to add comments on percentage of components and their possible role in bioactivities.

Response: Thanks for this suggestion, and these sentences have already removed in the revised manuscript.

Question 13: Chromatograms and mass spectra of 32 compounds should be provided as supplementary material.

Response: Thanks for this suggestion, and the Chromatograms and mass spectra of 32 compounds have already removed in Figure S1.

Question 14: There are serious grammatical errors, language should be improved, repetition should be avoided

Response: Thanks for this suggestion, and English revision have already performed by Editage Company.

Response to reviewer #2

General comments: while going through the manuscript, the manuscript did not have much interesting finding and lacking novelty as far as my knowledge is concerned. The manuscript should include some biological activities like antimicrobial activity, anticancer etc.

Response: Thanks for this suggestion. The current study aimed to assess the major nutrients and antioxidant properties of B. heteropoda fruits, while the biological activities were not illustrated. We have already addressed this comment in Discussion section.

Response to reviewer #3

General comments: The subject of this manuscript falls within the general scope of the journal, and the study of Nutritional Composition and Antioxidant Properties of the Fruit of Berberis heteropoda Schrenk is relevant.

Response: As behalf of all co-authors, I would like to appreciate this referee due to thoughtful comments proposed by the peer review. After we revised the manuscript, those significant issues could be changed.

Question 1: Keywords: the words Berberis heteropoda Schrenk; nutritional composition and antioxidant properties are in the title too. These keywords must be replaced by other ones.

Response: Thanks for this suggestion, and the keywords have already changed into: “Amino acids; minerals; fatty acids; total phenol; flavonoid; anthocyanin”.

Question 2: Abstract: the precise units for the total content of phenol, flavonoid, and anthocyanin are not found this section, i.e. “…68.55 mg gallic acid equivalentsg…”. g of fresh fruit weight or dry fruit weight?

Response: Thanks for this suggestion, and the results in Abstract section have already changed into: “The analytical results showed that average water, total fat, total protein, ash, and carbohydrates contents in B. heteropoda fruits were 75.22 g/100 g, 0.506 g/100 g, 2.55 g/100 g, 1.31 g/100 g, and 17.72 g/100 g fresh fruit, respectively. The total phenol, flavonoid, and anthocyanin contents of B. heteropoda fruits were 68.55 mg gallic acid equivalents/g, 108.42 mg quercetin equivalents/g, and 19.83 mg cyanidin-3-glucoside equivalent/g, respectively. The UPLC-Q-TOF-MSE analysis of polyphenols of B. heteropoda fruit revealed 32 compounds. ”

Question 3: Introduction: this section contains the information that justifies this work. The antecedents on B. heteropoda Schrenk fruits are described, although briefly. The objective of the manuscript is correctly stated.

Response: We appreciate the reviewer given this kindly comment.

Question 4: Line 47: “…The roots, bark, stems, and fruits of Berberis heteropoda Schrenk…”. Delete the term Schrenk in this sentence and in the following ones.

Response: Thanks for this suggestion, and these changes have already performed in the revised manuscript.

Question 5: Materials and Methods: even though this section includes details about the methods employed, several topics remain unclear.

Response: As behalf of all co-authors, I would like to appreciate this referee due to thoughtful comments proposed by the peer review. After we revised the manuscript, those significant issues could be changed.

Question 6: Line 74: “…Mature Berberis heteropoda Schrenk fruits were collected…”. The correct term is ripe rather than mature.

Response: Thanks for this suggestion, and this term have already changed into: “ripe”.

Question 7: Line 76: “…Fig. 1 shows its distribution in Xinjiang…”. Show the site where the fruits were collected. Site 1, 2 or 3 or all? This Figure must show the geographical position.

Response: Thanks for this suggestion, and the Figure 1 have already removed in the revised manuscript, and the details of geographical position have already described.

Question 8: Line 79: “…High-quality Berberis heteropoda Schrenk fruit were selected…” The term “High quality” must be more precise. Nothing is said about the sample size and the number of shrubs selected for the fruit harvest.

Response: Thanks for this suggestion, and the following sentences have already added in the revised manuscript: “B. heteropoda fruit were selected after combed, and all of fruit were evenly and full, without rotten and miscellaneous, then the stem and seeds were removed. A total of 3 kg B. heteropoda fruit were collected from dozens of shrubs in a ravine.”

Question 9: Line 125-132. The different solvents used for the fruit extraction is not described in this section, i.e. methanol, acetone and ethanol. It is necessary to add this information here.

Response: Thanks for this suggestion, and this information have already added in the revised manuscript.

Question 10: Line 140: “…TPC was denoted as milligram of gallic acid equivalent per gram of plant mass…”. Plant mass must be replaced by fruit mass. Fresh or dry fruit weight?

Response: Thanks for this suggestion. We have already made this change in the revised manuscript.

Question 11: Line 149-150: The same comment for Line 140.

Response: Thanks for this suggestion. We have already made this change in the revised manuscript.

Question 12: Results: this section is presented in 2 Figures and 3 Tables. Legends of Figures 2 and 3 do not content sufficient information about the statistical analysis of the results, i.e. the meaning of the asterisks above the bars.

Response: Thanks for this suggestion, and the meaning of the asterisks have already added in Figures legends.

Question 13: Line 266-267: “…The regression equations used were y = 0.0109x + 0.0157 and y = 0.067x - 0.0173…”. Rewrite as “…The regression equations used were y = 0.0109x + 0.0157 and y = 0.067x - 0.0173, respectively…”.

Response: Thanks for this suggestion, and this changes have already performed in the revised manuscript.

Question 14: Line 270: “…the TFC, TPC, and TAC values were 108.42, 68.55, and 19.83 mg/g fruit, 271 respectively…” Please add the term of expression referred, i.e. mg/g fresh fruit weight.

Response: Thanks for this suggestion, and this change have already performed in the revised manuscript.

Question 15: Discussion:

Lines 317-318: this sentence is more suitable for the Introduction section.

Response: Thanks for this suggestion, and this sentence have already removed in the revised manuscript.

Question 16: Lines 327-328: the term “this study” means the study of reference 3 or the study of this manuscript? Please, be clearer.

Response: Thanks for this suggestion, and “this study” have already changed into: “our study”.

Question 17: Lines 362-364: “…The anthocyanin content of Berberis heteropoda Schrenk fruit found in this study was slightly inconsistent with a prior study, possibly because the previous samples were obtained from Yili, Xinjiang [3]…”. The meaning of this sentence must be clearer.

Response: Thanks for this suggestion, and this sentence have already revised in the revised manuscript.

Question 18: As a general comment of the Discussion section, the authors do not discuss the obtained results with other Berberis fruits species, in particular with respect to TPC, TFC and TAC.

Response: Thanks for this suggestion, and several sentences have already added in the revised manuscript.

Question 19: Conclusions: the conclusions are well stated.

Response: We appreciate the reviewer given this kindly comments.

Question 20: References: the inclusion of references of other Berberis fruit species is suggested to be discussed.

Response: Thanks for this suggestion, and the inclusion of references have already discussed in the revised manuscript.

Question 21: Final comment: this manuscript needs to incorporate the corrections suggested before its publication in PLOS ONE.

Response: We appreciate the reviewer given this kindly comments, and all of changes have already performed in the revised manuscript with tracked.

Submitted filename: Response to reviewer.docx

Click here for additional data file.

15 Dec 2021

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We look forward to receiving your revised manuscript.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The authors need to thoroughly reorganize the Discussion section, since some Reviewers' concerns were not properly addressed. Up-to-date literature that deals with the same topic, basically with bioactivity of other Berberis taxa must be consulted. Moreover, the authors probably missed to see the Academic Editor's comments in the previous round, since they were left unaddressed as well. I will repeat them:

"The authors have presented the whole manuscript in a rather indistinct fashion, gaps in the content are visible and the whole text lacks flow. Moreover, there are numerous places in the M&M section as well as in Results containing inconsistent methodology and vaguely presented results and conclusions. The manuscript would hugely benefit if being proofread by a senior researcher of the similar expertise, which would steer the authors how to properly present their experiments. It is also highly recommended to have the manuscript copyedited by a native English speaker or a professional editing agency towards gaining clarity and better readability."

Therefore, language polishing is much needed throughout the text. In L27 please do not introduce the abbreviated plant species name in parenthesis. It is a common scientific practice to abbreviate the genus name once being introduced in full.

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

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: All comments have been addressed

Reviewer #3: (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: Partly

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #3: 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: No

Reviewer #3: Yes

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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: nothing special, ,most of the corrections are addressed but language still needs serious revision. In my first review, I strongly suggested to edit language by a native English speaker.

Reviewer #3: PONE-D-21-29715R1. Nutritional Composition and Antioxidant Properties of the Fruit of Berberis heteropoda Schrenk

The authors have incorporated most of the suggestions made by the reviewers. However the following items must be improved:

Discussion:

Lines 344-346: “…Our study found the anthocyanin content in B. heteropoda fruit was inconsistent with a prior study [3], which indicated the TAC was 20.37 mg/g fresh weight of B. heteropoda...".Are the differences between 19.83 and 20.37 mg cyanidin-3-glucoside equivalent/g fresh weight significant?

As a general comment of the Discussion section, the authors do not discuss the obtained results with other Berberis fruits species, in particular with respect to TPC, TFC and TAC. New bibliography on other Berberis species were not included in the revised manuscript.

Final comment: this manuscript needs to incorporate the corrections suggested before its publication in PLOS ONE.

**********

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Reviewer #1: No

Reviewer #3: No

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28 Dec 2021

Point-By-Point Response

Response to editor

General comments: The authors need to thoroughly reorganize the Discussion section, since some Reviewers' concerns were not properly addressed. Up-to-date literature that deals with the same topic, basically with bioactivity of other Berberis taxa must be consulted. Moreover, the authors probably missed to see the Academic Editor's comments in the previous round, since they were left unaddressed as well. I will repeat them:

"The authors have presented the whole manuscript in a rather indistinct fashion, gaps in the content are visible and the whole text lacks flow. Moreover, there are numerous places in the M&M section as well as in Results containing inconsistent methodology and vaguely presented results and conclusions. The manuscript would hugely benefit if being proofread by a senior researcher of the similar expertise, which would steer the authors how to properly present their experiments. It is also highly recommended to have the manuscript copyedited by a native English speaker or a professional editing agency towards gaining clarity and better readability."

Therefore, language polishing is much needed throughout the text. In L27 please do not introduce the abbreviated plant species name in parenthesis. It is a common scientific practice to abbreviate the genus name once being introduced in full.

Response: As behalf of all co-authors, I would like to appreciate this referee due to thoughtful comments proposed by the peer review. After we revised the manuscript, those significant issues could be changed.

As compared with other Berberis taxa have already added in Discussion. Moreover, the methods and results section were adjusted to address your comment. Furthermore, the English revision in the revised manuscript have already performed by Editage Company. Finally, the abbreviated plant species name in parenthesis have already removed in the revised manuscript.

Response to reviewer #1

General comments: nothing special, ,most of the corrections are addressed but language still needs serious revision. In my first review, I strongly suggested to edit language by a native English speaker.

Response: Thanks for this suggestion, and the English revision is provided by the Editage Company.

Response to reviewer #3

General comments: The authors have incorporated most of the suggestions made by the reviewers. However the following items must be improved:

Response: As behalf of all co-authors, I would like to appreciate this referee due to thoughtful comments proposed by the peer review. After we revised the manuscript, those significant issues could be changed.

Question 1: Lines 344-346: “…Our study found the anthocyanin content in B. heteropoda fruit was inconsistent with a prior study [3], which indicated the TAC was 20.37 mg/g fresh weight of B. heteropoda...”. Are the differences between 19.83 and 20.37 mg cyanidin-3-glucoside equivalent/g fresh weight significant?

Response: Thanks for this suggestion. According to the standard deviation of TAC in our study and prior study, we noted the difference was associated with statistically significant.

Question 2: As a general comment of the Discussion section, the authors do not discuss the obtained results with other Berberis fruits species, in particular with respect to TPC, TFC and TAC. New bibliography on other Berberis species were not included in the revised manuscript.

Response: Thanks for this suggestion, and several sentences have already added in Discussion section to compare the obtained results with other Berberis fruits.

Question 3: Final comment: this manuscript needs to incorporate the corrections suggested before its publication in PLOS ONE.

Response: Thanks for this suggestion, and all of changes have already performed to address your comments.

Submitted filename: Response to reviewer.docx

Click here for additional data file.

31 Dec 2021

Nutritional Composition and Antioxidant Properties of the  Fruit of Berberis heteropoda Schrenk

PONE-D-21-29715R2

Dear Dr. Li,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

29 Mar 2022

PONE-D-21-29715R2

Nutritional Composition and Antioxidant Properties of the Fruit of Berberis heteropoda Schrenk

Dear Dr. Li:

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.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Branislav T. Šiler

Academic Editor

PLOS ONE