1. Introduction
Plant seeds are a source of food, food ingredients, and supplements for human dietary needs in daily life [
1]. Most of the seeds produce proteins, carbohydrates, numerous secondary metabolites, and can be used for edible oils [
2,
3]. Edible oils, such as olive oil, corn oil, and soybean oil are used for human diets and contain lipids in the form of fatty acids (FAs). Among FAs, α-linolenic acid (ALA), a kind of unsaturated fatty acid (UFAs), is an essential human dietary nutrient with health promoting effects that cannot be synthesized independently within the body [
4]. Seeds from various plant species were detected with high levels of ALA, including
Ocimum basilicum (50.10%–67.61%) [
5,
6],
Lasiococca comberi (65.30%) [
7],
Linum usitatissimum (46.33%–55.47%) [
8],
Eucommia ulmoides (56.51%) [
9], and
Paeonia suffruticosa (21%–54%) [
10]. ALA is the most important n-3 fatty acid from vegetables from a dietary perspective. However, in traditional vegetable oils, such as soybean (8.95%), canola (9.74%), corn germ (0.55%), sesame (0.32%), and olive (0.57%), n-3 fatty acids represent less than 10%, and hence do not meet human dietary needs [
11]. Therefore, with improvements in living standards and the pursuit of healthy diets, the development of special edible oils rich in ALA from oil seed plants has become a worldwide demand.
Paeonia ostii (Paeoniaceae,
Paeonia, Sect.
Mutan DC) is an emerging woody oil crop that originates in China, and is one of the most important plants, traditionally grown for the root bark that is used as an antispasmodic medicine throughout Asia [
12]. It has been listed among the new promising woody crops for oil production in temperate regions due to its wide adaptability, low input requirement, and other advantages [
13]. The legume of
P. ostii has a star-shaped fruit, which contains dark oval seeds. In the industry, shelled seeds are used for oil production through cold pressed or subcritical extraction. Its seeds are an important source of edible oil for humans and possess economic potential due to their high amount of oil content (20%) and high yield (3.75 t/ha) [
14]. However, it was long neglected for food industrial purposes until its seed oil was authenticated as a new food resource by the Ministry of Health of China in 2011 [
15]. Its seed oil has been considered as a valuable vegetable oil and is enriched in UFAs (>90%) and ALA (40%) [
10]. Currently, it is regarded as a rare source of ALA supplementation, particularly in comparison to commonly consumed vegetable oil, and has been widely planted in large numbers as a woody oil crop throughout China. However, in comparison to industrial needs, seeds used as raw material for edible oil must meet a high-quality standard. Therefore, it is necessary to conduct a comprehensive quality evaluation of seeds from different cultivated areas of China.
Chemical composition and nutritional evaluation of seeds and seed oils has been an important research field in the past few years, which has been focused on FA composition, antioxidant activities, protein, antinutrient, squalene, phytosterols, moisture, tocotrienol, and mineral contents in
Dalbergia odorifera [
16],
Acacia saligna [
17],
Salvia hispanica [
18],
Camellia oleifera [
19], and
Phoenix dactylifera L. [
20]. Previous studies have focused on systematic assessments of fatty acids of seeds from 60
Paeonia cultivars, which suggest that these detected cultivars could be good candidates as an oil resource for practical oil production [
10]. More recently, attention has been focused on the analysis of FA composition in
Paeonia plants [
10,
21], but few studies have assessed the chemical characterization and bioactive compounds.
In this study, the main purpose is to evaluate the proximate composition, main nutritional component, secondary metabolites, and antioxidant activity of P. ostii seed kernels (PSK) and seed coat (PSC) from 11 main production areas. This study can provide important standards for evaluating the seed quality of P. ostii to be used as a valuable source of vegetable oil, and can potentially identify interesting components for the processing and development of functional foods with high contents of ALA, crude protein, or tocopherol.
2. Materials and Methods
2.1. Collection of Samples
Mature seeds of
P. ostii were collected in 2017 from eleven different production areas (
Figure 1 and
Table S1). The PSK and PSC from each location were separately grounded using a high-speed multi-purpose disintegrator (Zhejiang, China) before using them in further analyses.
2.2. Chemicals and Reagents
Fatty acid methyl esters (FAMEs) mixtures with 37-components and six FAMEs standard mixtures including palmitic acid (C16: 0, PA), methyl heptadecanoate (C17: 0), methyl stearate (C18: 0, stearic acid (SA)), methyl oleate (C18: 1∆9c, oleic acid (OA)), methyl linoleate (C18: 2∆9c, 12c, linolenic acid (LA)), ALA (C18: 3∆9c, 12c, 15c), and α-, β-, γ- and δ tocopherols standard were purchased from Sigma-Aldrich (St. Louis, MO, USA). Among them, methyl heptadecanoate was used as an internal standard (IS). Standard compounds of gallic acid and paeoniflorin were purchased from ANPEL Laboratory Technologies Inc. (Shanghai, China). All standards and stock solutions were kept in dark at 4 °C, while ALA was kept at −20 °C.
2.3. Seed Proximate Composition Analysis
The 100-seed weight, moisture content, and neatness were investigated according to Chinese standards of GB/T 3543.7-1995, GB/T 3543.6-1995, and GB/T 3543.3-1995. Crude protein content was determined by the micro-Kjeldhal method [
22]. The seed coat rate was calculated by the following equation:
2.4. Fatty Acid Composition and Content Analysis
The seed lipids were extracted, and FAMEs analyses were prepared according to the procedures described previously [
23]. Fatty acid analysis was performed using a gas chromatograph-mass spectrometer (GC-MS, GC6890N/MS5973, Aglilent Technoloties, Willmington, DE, USA) equipped with a flame-ionization detector (FID) and a capillary column (HP-88; 30 m × 0.25 mm, film thickness: 0.20 μm). The column temperature was maintained at 100 °C for 2 min, then elevated to 230 °C for 5 min at a rate of 15 °C/min. Ultra-high purity helium was used as the carrier gas at a flow rate of 1.0 mL/min. The injector temperature was set at 230 °C for split injection at a split ratio of 10:1. Identification of the peaks was achieved by retention time and comparing them with external standards analyzed under the same conditions.
2.5. Determination of the Components and Content of Tocopherol
The occurrence and content of four components of tocopherol (α-, β-, γ-, and δ tocopherols) in seeds of P. ostii was investigated according to GB 5009.82-2016 with modifications. Approximately 0.3 g of the sample and 0.05 g of amylase, together with 1.0 mL of ultrapure water, were added to a 10 mL glass tube at 60 °C for 45 min. After this, 1.5 mL of 100% ethanol containing 0.01 g of butylated hydroxytoluene (BHT), 0.1 g of ascorbic acid, and 1.5 mL of potassium hydroxide were added to the enzymatic hydrolysate and then kept at 80 °C for 60 min. Then, 2.5 mL of the extraction solvent mixture (petroleum ether:ethyl ether, 1:1) was added and mixed for 5 min and then followed by centrifugation. The ether layer was transferred into a tube, washed with distilled water at least 3 times until it had a slightly less than neutral pH value, and then the water phase was removed and the ether layer evaporated under nitrogen gas flow.
For the analysis, the ether layer was recovered in 1.0 mL of high-performance liquid chromatography (HPLC) grade methanol and filtered using a 0.22 μm membrane; 50 μL of the liquid was then injected into the chromatographic column for analysis. HPLC program: Acetonitrile-water (95:5) was applied for gradient elution for 40 min at a flow rate of 1.0 mL/min with the column temperature at 25 °C. Four tocopherols (α-, β-, γ- and δ-tocopherol) were used as external standards to generated calibration curves of peak area versus concentration.
2.6. Extraction and Assay of Phytochemical Compounds and HPLC-MS Analysis
Approximately 0.2 g of ground seed coat or kernel and 1.5 mL of extract solution (methanol:water = 70:30, v/v) were added into a 5 mL centrifuge tube and placed in an ultrasonic cleaner at 20 °C for 20 min. The supernatant was then transferred into another tube after centrifugation. The above procedure was repeated twice. The extract was filtered through a membrane (0.22 μm) and stored at −40 °C for further analysis.
For further HPLC analysis, the following solvent and gradient were used: A, 2% aqueous formic acid (v/v); B, 0.2% formic acid in acetonitrile (v/v); constant gradient from 5% to 64% of B within 80 min; a flow rate of 0.6 mL/min; and 10 μL of extract solution, injected for detection. The column temperature was maintained at 30 °C. DAD data were recorded at 280 nm.
An Agilent 6520 Accurate-Mass Q-TOF LC/MS was used for the qualitative analysis of the compounds. The electrospray ionization mass spectrometry method was applied with positive or negative ion modes. The scanning range (m/z) was 100–1200 u, the sprayer pressure was 35 psi, and the capillary voltage was 3500 V. Dry gas at 350 °C was carried at a flow rate of 12 L/min. The data were analyzed by Masshunter Qualitative Analysis Software B. 04. 00.
2.7. Total Phenolic Content and Antioxidant Activity Analysis
The total phenol content of the extracts was measured using the Folin-Ciocalteu (FC) reagent, as described by Li et al. [
23], with some modifications. Quantities of 100 μL of sample solution, 2500 μL of ddH
2O, and 100 μL of Folin-Ciocalteu reagent were added and mixed for 5 min, and then 300 μL of 20% Na
2CO
3 solution was added. The mixture was shaken and incubated at 37 °C in the dark for 2 h until the reaction reached a plateau. The absorbance was recorded at 750 nm using a spectrophotometer.
The antioxidant capacities of PSK and PSC were assessed by total phenol, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) and ferric reducing antioxidant power (FRAP) assays, which was established in our lab and described by Li et al. [
23]. The identification of the radical scavenging activities and total phenolic content of samples was achieved using gallic acid as the standard [
23,
24].
2.8. Antioxidant Potency Composite (APC) Index Analysis
The APC index was used to evaluate the overall antioxidant activity of PSK and PSC [
25]. The APC index was calculated by the followed formula:
2.9. Statistical Analysis
All of the chemical analyses were carried out in triplicate and values are expressed as the mean ± SD. Statistical significance was examined in GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA, USA) through one-way analysis of variance (ANOVA) and Duncan’s test at p ≤ 0.05. Correlation and clustering analysis was conducted by R version 3.5.3.
4. Discussion
The present study focused on the characterization of nutritional data, including fatty acids, crude proteins, tocopherol, phytochemical compounds, and antioxidants of PSK and PSC from 11 production areas. The data from this study will provide a direction for the selection of high-quality seeds for oil production and a strategy for making full use of seeds in the food industry.
Phenotypic related traits have a certain relationship with seed quality. Seed neatness, moisture content, 100-seed weight, and seed coat rate were characterized in the present study. Of these traits, 100-seed weight is an index that reflects the size and fullness of the seeds and has been shown to be a predictor of production [
29]. In the present study, the 100-seed weight ranged from 16.08 g to 32.89 g among sampled seeds from 11 production areas, which suggested variation did exist, and would provide a basis for target region selection. Seed coat rate is directly related to oil production, and high seed coat rate (35.17%) accounted for about 1/3 of the total seed mass obtained in the present study, which provides a potential use for remnants after oil processing. In future studies, the breeding of new cultivars with lower seed coat rate would be a promising prospect in view of edible oil production. Similar results of seed coat rate have been obtained in
Lupinus angustifolius [
30], rapeseed [
31], and beans [
32], which would function as good references for tree peony breeding with the intent of producing higher oil content. In addition, the relatively high level of crude protein in seed kernel (20.21%) indicates that the seed could be included in food formulations as a source of protein after oil processing.
FAs consisted of SFAs and UFAs, and the latter are divided into monounsaturated (MUFAs) and PUFAs, which are the most important nutritional components of edible oil or other functional foods [
33]. PUFAs are essential fatty acids (EFAs) and present as the predominant part of FAs in
P. ostii seeds, among which ALA was significantly abundant. In contrast, compared with other common edible oils, ALA was less than 10%, such as in soybean oil (8.95%), canola oil (9.74%), corn germ oil (0.55%), sesame oil (0.32%), and olive oil (0.57%) [
11], which indicated that
P. ostii seed oil is a good source for ALA supplementation. ALA is also a precursor for docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), which have been demonstrated as protection agents against chronic daily headaches, aging and dementia, and metal disorders [
34,
35,
36].
In addition, seed oil of
P. ostii is unique for its n-6:n-3 FA ratio (LA/ALA), which is lower than 1.00. Since n-6 and n-3 PUFAs demonstrated significant differences in biological function, the n-6:n-3 FA ratio has been suggested to be a key factor for the balance of dietary patterns and synthesis of eicosanoids, which is of significant nutritional importance [
35,
37]. Due to traditional dietary habits, the ratio of n-6:n-3 FA reached 15–20:1 as a result of a decreased supply of n-3 FAs, which have been related to a series of chronic diseases, such as atherosclerosis, essential hypertension, obesity, diabetes, arthritis, and other autoimmune diseases, as well as cancers of the breast, colon, and prostate [
10,
34]. Therefore, a lower n-6:n-3 FA ratio is preferred for modern diets when considering nutritional and health benefits [
34]. Thus, edible oil like
P. ostii seed oil with a higher content of ALA and a lower ratio of n-6:n-3 FA would be more desirable for diets to reduce the risk of many chronic diseases.
Tocopherols have commanded the most interest because of their availability and overall health impact, and their central role in preventing oxidation at the cellular level [
38]. Tocopherols are a group of fat-soluble antioxidants and can be divided into α-, β-, γ-, and δ- forms [
39]. In present study, the α-tocopherols were not detected in tested
P. ostii seed. Similarly to previous studies, γ-tocopherols were the main type in PSK, with significantly higher levels than that of other tocopherol components [
40,
41]. The largest extracted amount of the total tocopherols in
P. ostii seed was about 276.7 μg g
−1 DW (P1) in this study, which is lower than that in soybean (664 μg g
−1 DW) [
42], rapeseed (460.07 μg g
−1 DW) [
43], and sunflower seed (555.2 μg g
−1 DW) [
44], but higher than that in
Triticum species, such as
T. thaoudar,
T. aegilopoides,
T. monococcum, and
T. urartu (75.1, 70.8, 66.8 and 63.9 μg g
−1 DW, respectively) [
45,
46]. Although the content of tocopherols is moderate,
P. ostii seed would be a good source of tocopherol supply.
Oxidative stability of vegetable oils is significantly impacted by the FA composition, and the oxidation rates of individual FAs have been confirmed to be related to the degree of unsaturation. For OA (C18: 1), LA (18: 2), and ALA (C18: 3) in normal autoxidation reactions, the ratio of oxidation rates has been shown to be 1:12:25 [
47]. Tocopherols are essential for the protection of PUFAs against peroxidation in plants and animals due to their action in scavenging active oxygen species and free radicals. They also perform as efficient terminators in the lipid autoxidation reaction process [
48]. Vegetable oils are notable as major sources of dietary PUFAs and tocopherols. In sunflower oil, OA and LA content accounted for 90.2% of total FAs [
49], and three kinds (α, β, and γ) of tocopherols were detected, and α-tocopherols (671 ppm) were abundant. In contrast, in soybean oil, the total content of OA and LA was 76% of total FAs, and four kinds of tocopherols were obtained [
50] (β + γ- (595 ppm), and δ- tocopherols (263 ppm) were abundant. In olive seed oil, OA and LA were rich and accounted for 80.6% [
51], and α, β and γ-tocopherols were detected (α-(96 ppm) was abundant and β + γ- was 18 ppm). In perilla seed oil, ALA was 62.6%, and the abundant tocopherols were the γ- ones (526 ppm, β + γ-) [
52]. It has been demonstrated that a positive correlation between ALA and γ-tocopherol existed, and oils rich in ALA had low α-tocopherol content [
53]. Similarly, in this study, OA, ALA, and LA accounted for 95.16% of total FAs in
P. ostii seed, and α-tocopherol was almost undetected, while β + γ tocopherol content ranged from 16.08 to 26.81 mg g
−1. This would be a good protector for
P. ostii seed oil from the oxidative deterioration of the essential components of nutrients, since fat-soluble vitamins could prevent or delay lipid oxidation in seed oils [
47].
The presence of natural antioxidants in plants is well known and has various uses. The antioxidant effects of several substances in plants, such as polyphenols, have been reported to have multiple biological effects [
53]. In the present study, total phenols were significantly higher in seed coats and had a positive correlation with DPPH (
r = 0.61), FRAP (
r = 0.69), and ABTS (
r = 0.93) (
Figure 6E), which suggested that they contributed significantly to the antioxidant activity. The phenolic compounds are increasingly of interest in the food industry because they retard the oxidative degradation of lipids and thereby improve the quality and nutritional value of food [
54]. High amounts of secondary metabolites were also detected in seed coat, and we putatively identified nine chemicals. Similar results were also obtained in root bark in our former study [
55], which demonstrated high activities as an analgesic, sedative, and anti-inflammatory agent and performed as a good remedy for cardiovascular disease, stagnated blood, and brain injury in traditional oriental medicine [
56,
57,
58]. Therefore, the seed coat of
P. ostii could be a potential source of natural antioxidants in food and pharmaceutical applications.
High quality seeds are the most basic and important element of agricultural and industrial production. P. ostii is a new emerging woody oil crop, and the seed quality is a decisive factor for the development of the industry. In the present study, seeds from P9 showed the greatest amount of OA, LA, ALA, UFAs, PUFAs, and TFAs, followed by P2, both in PSC and PSK, which indicated that the P9 would be the most promising high-quality production area for the industrial purposes of the P. ostii seed. According to the above results of this study, the use of P. ostii seeds should be encouraged, since they present a high content of crude protein, ALA, PUFAs, and tocopherols, which are beneficial to human health. This study can guide producers to make a strategy for the full use of P. ostii seeds, not only for edible oil, but also for functional food.