1. Introduction
Alkylresorcinols (ARs), also called 5-
n-alkylresorcinols, are present in large amounts in cereals [
1,
2,
3]. Among the common crops used for human consumption, wheat (
Triticum aestivum L.) generally has a high ARs content with odd-numbered homologues from C15 to C25 [
4,
5]. ARs are of interest in nutrition science, which have been suggested as markers for whole-grain wheat in food and as biomarkers for human intake of whole-grain wheat [
6,
7,
8,
9].
The positive health effects of whole grains have been studied extensively in recent years, and whole grain intake has been consistently linked to decreased risk of developing type 2 diabetes [
10,
11,
12]. Emerging in vitro data suggest that ARs may have bioactivities related to enzyme inhibition [
13]. Previous reports also suggested that ARs could inhibit low-density lipoprotein oxidation, prevent cardiovascular diseases, and increase fecal cholesterol excretion [
14,
15,
16]. Scientific organizations believe that humans would benefit from increased consumption of whole grain foods, as they can reduce the risk of developing chronic diseases [
17,
18,
19,
20]. Being able to breed wheat that is rich in ARs, with enhanced health benefits, is of great importance.
A substantial amount of direct and indirect evidence indicates that the majority of ARs are located in an intermediate layer of the caryopsis (including the testa, hyaline layer and inner pericarp), while fine wheat flour contains essentially little ARs [
1,
21,
22,
23]. Therefore, to determine cereals’ ARs content, they are always milled into powder [
23,
24]. To date, there have been many methods reported for extracting ARs from whole grain flour and bran. In most cases, these extraction methods tend to require long extraction times (6 to 24 h) [
25,
26,
27], complicated operations, and high chemical-analytics costs [
28]. After extraction, cereal samples were not recycled, and thus the materials were wasted. It is important to note that, screening and breeding of wheat varieties with high AR content tends to be one of the major wheat breeding targets [
19,
29]. In breeding studies, it is necessary to screen large numbers of lines; however, during the early stages of the breeding process, only small quantities of grain are available [
29]. However, the lack of a non-destructive, simple-operation, cost-effective, and time-saving extraction method for the AR content determination limits trait selection in wheat breeding programs. Therefore, developing a non-destructive method that can help breeders efficiently and quickly analyze ARs in large populations of wheat materials is necessary.
The surface of above-ground plant organs is covered with a cuticle, a protective lipid structure sealing the tissue against the environment. The surface lipid serves as the major barrier preventing nonstomatal water loss, protects plant surfaces against pathogens and ultraviolet radiation, and affects plant-insect interactions [
30,
31,
32]. However, seed surface lipid (SSL) extraction methods are rarely reported in wheat, and there has been little focus on the chemical composition of lipid on wheat seed surface. Lipids extraction from tissue surface using chloroform has been extensively used in many studies [
2,
33,
34,
35]. Furthermore, the chloroform extraction method is fast, simple in operation, and highly-efficient [
36,
37]. Previous use of chloroform (with 30-s dropping in chloroform) to extract surface lipids (including ARs) from wheat and rye leaves also highlights the method’s possible use for intact ARs extraction from other wheat organs [
33,
37]. To the best of our knowledge, there have been no reports on the relationship between the SSL and whole grain lipid (WGL). In particular, little is known about the correlations of ARs and AR homologues on seed surface and that in whole grains.
The aim of this study was to investigate the chemical composition of the SSL and WGL from 36 cultivars of wheat in China, and develop a non-destructive method for AR content analysis on seed surface and whole-grain flour. Therefore, both chloroform and ethyl acetate were tested for extracting lipids from wheat samples. At the same time, the relationships between the content of ARs and AR homologues on seed surfaces and in the whole grains were studied in detail. In addition, wheat seed germination tests were carried out to confirm that it is a non-destructive method. In this study, we provide a non-destructive method for efficiently predicting total ARs content in whole grains, to help breeders select wheat varieties (lines) with high ARs content quickly for future breeding studies.
3. Discussion
This is the first time quantitative analysis of the lipids in whole grains and seed surfaces is reported. Furthermore, correlation analyses were conducted to determine the relationship between ARs on seed surfaces and in whole grains.
Both content of total ARs and AR homologues on seed surfaces and that in whole grains varied dramatically among the 36 wheat cultivars, while the relative composition of AR homologues in whole grains showed smaller variations. In whole grains, the average AR 17:0/AR 21:0 ratio was 0.1. Ratios of 0.1, 0.01 and 1.0 were reported for common wheat, durum wheat, and rye, respectively. These ratios have been suggested as an index for determining the source of cereal products [
46,
48]. Our study demonstrates that all tested samples were common wheat rather than rye or durum wheat. In fact, variations in ARs, AR 17:0/AR 21:0 ratio, and AR 19:0/AR 21:0 ratio were very low compared to the general variability of plant secondary metabolites [
45], which supports their use as biomarkers. This study also indicates that minor amounts of ARs were present on seed surfaces (in the outer cuticle of pericarp), as suggested by Landberg et al. [
21], meaning that the majority of ARs were present in the intermediate layer of seeds. Based on the above discussion, ARs can be used as a selective marker of the intermediate layer or testa in cereal fractions.
A low AR content was present on seed surfaces, and it showed a different homologue composition, indicating that AR compositions were different in different parts of wheat seed. Dayan et al. suggested that the formation of ARs with different chain lengths is highly dependent on the specificity of the appropriate enzyme for the substrates [
49,
50]. We speculated that the reason for these differences was that the enzyme activities were not equal in and outside the pericarp of wheat seeds. Although ARs have been found in an increasing number of organisms [
28,
37,
38,
51], a broader understanding of their bioactivities and underlying mechanisms is lacking. Further studies should be conducted to elucidate the related enzymes and substrates. More physiological experiments are needed in future studies.
On the other hand, the chloroform extraction procedure is a time-saving and simple extraction method, which can meet the need to efficiently screening large numbers of lines. Without affecting the seed germination rate and damaging seed structure, this method is suitable for breeders and nutritionists.
In summary, in this work, we analyzed the composition of SSL and WGL. Although ARs were the major compounds in both SSL and WGL, there were many differences between their compositions. Moreover, there was a highly significant correlation (p < 0.001) between total AR concentrations in whole grains and that on seed surfaces. Along with a substantially shorter sample extraction time, the major advantage of this method was that using chloroform allowed easy and non-destructive extraction of the ARs from seed surfaces. Therefore, results from this study could provide a non-destructive extraction method for determining ARs contents on seed surfaces, so as to predict the contents in whole grain flour. Using the methods developed in this paper can therefore help breeders select appropriate recombination events to accelerate the selection process without destroying seed structure or affecting seed germination rates.
4. Materials and Methods
4.1. Plant Materials and Reagents
Thirty-six wheat samples collected from different parts of China were kindly provided and identified by Crop Molecular Biology and Breeding laboratory, College of Agronomy, Northwest A&F University, China. The corresponding information of 36 wheat samples are listed in
Appendix A Table A1. All wheat samples were grown in the field during the 2017–2018 wheat-growing seasons in China. A total of 90 seeds per variety were individually hand-planted in a 1.5-m row at 10-cm spacing. Standard cultural practices for wheat were followed during the cultivation. All wheat materials were harvested in June 2018.
Chloroform and ethyl acetate were purchased from Xilong Scientific (Shantou, China) and used for lipid extraction from wheat samples. Pyridine, n-tetracosane (C24) and N,O-bis (trimethylsilyl)- trifluoroacetamide (BSTFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). GC-MS (GCMS-QP2010, SHIMADZU, Tokyo, Japan), and GC-FID (GC-2010 PLUS, SHIMADZU, Tokyo, Japan) were applied for identifying wheat lipid components.
4.2. Developing a Suitable Method for Extracting Lipids from Seed Surfaces
Surface lipids were operationally defined as the lipid material extracted by quickly immersing these organs in chloroform [
27,
37,
39]. In this paper, different extraction times were tested (10 s, 30 s, 60 s, 90 s, 120 s,180 s, 300 s, 600 s, and 1800 s), and it was found that SSL were fully extracted after 4 min (240 s) (
Figure 1). The final protocol used was as follows: Wheat seeds (3 g) were immersed in a glass beaker containing 20 mL chloroform and 10 µg C24 as an internal standard, then shaken four times for 4 min at room temperature. Each lipid sample was filtered through a paper filter, then transferred to a GC autosampler vial and dried under a stream of nitrogen gas.
4.3. Isolation of Lipids from Whole Grain Flour
The sample extraction procedure followed a previously established method for grains [
21,
46,
47]. Ross et al. confirmed that lipids could be extracted completely from whole grain flour in 24 h [
27]. The extraction programs were as follows: Wheat seeds were air-dried and knife-milled, then stored in polyethylene pouches at −20 °C before extraction. Grain samples (3 g) were immersed in a glass beaker containing 40 mL ethyl acetate and 40 µg C24 (internal standard); lipid was extracted by continuous stirring for 24 h at room temperature. Each lipid sample was filtered through a paper filter and dried under a stream of nitrogen gas.
4.4. Derivatization Reactions
For GC analysis, each lipid sample was derivatized with 40 μL pyridine and 40 μL BSTFA for 1 h at 70 °C, and each lipid sample was vortexed every 20 min [
33,
37,
39]. The purpose of this step was to transform hydroxyl (OH-) containing compounds into their corresponding trimethylsilyl derivatives [
33]. Then, the sample was dried under nitrogen gas. After that, 700 μL of chloroform was added for the next analysis.
4.5. Chemical Analysis of Lipids
After derivatization, lipid composition was analyzed on a capillary GC column (30 m long, i.d. = 0.32 mm, df = 0.25 μm; Restek, Bellefonte, PA, USA) and attached to an MS; helium (He) was used as carrier gas at a rate of 1.5 mL/min, programmed to temperature as follows: Set at 50 °C for 2 min, ramp 20 °C/min to 220 °C, hold for 2 min, ramp 1.6 °C/min to 310 °C, and hold for 18 min at 310 °C. The MS operated in full-scan mode applying a mass in the range of 35 to 700
m/
z. Compounds were identified by comparing their retention times and mass spectra with authentic standards and literature data [
36,
38,
39,
40].
The GC equipped with a flame ionization detector (FID, SHIMADZU, Tokyo, Japan) was used for composition quantitative analysis. GC-FID was carried out on the same GC conditions as above, but with Nitrogen (N2) carrier gas inlet pressure regulated for a constant flow of 2.0 mL/min. Individual lipid compounds were quantified against the C24 (internal standard) by automatically integrating peak areas. All values were reported on a dry matter (DM) basis. DM was determined by drying samples in an oven at 105 °C overnight, cooling in a desiccator, and then weighing. All wheat samples were analyzed in triplicate; the coefficient of variance (C.V.) was less than 10% for the three samples.
4.6. Wheat Germination Tests
YM1, HM608, and LY3 were chosen randomly from the 36 wheat cultivars to perform the germination tests. Both the normal seeds and the chloroform-soaked (5 min) seeds were in the same germination conditions. The germination test was performed in a petri dish with two layers of filter papers moistened with distilled water, at 25 °C in the dark. Each petri dish contained 100 seeds, 5 mL of sterilized water was added to meet the conditions of germination humidity, and the number of statistical seed germinations was observed every day. A seed was regarded as germinated when the radicle had pierced the seed coat. The germination rate was counted on the 7th day. The results presented here are the means of the germination rates obtained from four replicates; the coefficient of variance (C.V.) was less than 10% for the four samples.
4.7. Statistical Analysis
All results in our study denote the means ± SD. Pictures were drawn using Sigma plot 14.0 software, except for chemical structures, which were drawn using ChemDraw Professional 15.1 software. Correlation and linear analyses were performed using SPSS 19.0 software. Method agreement was assessed by a Bland–Altman plot [
52]. A
p-value less than 0.05 was considered to be statistically significant.