Fatty acid analysis in the seeds of 50 Paeonia ostii individuals from the same population

WEl Xiao-bao , XUE Jing-qi, WANG Shun-li, XUE Yu-qian LlN Huan SHAO Xing-feng, XU Donghui ZHANG Xiu-xin

1 Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture/Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

2 Department of Food Science and Engineering, Ningbo University, Ningbo 315211, P.R.China

Abstract Tree peony seeds are rich in α-linolenic acid (ALA), and the peony seed oil is now being produced in China. Paeonia ostii is the most widely used tree peony species for oil extraction, which is commercially called Fengdan and treated as a single cultivar. Here, 50 P. ostii individuals from the same population in northern China were randomly selected for fatty acids (FAs) analysis. Thirteen FAs were isolated, and the most abundant five were palmitic acid (5.31–6.99%), stearic acid(1.22–2.76%), oleic acid (18.78–28.15%), linoleic acid (11.86–26.10%), and ALA (41.11–57.51%). There were significant individual differences of plants in FA quality and quantity and the linoleic acid content in Plant No. 48 even exceeded the scope of 1–99%. Further statistical analysis indicated that most of the individual FAs, saturated FAs, unsaturated FAs, and total FAs levels showed significant positive correlations to each other, whereas the seed yield per plant was independent and not correlated to the factors mentioned above. Ward’s hierarchical clustering results grouped the 50 plants into four clusters based on FA contents and seed yield, and the seven plants in Cluster IV were identified as good candidates for oil production. Our results confirmed that the individual differences did occur in P. ostii and Fengdan cannot be simply treated as one uniform cultivar. Also, these results may help simplify the selection of plants for oil peony breeding and accelerate the development of the oil peony industry.

Keywords: Paeonia ostii, seed oil, fatty acids, α-linolenic acid, individual difference

1. lntroduction

Tree peony (Paeonia ostii) is a shrub in thePaeoniasectionMoutanDC. of the Paeoniaceae family (Xueet al. 2015).Tree peonies are traditional ornamental plants and the seeds have significant nutritional values. The root bark is a traditional Chinese medicine that contains nutrients and has therapeutic effects. Peony seed oil is now being produced in China. The unsaturated fatty acids (UFA) content in peony seed oil ＞90%, especially α-linolenic acid (ALA), whose content ＞40%. This ALA level is much higher than the level in other oil plants such as rapeseed (9.46%), sunflower(1.00%), olive (0.72%), and sesame (0.29%) (Sammanet al. 2008; Liet al. 2015b; Rincón-Cerveraet al. 2016).ALA is an essential omega-3 fatty acid that helps prevent cardiovascular disease, reduces blood fat and borderline hypertension which cannot be synthesized by the human body (Wanget al. 2015; Suet al. 2016). Besides UFA,peony seed oil is also rich in proteins, amino acids, trace elements, vitamins A, B2, B6, and carotenoids (Gao 2012;Zhaiet al. 2013). Due to these health benefits, peony seed oil has been regarded as a new nutrition resource by the NHFPCC (2011).

Two tree peony species are suitable for oil production mainly because of their high seed yield.P.rockiigenerally grows at high altitudes in northwest China, and has several cultivars (Li 2011).P. ostiihas a rapid maturation rate and high resistances to high humidity, pests and diseases,which makes it a well adapted species to most of the growth conditions. It is distributed in most parts of China (Li 2005).P.ostiihas many different genotypes or populations.Commercially, they are all called Fengdan and treated as a single cultivar. The fatty acids (FAs) content of Fengdan exhibit genotype differences (Hanet al. 2014; Hanet al.2016), but the details of these differences are not known.Although Fengdan is mostly propagated by seeds, there are few reports of intrapopulation variation in FAs.

We systematically studied FAs variation within one population of Fengdan. A total of 50 individual plants were randomly selected from the same area, and the seed yield,FAs composition, and content were measured for each plant. The results showed that the significant individual difference did exist in the same population. In addition,based on the correlation and cluster analysis, the selection strategies for oil peony promotion were discussed and some good candidates were also recommended in this study. These results may offer some references for oil peony breeding and finally accelerate the development of oil peony industry.

2. Materials and methods

2.1. Plant materials

The seeds of 50 individualP.ostiiplants were randomly collected from the peony germplasm resources nursery of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China (116°15′51′′N,40°33′32′′E, 673 m a.s.l.). The mean annual precipitation at this location is 450 mm and the mean daily temperature is 8°C. All the plants were five-year-old seedlings which were sowed in the same environment under field conditions,and the seeds were harvested when the pods became hard and seed color was brown (Fig. 1) (Xueet al. 2015). The pods were air-dried at room temperature until they cracked and the seeds were collected. Seeds were kept at 60°C for 48 h in an oven and dried to constant weight. They were then shelled, frozen in liquid N2, and stored at –80°C for future use. The seed yield per plant was the mean of three years of production. The oil analysis was based on seeds harvested in 2015.

2.2. lnstrumentations and methods

lnstrumentationsA gas chromatograph-mass spectrometer(GC/MS-QP5050A, SHIMADZU, Japan) was used to determine FAs composition and content. In addition, a TTL-DCII Nitrogen Blowing Instrument (Beijing Tongtailian Technology Co., Ltd., China), a centrifuge (3K15, Sigma,Germany), an analytical balance (AR1140, Mettler Toledo, Switzerland), an SSW-420-2S Water Bath (Beijing Tianlinhengtai Technology Co., Ltd., China), a THZ-D Shaker(Taicang Experimental Equipment Factory, China), a Vortex Instrument (Vortex Genius 3, IKA, Germany), and a DHG-9140 Electro-Thermostatic Blast Oven (Shanghai Bluepart instruments Co., Ltd., China) were also used in this study.

Fig. 1 Tree peony blooming and seed setting. A, peony demonstration base of the Chinese Academy of Agricultural Sciences. B, flower of Paeonia ostii. C, full fruit pod of P. ostii.D, slightly opened fruit pod with exposed seeds. E, fully mature seeds analyzed in the future. F, over mature seeds.

Experiment methodsPretreatment and derivatization were done as described in previous reports (Liet al. 2013;2015b). Briefly, the seeds were shelled and frozen in liquid nitrogen. A freeze grinder was used to grind the seeds into a powder. Then 50 mg of powder was introduced into 10 mL centrifuge tubes to which was added 3.0 mL chloroform-methanol (1:2, v/v). The tubes were shaken for 1 h at 4°C. Then, 1.0 mL chloroform and 1.8 mL KCl (1 mol L–1) was added to the tubes, and they were centrifuged at 2 500 r min–1for 10 min. The chloroform layer (lower level of the solution) was taken out and dried under a stream of nitrogen delivered by a blowing instrument. The dried residue was dissolved in 1.0 mL of 5% H2SO4methanol solution, vortexed for 1 min, and put in an 80°C water bath for 1 h. Then 1.0 mL of distilled water was added to stop the reaction and 5 mL ofn-hexane was added to extract FAMEs and then placed in a centrifuge with 2 500 r min–1for 10 min. Additionally, we placed 0.1 mL of supernatant in 1.5-mL vials and added 20 μL of methyl heptadecanoate(1.0 mg mL–1in hexane) as the internal standard. At the same time, 0.88 mL ofn-hexane was added.

Chromatography and mass spectrometry conditions were as follows: An AOC-5000 Auto Injector (Shimadzu,Japan) was used. The column had dimensions of 100 m×0.25 mm×0.2 μm film thickness SP-2560 (Supelco, USA).Analysis was performed in constant flow mode. The initial oven temperature was maintained at 100°C for 5 min before it was increased by 4°C min–1to 240°C and maintained at that temperature for 15 min. The temperature of the injector, transfer line, ion source and quadrupole were 250,280, 250, and 150°C, respectively. The injection volume was 1 μL. Electron impact ionization (EI+, 70 eV) was used for all samples.

Data analysisThe identification of the different compounds was based on the comparison of their mass spectra with the NIST05 database. In addition, the retention time of FAs peaks depends on the 37-component FAME Mix (Merck KGaA, Darmstadt, Germany). An internal standard (methyl heptadecanoate) was used as the quantitative approach to measure the content of FAs. Three biological repetitions were used and experimental results were the mean value. SAS V8 was used for the clustering analysis, ANOVA, and correlation analysis. The figures were plotted using Origin 9.0.

Fig. 2 Total ion chromatograms (TIC) of mixed fatty acids methyl ester standards.

3. Results and discussion

3.1. Qualitative and quantitative determination of FAs

Using the chromatographic conditions described in section 2.2, SCAN mode was used to get the total ion flow diagram,and different FAs were distinguished by their retention time(Fig. 2). After that, the SIM (selected ion monitor) mode was used to determine the qualitative value of each kind of FA.In this mode, a quantitative fragment ion with large mass and two reference ions with high response were selected as the characteristic ions for qualitative confirmation.

Quantitative determination of single ions was analyzed by the internal standard (IS) method, and the IS was methyl heptadecanoate. Fig. 3 showed a representative result of the total ion chromatogram (Plant No. 5). Five major FAs and one IS peak can be found (other FA peaks were not shown here for reasons of scale). The quantitative value of each FA was calculated based on the IS content.

3.2. Composition and content analysis of FAs

Fig. 3 Total ion chromatograms (TIC) of the fatty acid methyl ester sample. Five major fatty acids (palmitic acid (c16:0), stearic acid (c18:0), oleic acid (c18:1 n9c), linoleic acid (c18:2 n6c) and α-linolenic acid (c18:3 n3)) and internal standard (IS) are marked.

Thirteen FAs were detected from seeds of the 50 plants,including five kinds of saturated fatty acids (SFA) and eight kinds of UFA. The UFA content ranged from 90.52 to 93.01%of the total fatty acids (TFA) (Table 1). Five major kind of FAs accounted for 99.49% of the TFA as follows: (1) palmitic acid (PA, c16:0), 5.31–6.99%; (2) stearic acid (SA, c18:0),1.22–2.76%; (3) oleic acid (OA, c18:1 n9c), 18.78–28.15%;(4) linoleic acid (LA, c18:2 n6c), 11.86–26.10%; and (5) ALA(c18:3 n3), 41.11–57.51% (Tables 1 and 2). Of the thirteen FA compounds, including dodecadienoic acid (c22:2), which was first reported in peony seed oil and first detected in this study, only nine to ten compounds were found in Fengdan in previous studies (Liet al. 2015b; Hanet al. 2016). In their research, HP-INNOWAX (30 m×0.25 mm, 0.25 μm film thickness, Agilent, USA) and HP-88 (30 m×0.25 mm, 0.20μm film thickness, Agilent, USA) capillary column was used,respectively, whereas in the present study, SP-2560 (100 m×0.25 mm, 0.20 μm film thickness, Supelco, USA) capillary column was used. The stationary phase of the column used here was a strong polar cyanopropyl siloxane that should be especially effective for FAs separation. This might account for the differences. Interestingly, it was reported that γ-linolenic acid did not exist in the seeds of 60 tree peony cultivars, which was probably due to lack of expression information of genes encoding for delta-6 desaturase through transcriptome analysis (Liet al. 2015a, b). Here in this study, γ-linolenic acid was detected in all the Fengdan individuals, although whose contents were extremely low,ranged from 0.05 to 0.54 mg g–1DW (Table 1). To answer this inconsistency, higher precise detection devices and more materials from various environments may be needed.In addition, more direct evidences, such as gene expression and function analysis may also be needed.

To better understand the distribution of different FAs,the five major FAs and TFA contents of the 50 plants were analyzed using a box plot. As shown in Fig. 4, the content of each FA, including TFA, showed a variable distribution among the different individuals. It also showed that ALA had the highest content followed by OA, LA, PA, and SA.ALA content was the highest in all 50 individuals, whereas OA content was higher than LA in 32 plants (Table 1).Furthermore, all the data were within the 1–99% range,except the LA content in Plant No. 48, which was much higher than that of other 49 plants (Fig. 4). Previous studies found similar exceptions in different tree peony cultivars,such as OA in Xuelicangjiao and ALA in Liuliguanzhu (Liet al. 2015a). Our results confirmed that within the same population of Fengdan, significant variation in FA content can occur. However, in commercial situations, Fengdan is always treated as a single uniform cultivar. Research seldom focuses on the evaluation of individual plants for FAs content or other factors. Thus, additional selection and evaluation research would be useful prior to large-scale development of the peony oil industry.

In June 2014, Chinese Nutrition Society released the 2013 version of Chinese Dietary Reference Intakes (DRIs).This version increased the recommended value of ALA and suggested consumption of 1 600 to 1 800 mg ALA per day especially for pregnant women (CNS 2013). ALA cannot be synthesized and the best way to obtain it is from edible oils.Although flaxseed has the highest ALA content compared to other crops, the total yield per unit area is low (Connor 1999; Yuet al. 2016). In other edible oils, ALA content is＜10%, and some products, such as peanut oil, contain no ALA (Carvalhoet al. 2006). In tree peony, the ALA content ranges from 26.1 to 54.7% depending on different cultivars and wild relatives. In Fengdan, ALA content was 39.6% (Liet al. 2015a). Considering the seed yield, environmental adaptability, and other factors, Fengdan is very suitable as an oil crop in China and provides high levels of ALA.However, since the ALA content in Fengdan can vary significantly among different individuals, careful selection of plants for cultivation will be necessary.

The Fengdan seed oil composition followed the order:polyunsaturated fatty acids (PUFA, 66.91–69.14%)＞monunsaturated fatty acids (MUFA, 22.06–23.22%)＞SFA(6.99–10.06%) (Table 3, Fig. 5). PUFA and MUFA are healthier than that of SFA for FAs intake, whereas PUFA has lower freezing point, and cannot be synthesizedde novo. Since the PUFA content in Fengdan seed oil is much higher than that in other oil crops (approximately 74% of FAs are MUFA in olive), it is highly recommended for daily diet requirements. Another important index for evaluating oil nutritional quality is the n-6/n-3 ratio. There are evidences that a low n-6/n-3 ratio (＜4.0) is beneficial to human health, and it can reduce the risk of cancer and cardiovascular disease (Simopoulos 1999; Hu 2001;Gebaueret al. 2006). In Fengdan seed oil, all the n-3 PUFA was ALA, and the n-6/n-3 ratios ranged from 0.21 to 0.64 (Table 3). Together, these data indicate that Fengdan oil has significant nutritional value.Since individual differences also occurred in the PUFA and n-6/n-3 ratio of Fengdan, more individual selection will be necessary to allow Fengdan to become an economically established oil crop.

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3.3. Correlation analysis of FAs and seed yield

We used correlation analysis to study the relationship between different FAs compounds and seed yields. Most of the FAs,including SFA, UFA and TFA,were positively correlated to each other at the 0.01 or 0.001 level, except dodecadienoic acid(c22:2) and lignoceric acid (c24:0),whose content was relative low(Table 4). Interestingly, all the correlations of the top five major compounds were positive at the 0.001 level, respectively, and the correlation of UFA and TFA was 1.000. However, the seed yield per plant showed no significant correlation with any other factors.

In oat (Avena sativaL.), some FA levels, such as stearate and oleate, were positively correlated with groat-oil content, whereas other FAs, including palmitate and linoleate, were negatively correlated with groat-oil content(Hollandet al. 2001). In different sesame (Sesamum indicumL.)landraces, the oil content was positively correlated with oil yield as expected, so as palmitic acid with stearic and oleic acid. Some compounds were negatively correlated, such as stearic acid with linoleic and linolenic acids(Uzunet al. 2008). Concerning the negative correlation, oleic acid and linoleic acids have been found in many oil plants, such as crucifer species (Mandalet al.2002), soybean (Rebetzkeet al. 1996; Patilet al. 2007),peanut (Andersenet al. 1998) and safflower (Knowles and Hill 1964). In our study, most of the FAs were positively correlated with TFA, and no negative correlations occurred,whereas seed yield was not significantly correlated with any specific compounds. These results may help us for peony oil breeding strategy. We may only need to focus on the TFA content and the seed yield. Compared to individual compounds, the TFA content and seed yield are much easier to measure, and this will simplify selection of progeny for those who are interested in ALA or other nutritional compounds.

Fig. 4 The range and distribution of five major fatty acids and total fatty acids content in 50 plants. Median values are the horizontal lines in the box, 50% data is within the box and 99% data is within the bar (±SD). The data outside the box are indicated by black dots. c16:0, palmitic acid; c18:0, stearic acid; c18:1 n9c, oleic acid; c18:2 n6c, linoleic acid; c18:3 n3,α-linolenic acid. TFA, total fatty acid.

Fig. 5 Proportions of average fatty acids in 50 Fengdan plants.A, n-3 (α-linolenic acid (C18:3 n3)) and n-6 (γ-linolenic acid(C18:3 n6), linossleic acid (C18:2 n6c) and dodecadienoic acid(C22:2)) fatty acid accounts for the proportion of polyunsaturated fatty acids (PUFA). B, sort by unsaturated fatty acids (UFA),including PUFA (C18:3 n3, C18:3 n6, C18:2 n6c, and C22:2),monunsaturated fatty acids (MUFA) (palmitoleic acid (C16:1),heptadecenoic acid (C17:1), oleic acid (C18:1 n9c), and eicosenoic acid, (C20:1)) and saturated fatty acids (SFA)(pentadecanoic acid (C15:0), palmitic acid (C16:0), stearic acid(C18:0), arachidic acid (C20:0), and lignoceric acid (C24:0)).

Table 3 Overall fatty acids composition in seeds of 50 individual Paeonia ostii seeds

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3.4. Ward’s hierarchical clustering analysis

To complete a systematic analysis of the relevance of FAs in individual plants, we used Ward’s hierarchical clustering method. According to the relevance and difference of the five major FAs and TFA content as well as seed yield per plant, the 50 Fengdan plants were divided into four clusters. Clusters contained 21, 16, 6 and 7 individuals in clusters I to IV, respectively (Fig. 6). One-way ANOVA analysis showed significant differences among the four clusters (P＜0.05), and supported the validity of the clustering result.

To further evaluate the variation among the four clusters, the five major FAs, TFA and seed yield per plant were compared (Fig. 7). In Cluster I, the mean TFA content and seed yield per plant were 79.90 mg g–1and 65.15 g, respectively. These values were lower than the mean values of all 50 plants, which were 103.22 mg g–1and 75.79 g, respectively. The ALA content in Cluster I was the lowest of the four clusters. Therefore, the 21 individuals distributed in Cluster I were poor candidates for oil breeding. In clusters II and III, both the ALA and TFA contents in the former were higher than in the latter(1.33 and 1.53 times, respectively), but the seed yield in Cluster III was 1.90 times than that of Cluster II. Therefore,compared to Cluster II, Cluster III had greater yield potential. In Cluster IV, all the FAs contents, including TFA, were the highest among the four clusters, but the OA content was slightly lower than that in Cluster II. The mean seed yield per plant in Cluster IV was also high. In summary, considering the FAs content and plant seed yield, the seven individuals in Cluster IV (No. 1, 7, 22,25, 28, 48 and 50) are the currently recommended best candidates for large-scale production.

4. Conclusion

Fig. 6 Ward’s hierarchical clustering analysis of fatty acids content in 50 Fengdan plants. These plants were divided into four clusters (Clusters I–IV) at the distance of 0.085.

Fig. 7 Variations of five major fatty acids and total fatty acid(TFA) in four plant clusters (mean±SD, n=3). c16:0, palmitic acid; c18:0, stearic acid; c18:1 n9c, oleic acid; c18:2 n6c, linoleic acid; and c18:3 n3, α-linolenic acid.

We analyzed the FAs composition and content of seeds from 50 individuals in one population of tree peony Fengdan. Using GC/MS, 13 FAs were detected. The five most abundant FAs accounted for 99.49% of the TFAs,and ALA had the highest content. The five major FAs,including TFA, varied significantly among 50 individuals,especially the LA content in Plant No. 48, which exceeded the range of 99%. The variability of these results indicates that Fengdan cannot be treated as one uniform cultivar in peony oil industry. Additional selection work should be done before large-scale propagation. Most of the FAs compounds were positively correlated with each other whereas the seed yield per plant was independent, and was not significantly correlated to other factors. These results may help us to simplify the selection strategy for oil peony breeding. Clustering analysis divided the 50 individuals into four clusters with significant differences (P＜0.05), and the seven members in Cluster IV are recommended as prime candidates for large-scale propagation considering the FAs nutrition value, contents and yield.

Acknowledgements

This study was funded by the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS),the Natural Science Foundation of China (31572156,31501800), the Special Fund for Agro-scientific Research in the Public Interest, China (201203071), and the Beijing Municipal Science and Technology Project, China(D161100001916004).