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Metabolic profile analysis based on GC-TOF/MS and HPLC reveals the negative correlation between catechins and fatty acids in the cottonseed of Gossypium hirsutum



The diversified and high value-added utilization of cotton by products can promote the sustainable development of modern agriculture. Differences in potential nutrients among varieties can be explained by variations in the composition and abundance of fatty acids, polyphenols, carbohydrates, amino acids, and organic acids. Therefore, the analysis of metabolite species and relationships in cottonseed is meaningful for the development of cotton byproducts.


In this study, the metabolomes of three representative cotton cultivars of different species were compared using untargeted GC-TOF/MS analysis. A total of 263 metabolites were identified from 705 peaks, and their levels were compared across cultivars. Principal component analysis and OPLS-DA clearly distinguish these samples based on metabolites. There were significant differences in the contents of amino acids, carbohydrates, organic acids, flavonoids, and lipids in G. hirsutum TM-1 compared with G. arboreum Shixiya1 and G. barbadense Hai7124. Notably, the bioactive nutrient compound catechin obtained from the differential metabolites significantly accumulated in TM-1. Furthermore, a comprehensive analysis using catechin and oil-related traits was conducted in core collections of Gossypium hirsutum. The results revealed the reliability of the GC-TOF/MS analysis, as well as that catechin content has a negative association with myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, arachidic acid, and total fatty acids.


These findings suggest that untargeted GC-TOF/MS analysis could provide a new method for investigating the underlying plant biochemistry of nutrient variation in cottonseed, and that catechin content has a negative association with oil-related traits in cottonseed. This study may pave the way to exploit the value of cotton byproducts.


Cotton is grown in over 75 countries mainly for its fiber or lint, the most important natural and renewable material for the textile industry. However, other parts of the cotton plant also have commercial potential, such as the seeds, stalks, hulls, and flowers (Egbuta et al. 2017; Xu et al. 2021; Yuan et al. 2016). These are commonly referred to as cotton byproducts, and are widely used to obtain edible oil and protein feed in cotton producing countries. The nutritional and functional quality of cottonseeds is far from meeting the requirements of the diversified development of agricultural products. Cottonseed is a non-fresh agricultural product with quality characteristics that often been ignored in the process of planting and primary processing. And the latest cutting-edge technology is rarely applied to cotton byproduct, resulting in its low level of utilization.

Plants can produce a variety of metabolites with structurally diverse, which play an essential role in growth and development (Saito 2009; Saito and Matsuda 2010; Wang et al. 2019). In addition, these metabolites provide necessary and sufficient resources for human and animal nutrition, bioenergy, and medicine (Jacobs et al. 2021; Sharma et al. 2021). Understanding plant biochemistry and phytochemistry is thus of fundamental importance for sustainable agriculture and resource conservation. Metabolomics technology can be used to analyze the types and contents of small molecule metabolites in samples, providing a new method for us to study metabolic diversity to evaluate the nutritional and functional quality of crops. The development of modern analytical instruments with high resolution and high sensitivity has elevated the rapid development of metabolomics. Gas chromatography/mass spectrometry (GC/MS), as the most mature method has been used to separate metabolites occurring in urine or tissue extracts, and was also widely used in plant metabolome analysis (Dalgliesh et al. 1966; Beale et al. 2018; Choudhury et al. 2022). Liquid chromatography/mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR) have also been developed as the main analytical techniques for metabolomics in recent years (Rochfort 2005; Razzaq et al. 2019; Patel et al. 2021). Due to its high resolution, high sensitivity, large number of mass spectral libraries, good reproducibility, and relatively low cost, GC/MS is often used for the analysis of volatile and semivolatile metabolites with relative low molecular mass, low polarity, low boiling point or volatile compounds after derivatization. High-resolution time-of-flight (TOF) mass spectrometry can not only obtain the mass spectrum of compounds, but also accurately detect each fragment ion.

There are only four Gossypium species producing commercially spinnable fibers in the world, among which allotetraploid cultivars of G. hirsutum (upland cotton) accounts for approximately 90% of global cotton production, with most of the remaining production coming from G. barbadense (sea island cotton) (Mansoor and Paterson 2012; Zhang et al. 2015). The diploid cultivars of G. arboreum and G. herbaceum currently have no agricultural output. At present, there are few literature reports on the use of metabolomics techniques to evaluate nutrients, bioactivity, and health benefits for cottonseed, and fewer comparative studies among cotton species. In this study, to better understand the metabolite variations among cotton species, untargeted GC-TOF/MS analysis was performed to identify and quantify metabolites including fatty acids, polyphenols, carbohydrates, organic acids, and amino acids in three representative cultivars. Then, classical chromatographic analysis was used to further verify the test results. Last, correlation analysis of micronutrient catechin with oil-related traits was conducted to explore the underlying relationship between nutritional quality in core collections of Gossypium. This study provides a reference for exploiting the value of cotton byproducts and the development of functional foods from cottonseed in the future.

Materials and methods

Plant material

Three cultivars Shixiya1, Hai7124, and TM-1, which belong to Gossypium arboreum (G. arboreum), Gossypium barbadense (G. barbadense) and Gossypium hirsutum (G. hirsutum), respectively, were used for gas chromatography combined with time-of-flight mass spectrometry (GC-TOF/MS) analysis. These representative cultivars have complete genome sequences and a clear genetic background; they have also been intensively studied. The cotton was planted at Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang, China, and all cultivars were cultivated under same conditions. The seeds of the three cotton cultivars were harvested in the same period. Then, the samples were freeze-dried and stored at − 80 °C until GC-TOF/MS analysis.

To illustrate the content distribution of catechin across different species, 47 accessions of G. arboreum, 37 accessions of G. barbadense, and 144 accessions of G. hirsutum were chosen from core collections of Gossypium used for measuring catechin content by HPLC analysis. Moreover, to explore the correlation between catechin and fatty acids in G. hirsutum, 144 accessions were measured for fatty acids by GC analysis.

Sample extract preparation and GC-TOF/MS analysis

Briefly, cottonseed sample powder (50 mg) was extracted with 0.48 mL 75% methanol containing 10 μL adonitol (0.5 mg·mL−1 stock in dH2O) as an internal standard. The resulting mixture was ultrasound treated for 5 min and then centrifuged (12 000 r·min –1) at 4 °C for 15 min. The supernatant (0.4 mL) was transferred to a new 2 mL GC/MS glass bottle and dried completely in a vacuum concentrator. Then, the extracts were oximated using 80 μL methoxyamine hydrochloride (20 mg·mL−1 in pyridine) at 80 °C for 25 min. Subsequently, 100 μL BSTFA reagent (1% TMCS, v/v) was added to the samples and incubated at 70 °C for 1.5 h. GC-TOF/MS was performed as described by Deng et al (2020). Briefly, the GC-TOF/MS analysis was performed using an Agilent 7890 gas chromatograph (GC, Agilent Technologies, USA) system coupled with a Pegasus HT time-of-flight mass spectrometer (Leco, USA). The system utilized a DB-5MS capillary column (J&W Scientific, Folsom, CA, USA). A total of 1 μL samples were injected by the autosampler into GC. Helium was used as the carrier gas, the front inlet purge flow was 3 mL·min−1, and the gas flow rate through the column was 1 mL·min−1. The initial temperature was kept at 50 °C for 5 min, then raised to 210 °C at a rate of 3 °C·min−1, and held for 3 min. The injection, transfer line, and ion source temperatures were set at 280 °C, 280 °C, and 250 °C, respectively. The energy was 70 eV in electron impact mode.

Data preprocessing and annotation

Chroma TOF 4.3X software (LECO Corporation) and the LECO-Fiehn Rtx5 database were used to preprocess and annotate the GC-TOF/MS analysis data. In addition, the mass spectrum match and retention index match were also noted in metabolite identification (Kind et al. 2009).

Catechin extraction and analysis by high performance liquid chromatography (HPLC)

Briefly, approximately 100 mg cottonseed powder was extracted using 3 mL of 80% acidified methanol (1% hydrochloric acid). After 30 min of sonication, the homogenate was incubated for 12 h at 25 °C. Then samples were centrifuged at 10 000 r·min –1 for 15 min. The supernatant was used by an Agilent 1100 series HPLC system (Agilent Technologies, USA) to determine the content of catechin (Peng et al. 2018). The elution contained A (0.2% formic acid/water) and B (methanol). The linear gradient program was as follows: 0–5 min, 5%20% B; 5–18 min, 20%25% B; 18–25 min, 25%42%; and 25–32 min, 42% B. The flow rate was 1 mL·min−1. 10 µL of supernatant sample was injected. The column temperature was 30 °C and the detection wavelength was set at 278 nm.

Fatty acids extracted and analyzed by gas chromatography (GC)

The extraction of fatty acids from mature cottonseeds was performed as described previously. Briefly, approximately 50 mg of cottonseed powder was added to 1 mL of 0.3 mol·L−1 potassium hydroxide methanol solution, and 1 mL of n-hexane containing 500 μg·mL−1 C11:0 was used as an internal standard. After shaking for 30 s and holding at 25 °C for 1.5 h, the homogenate was added 1.5 mL of 0.9% (w/v) sodium chloride solution, followed by centrifugation (10 000 r·min –1) at 25 °C for 5 min. The supernatant was separated by gas chromatography (Agilent 7890-FID) according to previously described procedures (Dowd et al. 2010). Briefly, the gas chromatography was fitted with a DB-23 capillary column (Agilent Technologies, USA). Injectors were operated in split mode with a split ratio of 1:100. The temperature of the injection port was set at 240 °C. Helium was used as the carrier gas, and the injection volumes were 1 µL. The column temperature was held at 170 °C for 5 min, and increased to 180 °C at 1 °C·min−1, and then increased to 240 °C with a rate of 4 °C·min−1, and held for 5 min.

Multivariate analysis and statistics

For better visualization and subsequent analysis, GC-TOF/MS data were used to perform Principal component analysis (PCA) and OPLS-DA analysis by SIMCA software (V14.1, MKS Data Analytics Solutions, Umea, Sweden). Student’s t-test and one-way ANOVA were used to compare differences between two groups and multiple groups, respectively. Pearson’s correlation coefficient analysis was performed by OriginPro 2021 ( to assess the correlation between catechin and fatty acid content. P values < 0.05 or < 0.01 or < 0.001 were considered statistically significant.

Results and discussion

Characterization and analysis of GC-TOF/MS data

Metabolomics analysis was employed to elucidate the potential chemical basis of different species of the Gossypium genus. Three representative cultivars of cotton species, Shixiya1, Hai7124, and TM-1, were used for untargeted metabolome analysis by GC-TOF/MS, each with six biological replicates. A typical total ion chromatogram (TIC) from these cottonseed samples is shown in Additional file 1: Fig. S1, representing the summed intensity of all mass spectral peaks at every point in the analysis. From this step, obvious differences could be found in some TIC peaks across samples. A total of 705 peaks were extracted; these are listed in Additional file 2: Table S1. Based on the local metabolite database, 263 metabolites were identified and classified into 15 different categories (Additional file 3: Fig. S2, Additional file 4: Table S2), including 53 organic acids, 44 carbohydrates, 43 amino acid derivatives, 34 other metabolites, 25 alcohols and polyols, 14 lipids, 10 benzene and substituted derivatives, 9 nucleotide and its derivates, 7 amines, 6 phenylpropanoids, 6 flavonoids, 3 sphingolipids, 2 quinates and their derivatives, 6 alkaloids, and 1 vitamin. The above results indicate that many types of metabolites were obtained, consistent with the characteristics of metabolomics data based on GC-TOF/MS.

Identification of differential metabolites from three cotton cultivars

Principal component analysis (PCA) is the most common dimensionality reduction method. It converts a large number of variables into principal components (PCs) that still contain most of the information found in the large set. PCA is an unsupervised mode; thus, it shows the distribution of the origin data and is used to evaluate the difference for intergroup comparisons. As illustrated in Additional file 5: Fig. S3, the PCA score scatter plot indicated significant differences in the three cotton cultivars, and all the samples were within the 95% confidence intervals (Hotelling's T-squared ellipse). PC1 and PC2 accounted for 29.1% and 14.7% of the total variation, respectively.

The orthogonal partial least square-discriminant analysis (OPLS-DA), as a supervised mode, was conducted to provide a precise level of group separation, as well as to test the correlation of samples. It is apparent from Fig. 1A that, OPLS-DA was better for separating the differences in the metabolic phenotypes between three varieties of Gossypium. Furthermore, sevenfold cross validation and permutation tests were used to estimate the effectiveness of the model. The R2 value was close to 1 and the Q2 value was negative, indicating that the model was reliable and that the risk of overfitting was low. These results suggested that there were significant differences in the metabolites of each cultivar.

Fig. 1
figure 1

OPLS-DA analysis and heatmap of hierarchical clustering analysis of differential metabolites for three cotton cultivars. A Score scatter plot of OPLS-DA between each two cultivars. B The differential metabolites from three cotton cultivars used to draw the heatmap of hierarchical clustering analysis (HCA). The horizontal axis and vertical axis represent the different cottonseed samples and different metabolites, respectively. Each relative metabolite content is listed in Additional file 10: Table S6

Subsequently, cutoffs for variable importance in the projection (VIP) ≥ 1 obtained from OPLS-DA and P value < 0.05 were used for screening the differential metabolites (DMs). A volcano plot was employed to visualize the differential metabolites (Additional file 6: Fig. S4). The results showed that there were 119, 131, and 128 DMs between the two cultivars, respectively. Specifically, the Shixiya1/Hai7124 comparison had 64 upregulated (red scatter points) and 55 downregulated (blue scatter points) DMs (Additional file 6: Fig. S4A, Additional file 7: Table S3). The Shixiya1/TM-1 comparison had 29 upregulated and 102 downregulated DMs (Additional file 6: Fig. S4B, Additional file 8: Table S4) and the Hai7124/TM-1 comparison had 26 upregulated and 102 downregulated DMs (Additional file 6: Fig. S4C, Additional file 9: Table S5). In addition, one-way ANOVA was also conducted for the comparative analysis of metabolic differences between multiple experimental groups, and hierarchical cluster analysis (HCA) of the selected differential metabolites (P value < 0.05) was used to classify metabolites with similar characteristics and further study intergroup variation (Fig. 1B). The relative contents of metabolites represented by colored segments at the corresponding locations are showed in Additional file 10: Table S6. We observed a significant enrichment of metabolites in TM-1 compared to Hai7124 or Shixiya1. Then, we classified these metabolites and found that they included carbohydrates, amino acids, lipids, organic acids and flavonoids. These results indicated that the seeds of G. hirsutum TM-1 may have high levels of these metabolites, and could be used as raw materials for processing and utilization in the future.

The seeds of higher plants can be described as valuable factories capable of converting photosynthetically derived sugars into a variety of storage compounds, such as oils, proteins, sugars, and secondary metabolites (Baud 2018). G. hirsutum is the most widely planted cotton cultivar and makes up the absolute majority of cotton fiber yield and cottonseed byproducts worldwide. Therefore, further nutritional evaluation research on the cottonseeds of TM-1 is needed. There have been many studies and evaluations of primary metabolites in cottonseed, and this study focuses on the evaluation of secondary metabolites proanthocyanidins. Notably, catechin and epicatechin were both detected, catechin significantly accumulated in TM-1, while epicatechin accumulated in Hai7124. Proanthocyanidins are a class of polyphenols produced by the flavonoid pathway, that have important biological functions. Numerous in vivo and in vitro experimental studies have demonstrated that proanthocyanidins are beneficial to human and animal health (Dixon et al. 2005; Gonzalo-Diago et al. 2013). Proanthocyanidins are compounds formed by the polymerization of flavan-3-ol structural unit. And common flavan-3-ol units include catechin and epicatechin. Therefore, we find that the main monomeric forms in TM-1 and Hai7124 are different, which will also lead to the differences in oligomers formed by varying amounts of catechin or epicatechin.

Correlation analysis of catechin and oil-related in core collections of Gossypium

To verify the reliability of the data obtained, we conducted confirmatory studies with different test methods and an increased sample size. A classic chromatographic target HPLC method was used to evaluate the content of catechin, and the results indicated that the catechin content in TM-1 was significantly higher than that in other two cultivars, which is consistent with the above analysis (Fig. 2A). However, interestingly, we found that Shixiya1 had slightly higher values than Hai7124, which is different from the GC-TOF/MS results. Similar results have also been verified in core collections of Gossypium (47 accessions of G. arboreum, 37 accessions of G. barbadense, and 144 accessions of G. hirsutum) (Fig. 2B) (Du et al. 2018; He et al. 2021). Based on a comparative analysis of the detection methods, we thus believed that the acid used in the pretreatment method for HPLC could effectively release bound catechins into free catechins, which could be the potential reason behind this phenomenon. In Arabidopsis, the regulatory network of proanthocyanidins (PAs) in the seed coat is mainly controlled by the TT2 gene. Previous studies have shown that TT2 can suppress fatty acid (FA) biosynthesis, and that the tt2 mutation results in significantly elevated seed oil content and an alteration in FA composition (Chen et al. 2012; Wang et al. 2014). Therefore, the correlation analysis was conducted between catechin and oil-related traits to evaluate their relationship in cottonseed. As indicated in Fig. 2C, catechin content has a negative association with myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), arachidic acid (C20:0) and total fatty acid content in the core collections of G. hirsutum (Pearson, P < 0.001). In addition, the results also showed that there was no correlation between the content of catechin and cyclopropene fatty acid (C19:1), an anti-nutritional factor in cottonseed that is used for feed (Pearson, r = 0.095, P > 0.05). Taken together, this work advances our understanding of PAs functions in seed FA accumulation, and provides technical support for the development of special cotton varieties rich in catechins or high-quality fatty acids in the future. Lastly, further research should be undertaken to investigate the other nutrient components for the comprehensive evaluation of cottonseed.

Fig. 2
figure 2

Assessment of differential catechins content and correlation analysis in core collections of Gossypium. A The contents of catechin in Shixiya1, Hai7124 and TM-1 measured by HPLC. B Distribution of the catechin content in three cotton species (G. arboreum, G. barbadense, and G. hirsutum) C Frequency distribution of phenotypic variation of catechin and oil-related traits, and correlation analysis in core collections of G. hirsutum. Myristic acid (C14:0); Palmitic acid (C16:0); Stearic acid (C18:0); Oleic acid (C18:1); Linoleic acid (C18:2); Cyclopropene fatty acid (C19:1); Linolenic acid (C18:3); Arachidic acid (C20:0) and Total fatty acids (Total FAs). Columns show the mean ± SD with three replicates. *, **, *** represent P < 0.05, P < 0.01, and P < 0.001, respectively


In this work, the differential metabolites of three representative cultivars were identified and quantified using untargeted GC-TOF/MS to compare different cotton species. Catechin, as a bioactive component, was chosen to further verify the test results by classical HPLC analysis. The results revealed the reliability of the GC-TOF/MS analysis, as well as that catechin content has a negative association with that of myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, arachidic acid, and total fatty acids. The findings of the present study provide a theoretical basis for the rational utilization of cottonseeds in the future.


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The authors thank Prof. DU Xiongming from Institute of Cotton Research, CAAS for supplying the cotton germplasm resources.


This work was supported by China Agriculture Research System (CARS-15-27) and Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences.

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Authors and Affiliations



Ma L and Tian XQ designed the experiments. Tian XQ, Chen YB and Xu SJ carried out the experiments with the help of Dong RD, and Wang YW. Fang D collected the samples and pretreatment; Ma L managed the main data analysis and wrote the manuscript. Ma L and Peng J conceived the project. All authors have read and approved the final manuscript.

Corresponding authors

Correspondence to MA Lei, PENG Jun or TIAN Xinquan.

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The authors declare that they have no competing interests.

Supplementary Information

Additional file 1: Fig. S1.

The typical total ion chromatogram (TIC) from all cottonseed samples by mass spectrometry detection.

Additional file 2: Table S1.

The total peaks were extracted from cottonseeds by using GC-TOF/MS analysis.

Additional file 3: Fig. S2.

The classification of all identified metabolites.

Additional file 4: Table S2.

The information of 263 identified metabolites.

Additional file 5: Fig. S3.

The PCA analysis of the metabolites based on GC-TOF/MS in Shixiya1, Hai7124 and TM-1. The X axis and Y axis represent the first principal component (PC1) and the second principal component (PC2), respectively.

Additional file 6: Fig. S4.

Volcano plot of differential metabolites among three cultivars. A Shixiya1 vs. Hai7124. B Shixiya1 vs. TM-1. C Hai7124 vs. TM-1. Significantly up- and down-regulated metabolites are indicated in red and blue.

Additional file 7: Table S3.

The differential metabolites between Shixiya1 and Hai7124 obtained from OPLS-DA. Up-regulated and down-regulated metabolites are shown in red and blue, respectively.

Additional file 8: Table S4.

The differential metabolites between Shixiya1 and TM-1 obtained from OPLS-DA. Up-regulated and down-regulated metabolites are shown in red and blue, respectively.

Additional file 9: Table S5.

The differential metabolites between Hai7124 and TM-1 obtained from OPLS-DA. Up-regulated and down-regulated metabolites are shown in red and blue, respectively.

Additional file 10: Table S6.

The information of differential metabolites in the three cotton varieties obtained from one-way ANOVA.

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MA, L., CHEN, Y., XU, S. et al. Metabolic profile analysis based on GC-TOF/MS and HPLC reveals the negative correlation between catechins and fatty acids in the cottonseed of Gossypium hirsutum. J Cotton Res 5, 17 (2022).

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  • Cotton
  • Metabolic profile
  • Catechin
  • Fatty acid