Identification of Compounds with Potential Therapeutic Uses from Sweet Pepper (Capsicum annuum L.) Fruits and Their Modulation by Nitric Oxide (NO)

Abstract

Plant species are precursors of a wide variety of secondary metabolites that, besides being useful for themselves, can also be used by humans for their consumption and economic benefit. Pepper (Capsicum annuum L.) fruit is not only a common food and spice source, it also stands out for containing high amounts of antioxidants (such as vitamins C and A), polyphenols and capsaicinoids. Particular attention has been paid to capsaicin, whose anti-inflammatory, antiproliferative and analgesic activities have been reported in the literature. Due to the potential interest in pepper metabolites for human use, in this project, we carried out an investigation to identify new bioactive compounds of this crop. To achieve this, we applied a metabolomic approach, using an HPLC (high-performance liquid chromatography) separative technique coupled to metabolite identification by high resolution mass spectrometry (HRMS). After chromatographic analysis and data processing against metabolic databases, 12 differential bioactive compounds were identified in sweet pepper fruits, including quercetin and its derivatives, L-tryptophan, phytosphingosin, FAD, gingerglycolipid A, tetrahydropentoxylin, blumenol C glucoside, colnelenic acid and capsoside A. The abundance of these metabolites varied depending on the ripening stage of the fruits, either immature green or ripe red. We also studied the variation of these 12 metabolites upon treatment with exogenous nitric oxide (NO), a free radical gas involved in a good number of physiological processes in higher plants such as germination, growth, flowering, senescence, and fruit ripening, among others. Overall, it was found that the content of the analyzed metabolites depended on the ripening stage and on the presence of NO. The metabolic pattern followed by quercetin and its derivatives, as a consequence of the ripening stage and NO treatment, was also corroborated by transcriptomic analysis of genes involved in the synthesis of these compounds. This opens new research perspectives on the pepper fruit's bioactive compounds with nutraceutical potentiality, where biotechnological strategies can be applied for optimizing the level of these beneficial compounds.

Keywords: HPLC-HRMS; L-tryptophan; fruit ripening; gingerglycolipid A; melatonin; nitric oxide; phytosphingosin; quercetin; transcriptomics.

Figure 1

Principal Component Analysis (PCA) of metabolites contained in sweet pepper fruits at different ripening stage and as a consequence of exogenous treatment with NO. A clear grouping of differential metabolites from analysed samples could be observed. The Quality Control (QC) of samples positioned around the same range, indicating their analytical stability. PV, green fruits. PR, red fruits. PE + NO, pepper fruits treated with 5 ppm NO. PE − NO, pepper fruits not treated with NO.

Figure 2

Molecular structure and MS/MS spectrum of quercetin from sweet pepper fruits with the highest intensity peak (303.0506 Da) corresponding to the aglycone form of the metabolite (M) plus one proton [M + H].

Figure 3

Transcriptomic analysis of the biosynthetic pathway of quercetin and derivatives from sweet pepper fruits. On the left, a heatmap indicating the differentially expressed genes depending on the ripening stage of pepper fruits (red (PR) vs. green (PV)) is depicted with FDR ≤ 0.05. On the right, a heatmap indicating the differentially expressed genes depending on the NO treatment of pepper fruits (treated (PE + NO) vs. untreated (PE − NO)) is depicted with FDR ≤ 0.05. PAL, phenylalanine ammonia-lyase; C4H, trans-cinnamate 4-monooxygenase; 4CL, 4-coumarate-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-dioxygenase; F3′5′H, flavanoid 3′,5′-hydroxylase; F3′H, flavonoid 3′-monooxygenase; DFR, dihydroflavonol 4-reductase; ANS, leucocyanidin dioxygenase; FLS, flavonol synthase; 3GT, flavonol 3-o-glucosyltransferase; FGT, flavonol-3-O-glucoside L-rhamnosyltransferase; UGT, UDP-glycosyltransferase. Asterisks indicate genes which are differentially expressed in the experimental conditions.

Figure 4

Molecular structure and MS/MS spectrum of L-tryptophan (205.0983 Da) from sweet pepper fruits. The loss of the amino group (-NH₂) renders the peak at 188.0712 Da, besides the peak (146.0598 Da) corresponding to carboxyl group (-COOH). M, metabolite.

Figure 5

Molecular structure and MS/MS spectrum of tetrahydropentoxyline from sweet pepper fruits. The loss of the 18-Da fragment corresponds to H₂O group. M, metabolite.

Figure 6

Molecular structure and MS/MS spectrum of the putative phytosphingosine from pepper fruits. M, metabolite.

Figure 7

Molecular structure and MS/MS spectrum of FAD from sweet pepper fruits. The peak with the highest intensity (348.0704 Da) corresponds to the adenine nucleotide (aden), whereas that of 430.1014 Da is a consequence of flavin nucleotide (fla).

Figure 8

Molecular structure and MS/MS spectrum of blumenol C glucoside from sweet pepper fruits. The peak with 193.1584 Da is generated after the loss of the glucose (glu) residue. M, metabolite.

Figure 9

Molecular structure and MS/MS spectrum of colnelenic acid from sweet pepper fruits. The peak with the highest intensity (275.2011 Da) was obtained after a decrease of 18 Da as consequence of a H₂O loss. M, metabolite.

Figure 10

Molecular structure and MS/MS spectrum of gingeglycolipid A [M + NH₄] from sweet pepper fruits. The peak at 515.3214 Da corresponds to the ion resulting from the loss of the first galactose (gal), and the peak at 335.2575 was generated after the second galactose is lost. M, metabolite.

Figure 11

Molecular structure and MS/MS spectrum of capsoside A from sweet pepper fruits. The peak at 497.3096 Da corresponds to the ion resulting from the loss of the first galactose (gal), and the peak at 353.2682 was generated after the second galactose was lost.

Figure 12

Experimental design to set four groups of pepper fruits for metabolomic analysis. PV, immature green. PR, ripe red, PE + NO, breaking point fruits incubated with 5 ppm NO for 1 h and further storage for 3 d at room temperature (RT). PE − NO, breaking point fruits not treated with NO for 1 h and further storage for 3 d at RT. (Reproduced with permission from González-Gordo et al. [122]).