Figures (6)  Tables (2)

    • Figure 1. 

      Classification of all identified metabolites into major groups and subgroups.

    • Figure 2. 

      Annual frequency of articles meeting the inclusion criteria for metabolomic studies in pre-defined leafy vegetables published between 1997 and 2024.

    • Figure 3. 

      Frequency distribution of each included leafy vegetable in the included 147 articles in this review.

    • Figure 4. 

      Distribution and occurrence of secondary metabolite groups across leafy vegetable families. (a) Absolute counts of reported secondary metabolite groups across different families; (b) relative composition (%) of secondary metabolite groups within each family, illustrating proportional distribution independent of total counts; (c) presence-absence heatmap showing whether specific secondary metabolite groups were reported in each family.

    • Figure 5. 

      Heatmap showing relative reporting frequency of metabolite groups across technical factors.

    • Figure 6. 

      Principal component analysis of metabolite reporting patterns across technical factors.

    • Category Sub-category No. of studies
      (n = 147)      		 % of total studies
      Leafy vegetable families Brassicaceae 68 46.26%
      Asteraceae 39 26.53%
      Amaranthaceae 17 11.56%
      Lamiaceae 5 3.40%
      Fabaceae 3 2.04%
      Convolvulaceae 2 1.36%
      Caprifoliaceae 2 1.36%
      Apiaceae 1 0.68%
      Malvaceae 1 0.68%
      Portulacaceae 1 0.68%
      Solanaceae 1 0.68%
      Mixed species 7 4.76%
      Analytical approaches used LC-MS 77 52.38%
      LC-UV 23 15.65%
      GC-MS 14 9.52%
      NMR 8 5.44%
      Combined/multi analytical approaches 25 17.01%
      Primary metabolite major groups reported Amino acids 4 2.72%
      Carbohydrates* 0 0.00%
      Lipids 5 3.40%
      Nucleotides* 0 0.00%
      Organic acids 8 5.44%
      Vitamins 6 4.08%
      Others (nitrogen-containing compounds) 1 0.68%
      Multiple primary metabolite groups reported 54 36.73%
      NA (did not report primary metabolites) 69 46.94%
      Secondary metabolite major groups reported Nitrogen- and sulfur-containing compounds 23 15.65%
      Phenolic compounds 47 31.97%
      Terpenoids 5 3.40%
      Tetrapyrroles** 0 0.00%
      Multiple secondary metabolite groups reported 64 43.54%
      NA (did not report secondary metabolites) 8 5.44%
      Scope of analysis Metabolite screening (without any factor) 14 9.52%
      Metabolite screening (across intrinsic factors) 60 40.82%
      Metabolite screening (across environmental factors) 73 49.66%
      The symbol * indicates that this group was always reported together with other primary metabolite groups in the included studies.
      The symbol ** indicates that this group was always reported together with other secondary metabolite groups in the included studies.

      Table 1. 

      Sub-category, number of studies, and percentage of total studies derived from each category of 147 included studies in this systematic review.

    • Bioactive effect Leafy vegetable(s) Bioassay
      Anti-Alzheimer's Goosefoot leaves[75] In vitro enzymatic assay: (1) Anticholinesterase assay[75]
      Antibacterial Lettuce and red lettuce[104], spinach[77] In vitro cell-based assay: (1) Staphylococcus aureus DSM 346 strain[104]
      (2) Strain H. pylori 51 (HPKTCC B0006) culture assay[77]
      Anti-cancer Cowpea leaves[101], endive[24],
      perilla leaves[73],
      red lettuce[105]      		 In vitro cell-based assay: (1) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay[24,105]
      (2) Cytotoxicity on skeletal muscle cell line[101]
      (3) Water-soluble tetrazolium Salt-1 (WST-1) assay (anti-prostate cancer activity)[73]
      (4) Trypan blue (TB) exclusion assay[105]
      In silico assay: (1) Network pharmacology[73]
      Antidiabetic Cosmos[106], cowpea leaves[101], ebolo[102], kangkung[66,67],
      lettuce[6],
      water mimosa[70]      		 In vitro biochemical assay: (1) Dipeptidyl peptidase-4 (DPP-4) inhibitory activity assay[6]
      In vitro cell-based assay: (1) GLP-1 secretory response in pGIP/Neo STC-1 cells[6]
      In vitro enzymatic assay: (1) Alpha-glucosidase inhibition assay[6,66,67,70,101,106]
      (2) Alpha-amylase inhibition assay[101,102]
      In silico assay: (1) Network pharmacology[102]
      Anti-genotoxicity Turnip greens[103] In vitro cell-based assay: (1) Trypan blue (TB) exclusion assay[103]
      In vivo assay: (1) Somatic mutation and recombination test (SMART)[103]
      Anti-inflammatory Endive[24], fennel[107] In vitro enzymatic assay: (1) Cyclooxygenase (COX) inhibitor screening assay[107]
      In vitro cell-based assay: (1) Enzyme-linked immunosorbent assay (ELISA)[24]
      Anti-microbial Chinese cabbage[95] In vitro cell-based assay: (1) Pathogens: Bacillus cereus (KCTC 3624), Escherichia coli (KCTC 1682), Pseudomonas aeruginosa (KCCM 11803), Staphylococcus aureus (KCTC 3881), and Salmonella paratyphi C (KCCM 41577) culture assay[95]
      Antioxidant Abyssinian cabbage[68], amaranth[108], blackjack[78], Chinese cabbage[85,89,95,109], choisum[82], cosmos[80], cowpea leaves[101],
      curly kale[2,3], endive[24], goosefoot leaves[75],
      jew mallow[110], kangkung[66], lettuce[4,5,64,86,104,109,111], pak-choi[91], perilla leaves[65], purslane[69], radish leaves[109], red lettuce[4,5,64,86,104,105], spinach[78,88,109,112,113], water mimosa[70], watercress[84]      		 In vitro chemical-based assay: (1) 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay[2,4,5,24,66,69,70,75,80,82,84,87−89,91,95,105,108]
      (2) 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay[2,3,5,24,64,65,78,86,89,104,105,108,111]
      (3) Ferric reducing antioxidant power (FRAP) assay[4,78,84,85,89,91,101,105,108]
      (4) Oxygen radical absorbance capacity[114] assay[78,82]
      (5) Hydrogen peroxide (H2O2) assay[3,4]
      (6) Cupric reducing antioxidant capacity (CUPRAC) assay[75]
      (7) Linoleic acid peroxidation inhibition assay[110]
      (8) Lipid peroxidation (LPO) inhibition assay[65]
      (9) Hydrophilic oxygen radical absorbance capacity (H-ORAC) assay[113]
      (10) Hydrogen peroxide–horseradish peroxidase (H2O2-HRP) assay[24]
      In vitro cell-based assay: (1) 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay[78]
      (2) Radioimmunoprecipitation assay[68]
      (3) OxiSelect cellular antioxidant activity assay[24]
      In vitro enzymatic assay: (1) Superoxide dismutase (SOD) assay[24,68,91,109,112]
      (2) Catalase (CAT) assay[24,68,109,112]
      (3) Peroxidase (POD) assay[109]
      (4) Ascorbate peroxidase (APX) assay[112]

      Table 2. 

      Bioactive effects reported in various leafy vegetables and corresponding validation methods.