Figures (8)  Tables (4)

    • Figure 1. 

      Geographical distribution of lotus accessions collected in the Wuhan National Lotus Germplasm Bank at the Wuhan Botanical Garden of the Chinese Academy of Sciences. The different colored dots indicate cultivars belonging to different categories.

    • Figure 2. 

      1-Benzylisoquinoline alkaloids reported in Nelumbo nucifera.

    • Figure 3. 

      Aporphine-type alkaloids isolated from Nelumbo nucifera.

    • Figure 4. 

      Bis- and tri- benzylisoquinoline alkaloids isolated from Nelumbo nucifera.

    • Figure 5. 

      BIA profiles in the leaf and plumule during development. (a) Lotus leaves showing the seven defined developmental stages. (b) BIA profiles in the seven leaf developmental stages. (c) Lotus plumules showing different developmental stages. (d) BIA content in the plumule at different developmental stages. S, leaf developmental stages; DAP, days post pollination. The figure is modified from previous reports [ 11, 24] .

    • Figure 6. 

      Analytical mass spectra of lotus leaf BIAs and their fragmentation pathways. The chemical structures and the corresponding peak numbers 1 to 5 represent signals for N-nornuciferine, O-nornuciferine, anonaine, nuciferine, and Roemerine, respectively. Figures are modified from Luo et al. [ 65 ] and Chen et al. [ 23 ].

    • Figure 7. 

      Analytical mass spectra of lotus leaf BIAs and their fragmentation pathways. The chemical structures and the corresponding peak numbers 6 to 8 represent signals for liensinine, isoliensinine, and neferine, respectively. Figures are modified from Chen et al. [ 64 ], Deng et al. [ 11 ], and Lai et al. [ 66 ].

    • Figure 8. 

      Proposed BIAs biosynthetic pathway in lotus. Steps marked with red, green, and purple background represent the common reactions for the biosynthesis of (R)-N-Methylcoclaurine, the lotus aporphine branch, and the bis-BIA biosynthesis branch, respectively. All biosynthetic enzymes are shown in red. The (R)-N-Methylcoclaurine is the branch point for aporphine and bis-BIA biosynthesis in lotus. Dotted arrows indicate multiple enzymatic or unknown steps.

    • No. Alkaloid Formula Enantiomer Organ Reference
      1-BENZYLISOQUINOLINE
      1 Lotusine C 19H 24NO 3 + E, S, F [ 25]
      2 Methyl lotusine L
      3 Armepavine C 19H 23NO 3 (−)-R and (+)-S S, F, L [ 21]
      4 Coclaurine C 17H 19NO 3 (+)-R S, L [ 26]
      5 N-norarmepavine C 18H 21NO 3 (+)-R L [ 27]
      6 N-methylisococlaurine C 18H 21NO 3 L [ 16, 28]
      7 N-methylcoclaurine C 18H 21NO 3 (−)-R L, S [ 16]
      8 Isococlaurine C 19H 24NO 3+ L
      9 Methylhigenamine S [ 17]
      10 Norcoclaurine-6- O-glucoside S
      11 Norcoclaurine C 16H 17NO 3 (+)-R and (+)-S S, L [ 26, 29]
      12 Argemexirine S, L
      13 6-demethy-4-methyl- N-methylcoclaurine C 18H 21NO 3 S [ 30]
      14 Nor- O-methylarmepavine C 20H 25NO 3 S [ 30]
      15 4’- N-methylcoclaurine C 19H 23NO 3 L, S [ 17]
      16 4’-methyl coclaurine L, S [ 17]
      17 Bromo methyl armepavine L, S [ 17]
      18 Methoxymethy lisoquinoline L, S [ 17]
      19 Higenamine P [ 31, 32]
      20 Higenamine glucoside P [ 33]
      APORPHINE
      21 Nuciferine C 19H 21NO 2 (−)-R S, F, L [ 34, 35]
      22 N-nornuciferine C 18H 19NO 2 (−)-R S, L [ 26]
      23 Roemerine C 18H 17NO 2 (−)-R S, F, L [ 16, 26, 32]
      24 O-nornuciferine C 18H 19NO 2 (−)-R S, F, L [ 32, 36]
      25 Anonaine C 17H 15NO 2 (−)-R S, L [ 16, 26]
      26 Lirinidine C 18H 19NO 2 (−)-R S, L [ 37]
      27 Nuciferine- N-Methanol F
      28 Nuciferine- N-Acetyl F
      29 Anonaine- N-Acetyl F
      30 Caaverine C 17H 17NO 2 (−)-R S, L [ 20, 38]
      31 Oxidation-nuciferine S, L, F [ 19, 30]
      32 Asimilobine C 17H 17NO 2 (−)-R S, F, L [ 17, 20]
      33 Methyl asimilobine S, L [ 17]
      34 N-methyl asimilobine S, L [ 16, 39]
      35 Roemerine- N-oxide S, L [ 16, 40]
      36 N-methyl asimilobine- N-oxide S, L, F [ 16, 19]
      37 Nuciferine- N-oxide S, L, F [ 16, 19]
      38 Dehydroanonaine C 17H 13NO 2 L [ 16]
      39 Dehydronuciferine C 19H 19NO 2 L [ 16]
      40 Dehydroaporphine C 18H 15NO 2 L [ 41]
      41 Nelumnucine S, L
      42 Dehydroroemerine L [ 16]
      43 Liriodenine C 17H 9NO 3 L [ 16, 40]
      44 7-hydroxydehy dronuciferine C 19H 19NO 3 L [ 16]
      45 Pronuciferine C 19H 21NO 3 (+)-R and (−)-S S, F, L [ 16, 26, 40]
      46 Glaziovine S
      47 Lysicamine C 18H 13NO 3 L [ 38, 42]
      48 Cepharadione L [ 38]
      BISBENZYLISOQUINOLINE
      49 Neferine C 38H 44N 2O 6 1R, 1'S S, F, E [ 17, 31]
      50 Liensinine C 37H 42N 2O 6 1R, 1'R S, F, E [ 35]
      51 Isoliensinine 1R, 1'S S, F, E [ 25]
      52 N-norisoliensinine C 36H 40N 2O 6 S, F, E [ 25]
      53 6-hydroxynorisoliensinine C 36H 40N 2O 6 S, F, E
      54 Methyl neferine S, E [ 10, 17]
      55 Nelumboferine C 36H 40N 2O 6 S, E [ 10, 17]
      56 Negferine C 38H 44N 2O 6 L [ 17, 26]
      57 Nelumborine F [ 17]
      58 Dauricine S, F [ 43]
      TRIBENZYLISOQUINOLINE
      59 Neoliensinine C 63H 70N 3O 10 1R, 1'S, 1''R E [ 44]
      L, Leaf; E, embryo; F, flowers; S, seeds; R, rhizome; LS: leaf sap; NS, not specified.

      Table 1. 

      BIAs detected in the lotus ( Nelumbo nucifera) tissues. Alkaloids are assigned with numbers as shown in Figures 2– 5.

    • Extraction method Content (mg/g dry weight) a Total
      1 2 3 4 5 6 7 8 9 10
      Methanol, reflux 1.76 (100) 1.75 (100) 0.07 (100) 0.63 (100) 0.69 (100) 0.83 (100) 1.45 (100) 5.73 (100) 1.30 (100) 0.75 (100) 14.96 (100)
      50% Methanol, reflux 1.09 (62) 1.35 (77) 0.05 (71) 0.50 (79) 0.61 (88) 0.78 (94) 1.35 (93) 3.79 (66) 0.94 (73) 0.56 (75) 11.02 (74)
      H 2O, reflux 0.24 (14) 0.35 (20) nd. b 0.21 (33) 0.18 (26) 0.38 (45) 0.78 (54) 2.57 (45) 0.66 (51) 0.29 (38) 5.66 (38)
      Methanol, sonication 0.88 (50) 1.11 (64) 0.03 (44) 0.39 (62) 0.33 (48) 0.47 (56) 0.97 (67) 2.77 (48) 0.70 (54) 0.42 (56) 8.07 (54)
      50% Methanol, sonication 0.98 (56) 1.27 (73) 0.04 (58) 0.49 (78) 0.47 (69) 0.80 (96) 1.38 (95) 3.93 (69) 0.97 (75) 0.59 (79) 10.92 (73)
      H 2O, sonication 0.14 (8) 0.21 (12) nd. 0.12 (20) 0.08 (11) 0.25 (30) 0.53 (37) 1.91 (33) 0.48 (37) 0.19 (26) 3.91 (26)
      1. nuciferine; 2. N-nornuciferine; 3. N-methylasimilobine; 4. Asimilobine; 5. Pronuciferine; 6. Armepavine; 7. norarmepavine; 8. N-methylcoclaurine; 9. coclaurine; 10. Norjuziphine. a. relative value (%) against the content obtained by methanol under reflux is given in parentheses. b. less than the quantitation limit.

      Table 2. 

      Extraction efficiency of alkaloids from lotus flower.

    • Solvent systems Mix ratios (v/v) Target BIAs Reference
      Light petroleum (60–90 °C)–ethyl acetate–tetrachloromethane–chloroform–methanol–water 1:1:4:4:6:2 Small scale
      Plumule BIAs       		[ 59]
      Ethyl acetate–tetrachloromethane–chloroform–methanol–water 1:6:4:1 Small scale
      Plumule BIAs       		[ 59]
      n-hexane–ethyl acetate–methanol–water 5:8:4:5
      0.5% NH 4OH       		Plumule BIAs [ 60]
      n-hexane–ethyl acetate–methanol–water 5:5:2:8
      10 mM triethylamine
      5 mM HCl       		Plumule BIAs [ 57]
      Diethyl ether – Na 2HPO 4/NaH 2PO 4 (pH = 7.2 – 7.5) 1:1 Plumule BIAs [ 62]
      Petroleum ether (60–90 °C)–ethyl acetate–methanol–water 5:5:2:8
      10 mM triethylamine
      5 mM HCl       		Leaf BIAs [ 61]
      n-hexane-ethyl acetate-methanol-water-[C 4mim][PF 6] 5:2:2:8:0.1
      10 mM triethylamine
      3 mM HCl       		Whole plant BIAs [ 35]

      Table 3. 

      Major two-phase solvent systems developed for preparative separation of lotus BIAs through counter-current chromatography (CCC) techniques.

    • Peaks T R a (min) Molecular weight m/ z
      [M + H] +       		Major fragment ions Alkaloids
      1* 7.56 281 282 251/219 N-nornuciferine
      2* 9.03 281 282 265/250 O-nornuciferine
      3* 10.17 265 266 249/219 Anonaine
      4* 12.43 295 296 265/250 Nuciferine
      5* 13.39 279 280 249 Roemerine
      6 ∆ 6.61 610 611 503/283/206 Liensinine
      7 ∆ 9.47 610 611 489/297/192 Isoliensinine
      8 ∆ 17.72 624 625 503/297/206 Neferine
      * The retention time (T R a (min) of peaks 1–5 as obtained by Chen et al. [ 23] .
      ∆ The retention time of peaks 6–9 as reported by Chen et al. [ 63] .

      Table 4. 

      Identification of major lotus leaf and plumule alkaloids and their HPLC-MS/MS ion characteristics.