Lotus (<i>Nelumbo nucifera</i>) benzylisoquinoline alkaloids: advances in chemical profiling, extraction methods, pharmacological activities, and biosynthetic elucidation

Xue Wei; Minghua Zhang; Mei Yang; Collins Ogutu; Jing Li; Xianbao Deng; Xue Wei; Minghua Zhang; Mei Yang; Collins Ogutu; Jing Li; Xianbao Deng

doi:10.48130/vegres-0024-0004

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	C₁₉H₂₄NO₃⁺		E, S, F	[25]
2	Methyl lotusine			L
3	Armepavine	C₁₉H₂₃NO₃	(−)-R and (+)-S	S, F, L	[21]
4	Coclaurine	C₁₇H₁₉NO₃	(+)-R	S, L	[26]
5	N-norarmepavine	C₁₈H₂₁NO₃	(+)-R	L	[27]
6	N-methylisococlaurine	C₁₈H₂₁NO₃		L	[16,28]
7	N-methylcoclaurine	C₁₈H₂₁NO₃	(−)-R	L, S	[16]
8	Isococlaurine	C₁₉H₂₄NO₃+		L
9	Methylhigenamine			S	[17]
10	Norcoclaurine-6-O-glucoside			S
11	Norcoclaurine	C₁₆H₁₇NO₃	(+)-R and (+)-S	S, L	[26,29]
12	Argemexirine			S, L
13	6-demethy-4-methyl-N-methylcoclaurine	C₁₈H₂₁NO₃		S	[30]
14	Nor-O-methylarmepavine	C₂₀H₂₅NO₃		S	[30]
15	4’-N-methylcoclaurine	C₁₉H₂₃NO₃		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	C₁₉H₂₁NO₂	(−)-R	S, F, L	[34,35]
22	N-nornuciferine	C₁₈H₁₉NO₂	(−)-R	S, L	[26]
23	Roemerine	C₁₈H₁₇NO₂	(−)-R	S, F, L	[16,26,32]
24	O-nornuciferine	C₁₈H₁₉NO₂	(−)-R	S, F, L	[32,36]
25	Anonaine	C₁₇H₁₅NO₂	(−)-R	S, L	[16,26]
26	Lirinidine	C₁₈H₁₉NO₂	(−)-R	S, L	[37]
27	Nuciferine-N-Methanol			F
28	Nuciferine-N-Acetyl			F
29	Anonaine-N-Acetyl			F
30	Caaverine	C₁₇H₁₇NO₂	(−)-R	S, L	[20,38]
31	Oxidation-nuciferine			S, L, F	[19,30]
32	Asimilobine	C₁₇H₁₇NO₂	(−)-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	C₁₇H₁₃NO₂		L	[16]
39	Dehydronuciferine	C₁₉H₁₉NO₂		L	[16]
40	Dehydroaporphine	C₁₈H₁₅NO₂		L	[41]
41	Nelumnucine			S, L
42	Dehydroroemerine			L	[16]
43	Liriodenine	C₁₇H₉NO₃		L	[16,40]
44	7-hydroxydehy dronuciferine	C₁₉H₁₉NO₃		L	[16]
45	Pronuciferine	C₁₉H₂₁NO₃	(+)-R and (−)-S	S, F, L	[16,26,40]
46	Glaziovine			S
47	Lysicamine	C₁₈H₁₃NO₃		L	[38,42]
48	Cepharadione			L	[38]
BISBENZYLISOQUINOLINE
49	Neferine	C₃₈H₄₄N₂O₆	1R, 1'S	S, F, E	[17,31]
50	Liensinine	C₃₇H₄₂N₂O₆	1R, 1'R	S, F, E	[35]
51	Isoliensinine		1R, 1'S	S, F, E	[25]
52	N-norisoliensinine	C₃₆H₄₀N₂O₆		S, F, E	[25]
53	6-hydroxynorisoliensinine	C₃₆H₄₀N₂O₆		S, F, E
54	Methyl neferine			S, E	[10,17]
55	Nelumboferine	C₃₆H₄₀N₂O₆		S, E	[10,17]
56	Negferine	C₃₈H₄₄N₂O₆		L	[17,26]
57	Nelumborine			F	[17]
58	Dauricine			S, F	[43]
TRIBENZYLISOQUINOLINE
59	Neoliensinine	C₆₃H₇₀N₃O₁₀	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
Extraction method	1	2	3	4	5	6	7	8	9	10	Total
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)
H₂O, 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)
H₂O, 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% NH₄OH	Plumule BIAs	[60]
n-hexane–ethyl acetate–methanol–water	5:5:2:8 10 mM triethylamine 5 mM HCl	Plumule BIAs	[57]
Diethyl ether – Na₂HPO₄/NaH₂PO₄ (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-[C₄mim][PF₆]	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.