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Fig. 7. Forrestline F in Diterpenoid alkaloids from Delphinium forrestii var. viride and their anti-inflammation activity

Fig. 7. Forrestline F decreased LPS-induced ROS generation and mitochondrial membrane potential loss. (A) Cells were cultured with LPS for 12 h after treated with forrestline F (5, 10 and 20 μM) for 3 h, except the control group and the group only treated with LPS. After cells were incubated with DCFH-DA (10 μM) for 20 min, images were acquired with a fluorescence microscope (scale bar: 200 μM). (B) The cells were processed in the same way as above, and collected to flow tube and monitored by the flow cytometry. (C) Statistical analysis of the fluorescence intensity of DCFH-DA. (D–E) For the measurement of MMP, JC-1 fluorescent dyes were loaded for 20 min and images were acquired with flow cytometry. The values are presented as mean SD of three independent experiments, n 3. ###p <0.001, vs. ± = the control group; **p <0.01, ***p <0.001, vs. LPS-stimulated group.

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Fig. 6. Forrestline F in Diterpenoid alkaloids from Delphinium forrestii var. viride and their anti-inflammation activity

Fig. 6. Forrestline F reduced LPS-induced inflammatory activity through the NF-κB/MAPK pathways and blocked the LPS-induced nuclear translocation of NF-κB. (A–B) The cells were exposed to forrestline F (5, 10 and 20 μM) for 3 h and cultured with LPS for 24 h, except for the control group. The protein expression of NF-κB pathway was determined by western blotting and the quantification of protein expression was performed by Image J. (C) The inhibitory effect of forrestline F on the nuclear translocation of NF-κB p65 induced by LPS via confocal laser scanning microscopy, in conjunction with nuclear DAPI stain (scale bar: 50 μM). (D–E) Protein expression of MAPK pathway, including p-ERK1/2 and p-p38 and p-JNK1/2, was measured by western blotting. The values are presented as mean ± SD of three independent experiments, n 3 ###p <0.001, vs. the control group; ***p <0.001, vs. LPS-stimulated group.

Fig. 6 in Isolation, identification, and activity evaluation of diterpenoid alkaloids from Aconitum sinomontanum

Fig. 6. The effect on the expression of Nrf2-regulated gene by compounds in PC12 cells transfected with ARE-luciferase expression plasmid, measured with a Firefly Luciferase Reporter Gene Assay Kit. Data are presented as the means ± SD from three independent tests.

Fig. 2 in Isolation, identification, and activity evaluation of diterpenoid alkaloids from Aconitum sinomontanum

Fig. 2. (a) HMBC (→) and 1H–1H COSY (bold lines) correlations of compound 1, (b) ORTEP drawing of compound 1.

Fig. 5. Compounds 2 and 17 in Isolation, identification, and activity evaluation of diterpenoid alkaloids from Aconitum sinomontanum

Fig. 5. Compounds 2 and 17 exhibited potent inhibitory effects on the capsaicin mediated activation of TRPV1 channels expressed in HEK-293 cells. (A) Inhibition rate by compounds on the capsaicin mediated activation of TRPV1 channels expressed in HEK-293 cells. Compounds 1, 2, and 17 were evaluated at the concentration of 10 μM, the other compounds were evaluated at the concentration of 100 μM. (B) Activation of TRPV1 by capsaicin (0.5 μM), and inhibition of TRPV1 current by compound 2. Data are presented as the means ± SEM from three independent tests.

Fig. 4 in Isolation, identification, and activity evaluation of diterpenoid alkaloids from Aconitum sinomontanum

Fig. 4. (a) HMBC (→) and 1 H– 1 H COSY (bold lines) correlations of compounds 10 and 11, (b) key NOESY () correlations of compounds 10 and 11.

Fig. 4 in Isolation and structure characterization of cytotoxic alkaloids from Micromelum integerrimum

Fig. 4. The calculated and experimental ECD spectra of compounds 2a and 2b in MeOH (a); the experimental ECD spectra of compounds 2a and 3a in MeOH (b).

Fig. 3 in Isolation and structure characterization of cytotoxic alkaloids from Micromelum integerrimum

Fig. 3. The chiral HPLC analysis (a) and calculated and experimental ECD spectra (b) of compounds 1a and 1b in MeOH.

Fig. 5 in Isolation and structure characterization of cytotoxic alkaloids from Micromelum integerrimum

Fig. 5. The calculated and experimental ECD spectra of compound 6 in MeOH (a); the experimental ECD spectra of compounds 4a, 5a, and 6 in MeOH (b).

Fig. 4 in Tropane alkaloids from the stem bark of Erythroxylum bezerrae

Fig. 4. Asymmetric unit of the crystalline structure only one of the two non-equivalent conformations of 3.

Fig. 1 in A single residue determines substrate preference in benzylisoquinoline alkaloid N-methyltransferases

Fig. 1. Involvement of N-methyltransferases in BIA biosynthesis. Coclaurine N-methyltransferase (CNMT) acts in the core BIA pathway and converts an unmethylated secondary amine to a mono-methylated tertiary amine. Reticuline N-methyltransferase (RNMT) and Pavine N-methyltransferase (PavNMT) act in several branch pathways and primarily convert mono-methylated tertiary amines into a di-methylated quaternary amines. Tetrahydroprotoberberine N-methyltransferase (TNMT) acts in other branch pathways and converts a bicyclic unmethylated tertiary amine into a quaternary amine. † denotes activities that have been demonstrated in vitro but not conclusively associated with the enzyme via in planta experimental evidence (e.g. gene silencing).

Fig. 5 in A single residue determines substrate preference in benzylisoquinoline alkaloid N-methyltransferases

Fig. 5. Sequence-based prediction and in vivo screening of putative CNMT and RNMT enzymes. (A) The identity of residue 204 (E, glutamic acid; G, glycine) was used to predict CNMT- or RNMT-like function, respectively. (B) Overall amino acid sequence identity does not distinguish CNMT- or RNMT-like enzymes from each other (Supplementary Dataset 5). (C) Formation of N-methylated products following supplementation of yeast cultures with (S)-coclaurine (white bars), (S)-N- methylcoclaurine (black bars) and (S)-reticuline (grey bars). For each substrate, the culture with the greatest mean product concentration normalized to optical density was set to 100% and all other values were scaled accordingly (30.7μM OD−1 (S)-N-methylcoclaurine, HCANMT1; 3.4μM OD−1 (S)-N,N-dimethylcoclaurine, SCANMT1; 2.0μM OD−1 (S)-tembetarine, SCANMT1). Error bars represent standard deviation of four replicate cultures. Western blot analysis of NMT expression is show in Supplementary Fig. 6.

Fig. 3 in A single residue determines substrate preference in benzylisoquinoline alkaloid N-methyltransferases

Fig. 3. Enzymatic activities of wild-type CNMT, wild-type RNMT and reciprocal 204 mutants. (A) Activity with canonical CNMT substrate (S)-coclaurine. Black bars indicate mono-methylated product (S)-N-methylcoclaurine and grey bars indicate di-methylated product (S)-N,N-dimethylcoclaurine. (B) Activity with tertiary amine substrate (S)-N-methylcoclaurine. (C) Activity with canonical RNMT substrate (S)-reticuline. Products were identified and quantified by LC-MS/MS. Error bars represent standard deviation of three replicates. WT denotes results corresponding to the wild-type enzyme.

Fig. 2 in A single residue determines substrate preference in benzylisoquinoline alkaloid N-methyltransferases

Fig. 2. Analysis of key active site residues in BIA N-methyltransferases. (A) Primary sequence alignment of functionally validated BIA NMTs showing that residue 204 is conserved within BIA NMT subtypes (i.e. E in CNMTs and TNMTs, G in RNMTs, A in PavNMTs) whereas residues previously suggested to form a catalytic dyad (E207 and H208) are universally conserved. (B, C) Docking of RNMT substrate (S)-reticuline (cyan) into the active site of (B) CjCNMT (PDB 6GKV) and (C) PsRNMT (homology model) reveals a steric clash between the N-methyl group and residue E204 but not G204. S-Adenosylhomocysteine is shown in pink. Spheres represent Van der Waals radii and the distance between key atoms is given in angstroms.

Fig. 4 in A single residue determines substrate preference in benzylisoquinoline alkaloid N-methyltransferases

Fig. 4. Superimposition of residue 204 identity on a maximum-likelihood phylogenetic analysis of N-methyltransferase gene candidates. The cladogram was adapted from that deposited in DRYAD by Hagel et al. (2015b) (https://doi.org/10.5061/dryad.bh276/18) and the corresponding sequences are described in Supplementary Dataset 4. CjCNMT was not included in the original analysis but is most similar to TfCNMT (86% amino acid identity). Key nodes are labelled with support values representing the number of 1000 bootstrapped trees in which the associated taxa clustered together. Putative BIA NMT sequences were aligned by Clustal Omega and the identity of the residue corresponding to E204 in CjCNMT was mapped onto each taxa (glutamic acid, green; glycine, purple; alanine, cyan; glutamine, yellow; serine; orange). BIA NMTs for which in vitro functional characterization data was available are indicated with black arrows and those subjected to yeast-based in vivo characterization in this work are indicated with white arrows. Prior to this report, sequence-based prediction of BIA NMT sub-functionalization was reliable for TNMT-like enzymes only due to their clustering as a single well-defined clade. CNMT-, RNMT- and PavNMT-like sequences are distributed amongst several less well-defined clades.

Fig. 7 in HRESIMS-guided isolation of aspidosperma-scandine type bisindole alkaloids from Melodinus cochinchinensis and their anti-inflammatory and cytotoxic activities

Fig. 7. Effect of BIAs 1 and 6 on LPS-stimulated NO (A), IL-6 (B) and TNF-α (C) production in RAW 264.7 macrophages. DXM was used as a positive control at 30 μM concentration. The values are presented as mean SD of triplicate replicates. Statically significant difference was determined by ANOVA and Tukey test (#p <0.05, ± compared with control group, *p<0.05, compared with model group).

Fig. 1 in HRESIMS-guided isolation of aspidosperma-scandine type bisindole alkaloids from Melodinus cochinchinensis and their anti-inflammatory and cytotoxic activities

Fig. 1. (A) Total ion chromatogram of the extract of M. cochinchinensis. (B) UV absorption bands of compounds 1–3. (C) HRESIMS data of melokhanines KM (1–3).

Fig. 6 in HRESIMS-guided isolation of aspidosperma-scandine type bisindole alkaloids from Melodinus cochinchinensis and their anti-inflammatory and cytotoxic activities

Fig. 6. Key HMBC (single arrows in red), 1 H– 1 H COSY (bold in blue) and ROESY (double-headed arrows in red) correlations of BIA 3. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3 in HRESIMS-guided isolation of aspidosperma-scandine type bisindole alkaloids from Melodinus cochinchinensis and their anti-inflammatory and cytotoxic activities

Fig. 3. Key HMBC (single arrows in red), 1H–1H COSY (bold in blue) and ROESY (double-headed arrows in red) correlations of BIA 1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5 in HRESIMS-guided isolation of aspidosperma-scandine type bisindole alkaloids from Melodinus cochinchinensis and their anti-inflammatory and cytotoxic activities

Fig. 5. Key HMBC (single arrows in red), 1H–1H COSY (bold in blue) and ROESY (double-headed arrows in red) correlations of BIA 2. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)