OBJECTIVE: To evaluate the anti-hepatocellular carcinoma (HCC) activity of total alkaloids from Gelsemium elegans Benth. (TAG) in vivo and in vitro and to elucidate their potential mechanisms of action through transcriptomic analysis. METHODS: TAG extraction was conducted, and the primary components were quantified using high-performance liquid chromatography (HPLC). The effects of TAG (100, 150, and 200 µg/mL) on various tumor cells, including SMMC-7721, HepG2, H22, CAL27, MCF7, HT29, and HCT116, were assessed. Effects of TAG on HCC proliferation and apoptosis were detected by colony formation assays and cell stainings. Caspase-3, Bcl-2, and Bax protein levels were detected by Western blotting. In vivo, a tumor xenograft model was developed using H22 cells. Totally 40 Kunming mice were randomly assigned to model, cyclophosphamide (20 mg/kg), TAG low-dose (TAG-L, 0.5 mg/kg), and TAG high-dose (TAG-H, 1 mg/kg) groups, with 10 mice in each group. Tumor volume, body weight, and tumor weight were recorded and compared during 14-day treatment. Immune organ index were calculated. Tissue changes were oberseved by hematoxylin and eosin staining and immunohistochemistry. Additionally, transcriptomic and metabolomic analyses, as well as quatitative real-time polymerase chain reaction (RT-qPCR), were performed to detect mRNA and metabolite expressions. RESULTS: HPLC successfully identified the components of TAG extraction. Live cell imaging and analysis, along with cell viability assays, demonstrated that TAG inhibited the proliferation of SMMC-7721, HepG2, H22, CAL27, MCF7, HT29, and HCT116 cells. Colony formation assays, Hoechst 33258 staining, Rhodamine 123 staining, and Western blotting revealed that TAG not only inhibited HCC proliferation but also promoted apoptosis (P<0.05). In vivo experiments showed that TAG inhibited the growth of solid tumors in HCC in mice (P<0.05). Transcriptomic analysis and RT-qPCR indicated that the inhibition of HCC by TAG was associated with the regulation of the key gene CXCL13. CONCLUSION TAG inhibits HCC both in vivo and in vitro, with its inhibitory effect linked to the regulation of the key gene CXCL13.
Animals ; Apoptosis/drug effects* ; Liver Neoplasms/genetics* ; Carcinoma, Hepatocellular/genetics* ; Humans ; Alkaloids/therapeutic use* ; Gelsemium/chemistry* ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Mice ; Xenograft Model Antitumor Assays

Gelsemium elegans, a vine plant of Loganiaceae, has both medicinal and forage values. However, it is susceptible to low temperatures during growth. Exploring low temperature response genes is of great significance for cold resistance breeding of G. elegans. Ethylene response factors (ERFs) are the transcription factors of the AP2/ERF superfamily and play a crucial role in plant stress response. In this study, based on the unigene GeERF involved in the response to low temperature stress in the transcriptome of G. elegans, a full-length cDNA sequence of the transcription factor GeERF4B-1 was cloned from the leaves of G. elegans by reverse transcription-polymerase chain reaction (RT-PCR). Bioinformatics analysis showed that GeERF4B-1 had an open reading frame of 759 bp, encoding a protein composed of 252 amino acid residues and with a relative molecular weight of 27 kDa. The deduced protein was predicted to be an unstable, alkaline, and hydrophilic protein. The phylogenetic tree showed that GeERF4B-1 was in the same clade as the B-4 subfamily of the ERF family. The results of the subcellular localization experiment revealed that GeERF4B-1 was located in the nucleus. Real time quantitative PCR (RT-qPCR) analysis indicated that GeERF4B-1 was expressed in the root, stem, and leaf of G. elegans, with the highest expression level in the root. Compared with the control, the treatments with a low temperature (4 ℃), methyl jasmonate (MeJA), and abscisic acid (ABA) up-regulated the expression level of GeERF4B-1, which reached the peak at 24-48 h. This result revealed that GeERF4B-1 actively responded to low temperature, MeJA, and ABA stresses. However, both sodium chloride (NaCl) and drought treatments down-regulated the expression of GeERF4B-1. In addition, a prokaryotic expression vector of GeERF4B-1 was constructed, and a fusion protein of approximately 52 kDa was yielded after induced expression. The results of the plate stress assay showed that compared with the control, the prokaryotic strain expressing GeERF4B-1 demonstrated enhanced tolerance to low temperatures and sensitivity to salt and mannitol stresses. This study provides theoretical references and potential genetic resources for breeding G. elegans varieties with stress resistance.
Transcription Factors/metabolism* ; Plant Proteins/metabolism* ; Gelsemium/metabolism* ; Acetates/pharmacology* ; Gene Expression Regulation, Plant ; Phylogeny ; Cold Temperature ; Amino Acid Sequence ; Cyclopentanes/metabolism* ; Oxylipins/metabolism* ; Stress, Physiological/genetics* ; Abscisic Acid/metabolism* ; Cloning, Molecular

Gelsemium elegans is a traditional Chinese herb of medicinal importance, with indole terpene alkaloids as its main active components. To study the expression of the most suitable housekeeping reference genes in G. elegans, the root bark, stem segments, leaves and inflorescences of four different parts of G. elegans were used as materials in this study. The expression stability of 10 candidate housekeeping reference genes (18S, GAPDH, Actin, TUA, TUB, SAND, EF-1α, UBC, UBQ, and cdc25) was assessed through real-time fluorescence quantitative PCR, GeNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that EF-1α was stably expressed in all four parts of G. elegans and was the most suitable housekeeping gene. Based on the coexpression pattern of genome, full-length transcriptome and metabolome, the key candidate targets of 18 related genes (AS, AnPRT, PRAI, IGPS, TSA, TSB, TDC, GES, G8H, 8-HGO, IS, 7-DLS, 7-DLGT, 7-DLH, LAMT, SLS, STR, and SGD) involved in the Gelsemium alkaloid biosynthesis were obtained. The expression of 18 related enzyme genes were analyzed by qRT-PCR using the housekeeping gene EF-1α as a reference. The results showed that these genes' expression and gelsenicine content trends were correlated and were likely to be involved in the biosynthesis of the Gelsemium alkaloid, gelsenicine.
Genes, Essential ; Gelsemium/genetics* ; Peptide Elongation Factor 1/genetics* ; Transcriptome ; Gene Expression Profiling/methods* ; Alkaloids ; Real-Time Polymerase Chain Reaction/methods* ; Reference Standards

This study aimed to comprehensively assess the difference of alkaloid components between old stems and tender stems of Gelsemium elegans by using ultra high-performance liquid chromatography coupled with photo-diode array and quadrupole time-of-flight mass spectrometry( UPLC-Q-TOF/MS~E) and high-performance liquid chromatography coupled with UV detector( HPLC-UV). Firstly,the different components in old stems and tender stems were analyzed by UHPLC-Q-TOF/MSEcombined with principal component analysis( PCA) and orthogonal partial least squares discriminant analysis( OPLS-DA),respectively. As a result,17 major different components were found. At the same time,the distribution of these alkaloids in old stems and tender stems was determined,and the alkaloids with higher polarity are relatively higher in the tender stems,while the old stems are in the opposite case. In addition,three main components in the G. elegans were quantified by HPLC-UV. The results showed that the contents of koumine and humantenmine in old stems were higher than those in tender stems,and the content of gelsemine in tender stems was relatively high. This study systematically evaluated the differences of alkaloids between the old stems and tender stems of G. elegans,and quantified the main three alkaloids. It laid the foundation of the safe and effective application of G. elegans.
Alkaloids ; analysis ; Chromatography, High Pressure Liquid ; Gelsemium ; chemistry ; Mass Spectrometry ; Plant Extracts ; Plant Stems ; chemistry

OBJECTIVETo explore the new method of discriminating Gelsemium elegans from Lonicera japonica and its close species by using specific PCR amplification.

METHODThirteen samples of the different G. elegans materials and 58 samples of L. japonica, L. macranthoides and L. dasystyla were collected. The total DNA of the samples were extracted, and the DNA of G. elegans, L. japonica and L. macranthoides water extracts were extracted. PsbA-rnnH sequence from G. elegans was amplified by PCR and sequenced unidirectionally, ClustulW was used to align psbA-trnH sequences of the G. elegans and L. japonica and its close species from GenBank database.

RESULTAll samples were amplified by PCR with specific primer, DNA from G. elegans would be amplified 97 bp whereas PCR products from all of Lonicera samples had not bands.

CONCLUSIONSpecific PCR amplification can be used to identify G. elegans from L. japonica and its close species successfully and is an efficient molecular marker for authentication of G. elegans and L. japonica and its close species.

DNA, Plant ; genetics ; isolation & purification ; Drugs, Chinese Herbal ; analysis ; Gelsemium ; chemistry ; genetics ; Lonicera ; chemistry ; genetics ; Phylogeny ; Plant Extracts ; chemistry ; genetics ; Polymerase Chain Reaction ; Water ; chemistry

OBJECTIVETo study the chemical constituents from the aerial parts of Gelsemium elegans.

METHODCompounds were isolated and purified by repeated column chromatography, as well as semiprep arative HPLC, and their structures were identified by physicochemical properties and spectroscopic methods, such as NMR and MS.

RESULTSixteen compounds were obtained and identified from G. elegans, including nine alkaloids: koumine (1), gelsenicine (2), 19-(Z)-akuammidine (3), gelsemoxonine(4), gelsemin (5), gelsevirine (6), humantenine (7), 11-methoxygelsemamide (8) and gelegamine D (9). Three megastigmane glycosides: (3R, 5S, 6S, 7E, 9R)-megastigman-7-ene-3, 5, 6, 9-tetrol-9-O-beta-D-glucopyranoside (10), (6R, 7E, 9R)-9-hydroxy-4, 7-megastigmadien-3-one-9-O-[alpha-L-arabinopyranosyl-(1 --> 6)-beta-D-glucopyranoside] (11) and (6S, 7E, 9R) -6, 9-dihydroxy-4, 7-megastigmadien-3-one-9-O-[alpha-L-arabinopyranosyl-(1 --> 6) -beta-D-glucopyranoside] (12). Two flavone C-glycosides: orientin (13) and isorientin (14); one iridoid glycoside, sweroside (15) and one fructoside, n-butyl-alpha-D-fructofuranoside (16).

CONCLUSIONCompounds 10-16 were isolated from the genus Gelsemium for the first time.

Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Gelsemium ; chemistry ; Mass Spectrometry ; Plant Components, Aerial ; chemistry

OBJECTIVETo study the non-alkaloid chemical constituents of Gelsemium elegans.

METHODCompounds were isolated and purified by repeated column chromatography, and their structures were elucidated by spectroscopic methods.

RESULTTen compounds were isolated and their structures were identified as tamarixin (1), tamarixetin 3-O-beta-D-galactopyranoside (2), scopolin (3), scopoletin (4), uradine (5), caffeic acid (6), caffeic acid ethyl ester (7), ferulic acid ethyl ester (8), ethyl-alpha-D-fructofuranoside (9), and ethyl-beta-D-fructopyranoside (10).

CONCLUSIONCompounds 1-3,5-10 are firstly isolated from this plant and compounds 1, 2, and 5-10 are isolated from the genus Gelsemium for the first time.

Alkaloids ; chemistry ; isolation & purification ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Gelsemium ; chemistry ; Mass Spectrometry

OBJECTIVETo investigate the antitumor effects of koumine in mice bearing H22 solid tumor and its effect on the immune system of the mice.

METHODSThe changes in spleen and tumor weights and blood cell count were observed after koumine treatment in BALB/c athymic mice bearing H22 solid tumor, using normal saline solution and 5-Fu as the controls.

RESULTSKoumine significantly inhibited the tumor growth in a dose-dependent manner. The spleen index and blood cell counts in koumine group showed no significant differences from those in the saline control group, but higher than those in 5-Fu group.

CONCLUSIONKoumine can significantly inhibit the growth of H22 solid tumor without obvious inhibitory effect on the immune system in mice.

Animals ; Antineoplastic Agents, Phytogenic ; therapeutic use ; Female ; Gelsemium ; chemistry ; Indole Alkaloids ; isolation & purification ; therapeutic use ; Liver Neoplasms, Experimental ; drug therapy ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Phytotherapy