(±)-Penicithrones A-D (1a/1b-4a/4b), four novel pairs of anthrone-cyclopentenone heterodimers characterized by a distinctive bridged 6/6/6-5 tetracyclic core skeleton, together with three previously identified compounds (5-7), were isolated from the crude extract of the mangrove-derived fungus Penicillium sp., guided by heteronuclear single quantum correlation (HSQC)-based small molecule accurate recognition technology (SMART 2.0) and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based molecular networking. The structural elucidation of new compounds was accomplished through comprehensive spectroscopic analysis, and their absolute configurations were determined using DP4+ ¹³C nuclear magnetic resonance (NMR) calculations and electronic circular dichroism (ECD) calculations. Compounds 1a/1b-4a/4b demonstrated moderate cytotoxicity against three human cancer cell lines HeLa, HCT116 and MCF-7 with half maximal inhibitory concentration (IC₅₀) values ranging from 15.95 ± 1.64 to 28.56 ± 2.59 μmol·L^-1.
Humans ; Penicillium/chemistry* ; Molecular Structure ; Cyclopentanes/isolation & purification* ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology* ; Tandem Mass Spectrometry ; Dimerization ; HeLa Cells ; Magnetic Resonance Spectroscopy

Anthoceros angustus Steph. is rich in phenolic acids such as rosmarinic acid (RA). Phenylalanine ammonia-lyase (PAL) is an entry enzyme in the phenylpropanoid pathway of plants, playing an important role in the biosynthesis of RA. To investigate the important role of PAL in rosmarinic acid synthesis, two PAL genes (designated as AanPAL1 and AanPAL2) were cloned from A. angustus, encoding 755 and 753 amino acid residues, respectively. The AanPAL deduced amino acid sequences contain the conserved domains of PAL and the core active amino acid residues Ala-Ser-Gly. The phylogenetic analysis indicated that AanPAL1 and AanPAL2 were clustered with PALs from bryophytes and ferns and had the shortest evolutionary distance with the PALs from Physcomitrella patens. Quantitative real-time PCR results showed that the expression of AanPAL1 and AanPAL2 was induced by exogenous methyl jasmonate (MeJA). HPLC results showed that the MeJA treatment significantly increased the accumulation of RA. AanPAL1 and AanPAL2 were expressed in Escherichia coli and purified by histidine-tag affinity chromatography. The recombinant proteins catalyzed the conversion of L-phenylalanine to generate trans-cinnamic acid with high efficiency, with the best performance at 50 ℃ and pH 8.0. The K_m and k_cat of AanPAL1 were 0.062 mmol/L and 4.35 s^-1, and those of AanPAL2 were 0.198 mmol/L and 14.48 s^-1, respectively. The specific activities of AanPAL1 and AanPAL2 were 2.61 U/mg and 8.76 U/mg, respectively. The two enzymes had relatively poor thermostability but good pH stability. The high activity of AanPAL2 was further confirmed via whole-cell catalysis with recombinant E. coli, which could convert 1 g/L L-phenylalanine into trans-cinnamic acid with a yield of 100% within 10 h. These results give insights into the regulatory role of AanPAL in the biosynthesis of RA in A. angustus and provide candidate enzymes for the biosynthesis of cinnamic acid.
Phenylalanine Ammonia-Lyase/metabolism* ; Cloning, Molecular ; Cinnamates/metabolism* ; Recombinant Proteins/metabolism* ; Rosmarinic Acid ; Depsides/metabolism* ; Escherichia coli/metabolism* ; Amino Acid Sequence ; Plant Proteins/metabolism* ; Phylogeny ; Acetates/pharmacology* ; Cyclopentanes ; Oxylipins

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

The well-known detrimental effects of cadmium (Cd) on plants are chloroplast destruction, photosynthetic pigment inhibition, imbalance of essential plant nutrients, and membrane damage. Jasmonic acid (JA) is an alleviator against different stresses such as salinity and drought. However, the functional attributes of JA in plants such as the interactive effects of JA application and Cd on rapeseed in response to heavy metal stress remain unclear. JA at 50 µmol/L was observed in literature to have senescence effects in plants. In the present study, 25 µmol/L JA is observed to be a "stress ameliorating molecule" by improving the tolerance of rapeseed plants to Cd toxicity. JA reduces the Cd uptake in the leaves, thereby reducing membrane damage and malondialdehyde content and increasing the essential nutrient uptake. Furthermore, JA shields the chloroplast against the damaging effects of Cd, thereby increasing gas exchange and photosynthetic pigments. Moreover, JA modulates the antioxidant enzyme activity to strengthen the internal defense system. Our results demonstrate the function of JA in alleviating Cd toxicity and its underlying mechanism. Moreover, JA attenuates the damage of Cd to plants. This study enriches our knowledge regarding the use of and protection provided by JA in Cd stress.
Brassica napus/metabolism* ; Cadmium/toxicity* ; Catalase/metabolism* ; Cyclopentanes/pharmacology* ; Oxylipins/pharmacology* ; Photosynthesis ; Plant Leaves/metabolism* ; Superoxide Dismutase/metabolism*

Hyoscyamine and scopolamine are important secondary metabolites of tropane alkaloid in Atropa belladonna with pharmacological values in many aspects.In this study, the seedlings of A.belladonna were planted by soil culture and treated with different concentrations of methyl jasmonate (MeJA). The contents of hyoscyamine and scopolamine,the upstream products in alkaloid synthesis,and the expression levels of key enzyme genes PMT, TR Ⅰ and H6H in secondary metabolites of A. belladonna seedlings were measured to clarify the mechanism of MeJA regulating alkaloids synthesis.The results showed that MeJA(200 μmol·L⁻¹) treatment was more favorable for the accumulation of alkaloids.The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine,the both increased first and then decreased with the increased MeJA concentration and the content of putrescine reached the highest at 200 μmol·L⁻¹ MeJA.Further detection of gene expression of PMT, TR Ⅰ and H6H in TAs synthesis pathway showed that no significant trend in PMT gene expression levels.The expression levels of TR Ⅰ and H6H in leaves and roots under 200 μmol·L⁻¹ MeJA were the highest.It can be speculated that the regulation of the formation of hyoscyamine and scopolamine by MeJA mainly through affecting the expression of key enzyme genes.Appropriate concentration of MeJA increased the gene expression of TR Ⅰ in both leaves and roots as well as H6H in roots,promoting the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine.
Acetates ; pharmacology ; Atropa belladonna ; drug effects ; genetics ; metabolism ; Cyclopentanes ; pharmacology ; Gene Expression Regulation, Plant ; Hyoscyamine ; metabolism ; Oxylipins ; pharmacology ; Plant Leaves ; metabolism ; Plant Roots ; metabolism ; Scopolamine ; metabolism

To study the effects of the extract of fungal elicitor, AgNO3, MeJA and yeast on the growth and content of secondary metabolites of adventitious roots in Tripterygium wilfordii. The above elicitors were supplemented to the medium, the growth and the content of secondary metabolites were measured. When the medium was supplemented with the elicitor Glomerella cingulata or Collectotrichum gloeosporioides, the content of triptolide was increased by 2.24 and 1.93-fold, the alkaloids content was increased by 2.02 and 2.07-fold, respectively. The optimal concentration of G. cingulata was 50 μg/mL for accumulation of triptolide, alkaloids and for the growth of adventitious roots. AgNO3 inhibited the growth of adventitious roots and the accumulation of the alkaloids, whereas it (at 25 μmol/L) increased the accumulation of triptolide by 1.71-fold compared to the control. The growth of adventitious roots, the contents of triptolide and alkaloids were increased 1.04, 1.64 and 2.12-folds, respectively when MeJA was at 50 μmol/L. When the concentration of yeast reached 2 g/L, the content of triptolide increased 1.48-folds. This research demonstrated that supplementation of AgNO3 and yeast enhanced the biosynthesis of triptolide in adventitious roots and the synergism of G. cingulata and MeJA could promote the biosynthesis of both triptolide and alkaloids.
Acetates ; pharmacology ; Alkaloids ; biosynthesis ; Colletotrichum ; Cyclopentanes ; pharmacology ; Diterpenes ; metabolism ; Drugs, Chinese Herbal ; Epoxy Compounds ; metabolism ; Oxylipins ; pharmacology ; Phenanthrenes ; metabolism ; Phyllachorales ; Plant Roots ; drug effects ; growth & development ; Secondary Metabolism ; Tripterygium ; drug effects ; growth & development ; metabolism

OBJECTIVETo provide a new material for producing the Rhodiolasachalinensis products, the effect of methyl jasmonate (MeJA) on callus biomass and effective compound accumulation of Rhodiolasachalinensis was studied.

METHODThe calluses-cultured in 3 L-air lift balloon type bioreactor were treated with MeJA after 20 d of bioreactor culture and the effect of MeJA concentration and treatment days on callus biomass, salidroside or polysaccharide accumulation and superoxide dismutase (SOD) and peroxidase (POD) activities were investigated.

RESULTThe callus biomass was not significantly different after MeJA treatment (125) for 0-6 d but obviously decreased after 6 d treatment. The maximum salidroside or polysaccharide contents and SOD or POD activities were found after 4 d treatment of MeJA. MeJA concentration significantly affected callus biomass and effective compound accumulation, biomass decreased at MeJA concentrations higher than 125 μmol x L(-1). However, the effective compound contents were determined at higher MeJA concentration, and the highest salidroside and polysaccharide accumulation was found at 225 and 275 μmol x L(-1) MeJA, respectively and the maximum SOD and POD activities was found at 225 μmol x L(-1) MeJA. The effective compound contents in callus were compared with field-grown plants. Salidroside contents in calluses were 1.1-fold and 2. 4-fold more than in plant roots and stem or leave, respectively. Polysaccharide content in calluses were 3. 6-fold and 8.0-fold more than in plant roots and stem or leave, respectively.

CONCLUSIONSalidorside and polysaccharide in Rhodiolasachalinensiscalluses improved by MeJA treatment, 225 μmol x L(-1) MeJA and 4 d treatment were optimal. The effective compound contents in callus were obviously higher than in field-grown plants. Therefore, bioreactor culture is efficient for obtaining mass effective compounds of Rhodiolasachalinensis by culturing calluses. This method could provide an alternative material source for production of Rhodiolasachalinensis products.

Acetates ; pharmacology ; Biomass ; Bioreactors ; Cyclopentanes ; pharmacology ; Glucosides ; metabolism ; Oxylipins ; pharmacology ; Peroxidase ; metabolism ; Phenols ; metabolism ; Polysaccharides ; metabolism ; Rhodiola ; metabolism ; Superoxide Dismutase ; metabolism

The tissue-specific and MeJA-induced transcriptional levels of BcUGT3 and BcUGT6 in Bupleurum chinense were analyzed in the present study. The transcriptional levels of BcUGT3 in root, leaf, flower and fruit were similar and they all were higher than those in stem. The transcriptional level of BcUGT6 was the highest in leaf and the lowest in flower among in all tested tissues. With non-treated adventitious roots as control, BcUGT6's transcriptional levels were elevated to nearly 2 folds for 2 h, 8 h, 24 h, 2 d and 4 d in MeJA-treated adventitious roots of B. chinense. It showed that the transcriptional level of BcUGT6 was slightly affected by MeJA. While, BcUGT3's transcriptional levels were gradually elevated, and till 4 d after MeJA treatment, the expression level was about 7 folds than that of non-treated control. Using pET-28a (+), the expressions of two genes was investigated. Induced by IPTG, the target proteins were expressed in E. coli and then purified. All the results obtained in the present study will be helpful for follow-up bio-function analysis of BcUGT3 and BcUGT6.
Acetates ; pharmacology ; Bupleurum ; cytology ; enzymology ; genetics ; Cell Membrane ; metabolism ; Cyclopentanes ; pharmacology ; Escherichia coli ; genetics ; Gene Expression ; Gene Expression Regulation, Plant ; drug effects ; Hexosyltransferases ; chemistry ; genetics ; isolation & purification ; metabolism ; Intracellular Space ; metabolism ; Oxylipins ; pharmacology ; Protein Sorting Signals ; Protein Structure, Secondary ; Protein Transport ; Sequence Analysis ; Transcription, Genetic ; drug effects

OBJECTIVETo observe the protective effect of the Weinaokang (WNK) and its active compound bilobalide on focal cerebral ischemia reperfusion, and their mechanisms.

METHODThe 60-minute middle cerebral artery occlusion (MCAO) was adopted to establish the 24 h-14 d reperfusion model. The expression of Beclin-1 was detected by the Western blotting technique. The transmission electron microscopy was used to observe ultrastructural changes. Neurogenesis was detected by the immunofluorescence staining.

RESULTWNK (20, 10 mg x kg(-1), ig) or its active compound bilobalide (10, 5 mg x kg(-1), ig) could promote the generation of mature neurons (BrdU(+) -MAP-2+) at the ischemic side, and inhibit expression of autophagy-related gene Beclin-1, so as to reduce the neuron injury induced by focal cerebral ischemia reperfusion.

CONCLUSIONWNK and its active compound bilobalide can inhibit neuron autophagy and improve neurogenesis in ischemic peripheral area, suggesting that neurogenesis may be the intervention target for WNK to promote self-repairing of ischemic area.

Animals ; Autophagy ; drug effects ; Brain Ischemia ; drug therapy ; pathology ; physiopathology ; Cyclopentanes ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Furans ; pharmacology ; Ginkgolides ; pharmacology ; Male ; Neurogenesis ; drug effects ; Neurons ; ultrastructure ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control

OBJECTIVETo study the effects of methyl jasmonate (MJ) on the accumulation and release of main secondary metabolites i. e. scopolamine and hyoscyamine in liquid cultures of Datura stramonium hairy roots.

METHODAfter 18 days liquid culture of D. stramonium hairy roots induced by agrobacterium rhizogenes C58C1, the chemical elicitor methyl jasmonate was added into 1/2 MS liquid cultures and scopolamine and hyoscyamine on the day 0, 3, 6, 9 and 12, after dealing with MJ, was determined by HPLC.

RESULTAfter dealing with MJ on the day 3, 6, 9 and 12,the concentration of scopolamine reached to 0.419, 0.439, 0.431, 0.374 mg x g(-1), respectively, the increase of scopolamine were 1.36, 1.42, 1.17 and 1.12 fold higher than that of the control, respectively. And hyoscyamine reached 1.493, 0.817, 0.723 and 0.698 mg x g(-1), respectively, the increase of hyoscyamine were 2.28, 1.11, 0.63 and 0.70 fold higher than that of the control, respectively.

CONCLUSIONMJ could stimulate the accumulation of scopolamine and hyoscyamine (3,6 d) in D. stramonium hairy root and have released them into the culture medium.

Acetates ; pharmacology ; Alkaloids ; analysis ; metabolism ; Cell Culture Techniques ; Cyclopentanes ; pharmacology ; Datura stramonium ; chemistry ; drug effects ; growth & development ; metabolism ; Oxylipins ; pharmacology ; Plant Roots ; chemistry ; drug effects ; growth & development ; metabolism ; Tropanes ; analysis ; metabolism