Isoxerophilusins A and B, Two Novel Polycyclic Asymmetric Diterpene Dimers from Isodon xerophilus: Structural Elucidation, Modification, and Inhibitory Activities against α-Glucosidase.

Isoxerophilusins A (1) and B (2), two unprecedented diterpene heterodimers biogenetically from ent-atisanes and abietanes, were isolated from the rhizomes of Isodon xerophilus. Their structures were determined by extensive spectroscopic analysis and single-crystal X-ray diffraction. Selective esterification of 1 generated 11 new derivatives. All derivatives showed excellent α-glucosidase inhibitory activity in comparison to acarbose. Compounds 12 and 13 demonstrated significant inhibition against α-glucosidase with IC50 values of 4.92 and 3.83 µM, respectively.

Comparative transcriptome analysis reveals genes involved in trichome development and metabolism in tobacco.

BACKGROUND: The glandular trichomes of tobacco (Nicotiana tabacum) can efficiently produce secondary metabolites. They act as natural bioreactors, and their natural products function to protect plants against insect-pests and pathogens and are also components of industrial chemicals. To clarify the molecular mechanisms of tobacco glandular trichome development and secondary metabolic regulation, glandular trichomes and glandless trichomes, as well as other different developmental tissues, were used for RNA sequencing and analysis. RESULTS: By comparing glandless and glandular trichomes with other tissues, we obtained differentially expressed genes. They were obviously enriched in KEGG pathways, such as cutin, suberine, and wax biosynthesis, flavonoid and isoflavonoid biosynthesis, terpenoid biosynthesis, and plant-pathogen interaction. In particular, the expression levels of genes related to the terpenoid, flavonoid, and wax biosynthesis pathway mainly showed down-regulation in glandless trichomes, implying that they lack the capability to synthesize certain exudate compounds. Among the differentially expressed genes, 234 transcription factors were found, including AP2-ERFs, MYBs, bHLHs, WRKYs, Homeoboxes (HD-ZIP), and C2H2-ZFs. These transcription factor and genes that highly expressed in trichomes or specially expressed in GT or GLT. Following the overexpression of R2R3-MYB transcription factor Nitab4.5_0011760g0030.1 in tobacco, an increase in the number of branched glandular trichomes was observed. CONCLUSIONS: Our data provide comprehensive gene expression information at the transcriptional level and an understanding of the regulatory pathways involved in glandular trichome development and secondary metabolism.

Isochromenes from the Nicotiana tabacum-derived endophytic fungus Aspergillus versicolor and their anti-tobacco mosaic virus activities.

Three new isochromenes, (5-methoxy-7-prenyl-1H-isochromen-3-yl)methanol (1), 3-(3-(hydroxymethyl)-5-methoxy-1H-isochromen-7-yl)propan-1-ol (2), and (5-methoxy-7-methyl-1H-isochromen-3-yl)methanol (3), along with three known analogues (4-6) were isolated from the fermentation products of a Nicotiana tabacum-derived endophytic fungus Aspergillus versicolor. Their structures were elucidated by spectroscopic methods, including extensive 1 D and 2 D NMR techniques. Compounds 1-3 and 6 were evaluated for their anti-tobacco mosaic virus (anti-TMV) activities. The results showed that compound 2 exhibited high anti-TMV activity with inhibition rate of 46.4%, and this rate is higher than that of positive control. Compounds 1, 3, and 6 also showed potential anti-TMV activity with inhibition rates of 28.6, 30.5, and 26.2%, respectively. The IC50 of compounds 1-3 and 6 were also tested, and showed IC50 values of 49.3, 22.4, 42.2, and 54.1 µM, respectively.

The anti-TMV potency of the tobacco-derived fungus Aspergillus versicolor and its active alkaloids, as anti-TMV activity inhibitors.

Nicotiana tabacum (tobacco) has attracted interest as one of the most economically important industrial crops widely cultivated in China, whose dried leaves are popularly consumed medicinally and recreationally by human societies. In this study, five undescribed alkaloids derivatives, isoaspergillines A-E, together with eight known alkaloids, notoamide D, (1R,4S)-4-benzyl-1-isopropyl-2,4-dihydro-1H-pyrazino-[2,1-b]quinazoline-3,6-dione, protuboxepin K, notoamide C, notoamide M, deoxybrevianamide E, cyclo (D-Pro-L-Trp), and versicolamide B, were obtained from the culture of the Nicotiana tabacum-derived fungus Aspergillus versicolor. Their structures were mainly elucidated through comprehensive analyses of spectroscopic data. Bioactivity evaluation of all isolated compounds revealed that isoaspergilline A and notoamide M exhibited anti-TMV activities with IC50 values of 20.0 and 22.8 µM, respectively. Molecular docking suggested that isoaspergilline A and notoamide M were well located into the active site of anti-TMV by interacting with SER138, SER143, and ASN73 residues. This study enlightens the therapeutic potential of the endophytic fungus A. versicolor and it is helpful to find undescribed anti-TMV activity inhibitors, as well as searching for new anti-TMV candidates from natural sources.

Isoindolin-1-ones from the stems of Nicotiana tabacum and their antiviral activities.

In previous studies, several isoindolin-1-one analogs that exhibited significant anti-tobacco mosaic virus (anti-TMV) activities were isolated from Nicotiana tabacum. Since gene-editing mutants provide a new sample for the discovery of active metabolites, we focused on the stems of YN-18-23 (a mutant N. tabacum for gene editing with the alkaloid metabolic pathway cultivated by Yunnan Tobacco Company), which led to the isolation of four new (1-4) and four known (5-8) isoindolin-1-ones. To the best of our knowledge, nicindole C (3) is the first subclass of isoindolin-1-one bearing a pentacyclic ketone, while nicindole D (4) is the first example of isoindolin-1-one bearing a methyl-pyridin-2-(1H)-one moiety. Compounds 1-4 were tested for their anti-TMV activities, and the results revealed that compounds 1, 3, and 4 exhibited high anti-TMV activities at concentrations of 20 µM with inhibition rates of 48.6, 42.8, and 71.5%, respectively. These rates are higher than the inhibition rate of the positive control (33.2%). The mechanistic study of compound 4, which had the highest anti-TMV activity revealed that increased potentiation of defense-related enzyme activities and downregulation of expression of the NtHsp70 protein may induce resistance in tobacco against the viral pathogen TMV. Molecular docking studies also revealed that the isoindolin-1-one substructure is fundamental for anti-TMV activity. The methyl-pyridin-2-(1H)-one moiety in compound 4 and the 2-oxopropyl groups in compounds 1 and 3 at the N-2 position may increase inhibitory activities. This study of the structure-activity relationship is helpful for finding new anti-TMV activity inhibitors. To study whether the isoindolin-1-ones have broader antiviral activities, compounds 1-4 were also tested for their anti-rotavirus activities. Compound 4 exhibited high anti-rotavirus activity with a therapeutic index (TI) value of 20.7. This TI value is close to that of the positive control (20.2).

Genetic polymorphisms of 16 X-STR loci in the Hani population from Southwest China.

X chromosomal short tandem repeats (X-STRs) have the characteristics of both autosomal and uniparental genetic markers and have been shown to be particularly useful in forensic casework. However, relevant research or reports have not focused on X-STRs in the Hani population. To investigate the genetic variation and forensic efficiency of 16 X-STR loci in the Hani ethnic minority, we calculated the allele frequencies and forensic parameters of 451 (116 males and 335 females) unrelated healthy Hani individuals from Yunnan Province, Southwest China. All these loci are highly polymorphic in Hani individuals in Yunnan Province except DXS6800. The combined power of discrimination in males (PDM) and power of discrimination in females (PDF) were found to be 0.999 999 998 433 993 and 0.999 999 999 999 998, respectively. Furthermore, a population genetic structure investigation between the Yunnan Hani population and another 18 populations was performed using a principal component analysis, multidimensional scaling plot and neighbouring-joining phylogenetic tree and the findings illustrated that neighbouring populations and different nationalities in the same area appeared to have a closer evolutionary relationship. This study provides the first batch of X chromosome genetic polymorphism data of the Hani population in Yunnan Province, Southwest China and enriches the reference database of the Chinese minority population.Key pointsThis is the first study of X-STR in the Hani population.We calculated the allele frequencies and forensic parameters of 451 unrelated healthy Hani individuals from Yunnan Province, Southwest China.All these loci are highly polymorphic in Hani individuals in Yunnan Province except DXS6800.The genetic relationship between the Hani and other 18 nationalities was analyzed.This study provides the first batch of X chromosome genetic polymorphism data of the Hani population in Yunnan Province, Southwest China and enriches the reference database of the Chinese minority population.

Indole Alkaloids and Chromones from the Stem Bark of Cassia alata and Their Antiviral Activities.

The Cassia (Leguminosae) genus has attracted a lot of attention as a prolific source of alkaloids and chromones with diverse structures and biological properties. The aim of this study is to screen the antiviral compounds from Cassia alata. The extract of the stem bark of this plant was separated using silica gel, MCI, ODS C18, and Sephadex LH-20 column chromatography, as well as semi-preparative HPLC. As a result, three new indole alkaloids, alataindoleins A-C (1-3); one new chromone, alatachromone A (4); and a new dimeric chromone-indole alkaloid, alataindolein D (5) were isolated. Their structures were determined by means of HRESIMS and extensive 1D and 2D NMR spectroscopic studies. Interestingly, alataindolein D (5) represents a new type of dimeric alkaloid with an unusual N-2-C-16' linkage, which is biogenetically derived from a chromone and an indole alkaloid via an intermolecular nucleophilic substitution reaction. Compounds 1-5 were tested for their anti-tobacco mosaic virus (TMV) and anti-rotavirus activities, and the results showed that compounds 2-4 showed high anti-TMV activities with inhibition rates of 44.4%, 66.5%, and 52.3%, respectively. These rates were higher than those of the positive control (with inhibition rate of 32.8%). Compounds 1 and 5 also showed potential anti-TMV activities with inhibition rates of 26.5% and 31.8%, respectively. In addition, compounds 1-5 exhibited potential anti-rotavirus activities with therapeutic index (TI) values in the range of 9.75~15.3. The successful isolation and structure identification of the above new compounds provided materials for the screening of antivirus drugs, and contributed to the development and utilization of C. alata.

Cyclopiazonic acid type indole alkaloids from Nicotiana tabacum-derived fungus Aspergillus versicolor and their anti-tobacco mosaic virus activities.

Indole alkaloids have attracted widespread attention of chemists and biologists. Therefore, the aim of this study is to screen more bioactivities indole alkaloids from the microorganisms. In this study, five undescribed CPA-type indole alkaloids, aspergillines F-J, and three known CPA-type indole alkaloids, aspergilline A, aspergilline C, and cyclopiamide E, were obtained from the Nicotiana tabacum-derived fungus Aspergillus versicolor. Notably, aspergillines F and G represent the first examples of indole alkaloids with a benzo[cd]indol-2(1H)-one skeleton, and aspergilline J is also the firstly obtained indole alkaloids bearing a N-1-(2-(1H-imidazole-5-yl)ethyl) moiety. Aspergillines F-J and cyclopiamide E were tested for their anti-TMV activities, and the results revealed that aspergillines G and J exhibited obvious anti-TMV activities with inhibition rates of 41.2 and 56.8% at the concentration of 20 µM, respectively. These rates are high than that of positive control (with inhibition rate of 32.5%). In addition, the molecular docking studies for the isolated CPA-type indole alkaloids may also reveal that the benzo[cd]indol-2(1H)-one substructure is the fundamental for anti-TMV activity and the oxygen-containing substituent groups at C-19 also increases the inhibitory activity. This study of structure-activity relationship is helpful to find new anti-TMV activity inhibitors.

Forensic genetic polymorphisms of 16 X-STR loci in the Yunnan Miao population and their relationship to other Chinese groups.

Allele frequencies for 16 X-chromosomal STR (X-STR) loci were obtained from a sample set of 440 unrelated Yunnan Miao individuals in China. A total of 117 alleles were observed in this group, with allele frequencies ranging from 0.0016 to 0.7565. The most informative marker for the studied population was DXS10134, with a polymorphism information content (PIC) of 0.8499, and the least polymorphic locus was DXS6810 (PIC = 0.3071). The power of discrimination (PD) varied from 0.4046 (DXS6800) to 0.8642 (DXS10134) in males and from 0.6188 (DXS6800) to 0.9673 (DXS10134) in females. The combined PDM and PDF were 0.999999989975990 and 0.999999999999949, respectively. The combined MECD and MECT were 0.999983301904059 and 0.999999915883733, respectively. Furthermore, population genetic structure investigation between the Yunnan Miao and 20 other populations using principal component analysis (PCA), multidimensional scaling plot (MDS), and neighboring-joining (NJ) phylogenetic tree analyses illustrated significant genetic difference between the Yunnan Miao and the other populations. This study is the first to provide X chromosome genetic polymorphism data of the Miao population in Yunnan Province and can be used as a supplementary reference to enrich the national database.

High-content screening of diterpenoids from Isodon species as autophagy modulators and the functional study of their antiviral activities.

Autophagy is a conserved lysosomal degradation process, and abnormal autophagy has been associated with various pathological processes, e.g., neurodegeneration, cancer, and pathogen infection. Small chemical modulators of autophagy show the potential to treat autophagy-associated diseases. Diterpenoids, nature products found in various plants, exhibit a wide range of bioactivity, and we have recently isolated and characterized over 150 diterpenoids from Isodon species distributed in China. Here, we applied a high-content fluorescence imaging-based assay to assess these diterpenoids' ability to affect autophagic flux in HeLa cells. We found that enanderinanin J, an ent-kauranoid dimer, is an autophagy inhibitor, manifested by its ability to increase lysosomal pH and inhibit the fusion between autophagosomes and lysosomes. Autophagy has been shown to be either positively or negatively involved in the life cycle of Zika virus (ZIKV), Japanese encephalitis virus (JEV), Dengue virus (DENV), and enterovirus-A71 (EV-A71). We found that enanderinanin J significantly inhibited the infection of ZIKV, DENV, JEV, or EV-A71. Interestingly, although ATG5 knockdown inhibited ZIKV or JEV infection, enanderinanin J further inhibited the infection of ZIKV or JEV in ATG5-knockdown cells. Taken together, our data indicate that enanderinanin J inhibits autophagosome-lysosome fusion and is a potential antiviral agent.

ent-Kaurane-Based Diterpenoids, Dimers, and Meroditerpenoids from Isodon xerophilus.

Eight new diterpenoids (1-8) with varied structures were isolated from the aerial parts of Isodon xerophilus. Among them, xerophilsin A (1) was found to be an unusual meroditerpenoid representing a hybrid of an ent-kauranoid and a long-chain aliphatic ester, xerophilsins B-D (2-4) are dimeric ent-kauranoids, while xerophilsins E-H (5-8) are new ent-kauranoids. The structures of 1-8 were elucidated mainly through the analyses of their spectroscopic data. The absolute configurations of 2, 6, and 8 were confirmed by single-crystal X-ray diffraction, and the configuration of C-16 in 7 was established through quantum chemical calculation of NMR chemical shifts, as well as modeling of key interproton distances. Bioactivity evaluation of all isolated compounds revealed that 2, 3, and 5 inhibited NO production in LPS-stimulated RAW264.7 cells.

Phenylpropanoid and Iridoid Glucosides from the Whole Plant of Hemiphragma heterophyllum and Their alpha-Glucosidase Inhibitory Activities.

Three phenylpropanoid glucosides (1:  - 3: ) and one iridoid glucoside (11: ), together with eleven known glucosides, were isolated from the ethanol extract of the whole plant of Hemiphragma heterophyllum. Their structures were elucidated by means of 1D and 2D NMR spectroscopy, HRMS, and chemical methods. All compounds except 11: and 13:  - 15: showed varying degrees of α-glucosidase inhibitory activity. Compounds 5, 9: , and 12: were marginally active in the bioassay, while compounds 1:  - 4: , 6:  - 8: , and 10: exhibited appreciable inhibitory activity with an IC50 value of 33.6 ~ 83.1 µM, which was much lower than that of the positive control acarbose (IC50 = 310.8 µM).

Chemical constituents from the whole herb of Hemiphragma heterophyllum.

Phytochemical investigation on Hemiphragma heterophyllum led to the isolation of two new compounds, heterophyllumin A (1) and heterophylliol (3), along with nine known compounds, (‒)-sibiricumin A (2), iridolactone (4), jatamanin A (5), dihydrocatalpolgenin (6), 25-hydroperoxycycloart-23-en-3ß-ol (7), 24-methylenecycloartanol (8), (+)-pinoresinol (9), hexadec-(4Z)-enoic acid (10), and 9,12, 15-octadecatrienoic acid (11). Their structures were elucidated on the basis of detailed spectroscopic analyses and by comparison with literature data. Further, the structure of compound 3 was unambiguously confirmed by single-crystal X-ray analysis. Some of those compounds showed moderate activity in the α-glucosidase inhibition assay.