Diverse neolignans and lignans from an aqueous extract of the Angelica sinensis root head.

Neolignans and lignans with diverse new chemical structures, including eleven pairs of separated chiral enantiomers [(+)-/(-)-1-(+)-/(-)-5, (+)-/(-)-8, (+)-/(-)-10, and (+)-/(-)-12-(+)-/(-)-15], two achiral compounds (6 and 9), and an unseparated racemate [(±)-11], together with a new natural product (7) and 21 known derivatives, were isolated from an aqueous extract of the Angelica sinensis root head (guitou). Among the chiral isolates, (+)-/(-)-13 and (+)-/(-)-15 were scalemic pairs with enantiomeric ratios of around 3:1 and 1.5:1, respectively, while others were enantiomeric equivalent pairs. This indicates that the diverse neolignans in A. sinensis are biosynthesized via different pathways with varying degrees of stereo-controlled manners.

Denudatine-type diterpenoid alkaloids from an aqueous extract of the lateral root of Aconitum carmichaelii.

Five new denudatine-type diterpenoid alkaloids (1-5), along with the known analogue aconicarmine (6), were isolated from an aqueous decoction of the lateral roots of Aconitum carmichaelii (fu-zi). Their structures were determined by spectroscopic data analysis and electronic circular dichroism (ECD) calculations. Compound 5 is the first denudatine-type diterpenoid alcohol iminium alkaloid, which could be partially transformed into the aza acetal form in pyridine-d5. Compound 5 inhibited mice writhing in an acetic acid-induced writhing assay.

Minor alkaloids from an aqueous extract of the hook-bearing stem of Uncaria rhynchophylla.

Seven new monoterpene alkaloids (1-7), along with 18 known analogues, were isolated from an aqueous decoction of the hook-bearing stems of Uncaria rhynchophylla (Gou-teng). Their structures were determined by spectroscopic data analysis and electronic circular dichroism (ECD) calculations. Compound 1 is the first monoterpene 22-norindoloquinolizidine alkaloid with a ketene unit, while 2 and 3 are unusual indoloquinolizidine alkaloids having an oxazinane ring.[Formula: see text].

Minor triterpenes from an aqueous extract of the hook-bearing stem of Uncaria rhynchophylla.

Six new triterpenes, uncarinic acids KP (1-6), along with 24 known analogues, were isolated as minor constituents of an aqueous decoction of the hook-bearing stems of Uncaria rhynchophylla (Gou-teng). By comprehensive spectroscopic data analysis, their structures were elucidated as derivatives of olean-12-en-28-oic acid and urs-12-en-28-oic acid with different oxidized forms at C-3, C-6, and/or C-23, respectively. Cell-based preliminary bioassay showed that the (E)-/(Z)-coumaroyloxy and (E)-/(Z)-feruloyloxy units at C-27 of olean-12-en-28-oic acid and urs-12-en-28-oic acid played roles in their bioactivities.[Formula: see text].

Insight into Medicinal Chemistry Behind Traditional Chinese Medicines: p-Hydroxybenzyl Alcohol-Derived Dimers and Trimers from Gastrodia elata.

From an aqueous extract of "tian ma" (the steamed and dried rhizomes of Gastrodia elata), ten new compounds gastrodibenzins A-D (1-4) and gastrotribenzins A-F (5-10), along with known analogues (11-20), having structure features coupling between two and three p-hydroxybenzyl-derived units via carbon- and/or ether-bonds, were isolated and characterized by spectroscopic data analysis. Meanwhile, the new compounds 5a, 6a, 8a, 22, and 23, as well as the known derivatives 13a, 14a, 15, 17-21, 24, 25, and p-hydroxybenzyl aldehyde were isolated and identified from a refluxed aqueous solution of p-hydroxybenzyl alcohol. Methylation of 5a and 6a in methanol and ethylation of 6a, 8a, 13a, and 14a in ethanol produced 5 and 6 and 7, 8, 13, and 14, respectively. using ultra-performance liquid chromatography high-resolution electrospray ionization mass spectrometry (UPLC-HRESIMS) analysis of the refluxed solutions of p-hydroxybenzyl alcohol and the refluxed extracts of the fresh G. elata rhizome and "tian ma" extracts indicated consistent production and variation of the dimeric and trimeric derivatives of p-hydroxybenzyl alcohol upon extracting solvents and refluxing time. In various assays, the dimeric and trimeric derivatives showed more potent activities than p-hydroxybenzyl alcohol itself and gastrodin, which are the main known active constituents of "tian ma". These results revealed for the first time that the more effective dimers and trimers can be produced through condensation of the co-occurring p-hydroxybenzyl alcohol during processing and decocting of the G. elata rhizomes, demonstrating insights into medicinal chemistry behind application protocols of traditional Chinese medicines.

A bibenzyl compound 20C protects rats against 6-OHDA-induced damage by regulating adaptive immunity associated molecules.

Parkinson's disease (PD) is a neurodegenerative disease with complicated pathogenesis. A novel bibenzyl compound 2-[4-hydroxy-3-(4-hydroxyphenyl)benzyl]-4-(4-hydroxyphenyl)phenol (20C) has been shown to have some neuroprotective effects, and its mechanism still needs further research. In this study, we used a 6-hydroxydopamine (6-OHDA)-induced PD rat model to evaluate the protective effect of 20C. Our study found that 20C could improve behavioral defects in 6-OHDA-lesion rats, decrease neuroinflammation and protect their DA neurons. It could inhibit the activity of inducible nitric oxide synthase (iNOS) induced by 6-OHDA, and lead to a decrease in the expression of nitrated-α-synuclein. When exposed to AMT-an inhibitor of iNOS, the nitrated-α-synuclein in PC12 decreased, and 20C demonstrated the same function on nitrated-α-synuclein as AMT. Besides, we also found that nitrated-α-synuclein was displayed in microglia. And 20C could decrease the expression of antigen-presenting molecule major histocompatibility complex I (MHC I) in dopamine (DA) neurons and MHC II in microglia induced by 6-OHDA. So, these imply that nitrated-α-synuclein might act as an endogenous antigen activating adaptive immunity, and the neuroprotection of 20C might be associated with inhibiting the activity of iNOS, decreasing the expression of the antigen molecule nitrated-α-synuclein and the antigen presenting molecule MHC. Our results indicated that inhibiting iNOS might be an effective strategy to protect neurons from oxidative stress.

Indole alkaloid glycosides with a 1'-(phenyl)ethyl unit from Isatis indigotica leaves.

Seven indole alkaloid glycosides containing a 1'-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl unit (1-7) were isolated from an aqueous extract of Isatis indigotica leaves (da qing ye). Their structures were determined by spectroscopic data analysis combined with enzymatic hydrolysis as well as comparison of their experimental CD (circular dichroism) and calculated ECD (electrostatic circular dichroism) spectra. Based on analysis of [ α ] D 20 and/or Cotton effect (CE) data of 1-7, two simple roles to assign location and/or configuration of ß-glycopyranosyloxy and 1'-(phenyl)ethyl units in the indole alkaloid glycosides are proposed. Stereoselectivity in plausible biosynthetic pathways of 1-7 is discussed. Compounds 3 and 4 and their mixture in a 3:2 ratio showed activity against KCNQ2 in CHO cells. The mixture of 5 and 6 (3:2) exhibited antiviral activity against influenza virus H1N1 PR8 with IC50 64.7 µmol/L (ribavirin, IC50 54.3 µmol/L), however, the individual 5 or 6 was inactive. Preliminary structure-activity relationships were observed.

Discovery, synthesis, and optimization of an N-alkoxy indolylacetamide against HIV-1 carrying NNRTI-resistant mutations from the Isatis indigotica root.

From an aqueous decoction of the traditional Chinese medicine "ban lan gen" (the Isatis indigotica root), an antiviral natural product CI - 39 was isolated as an NNRTI (non-nucleoside reverse transcriptase inhibitor) (EC50 = 3.40 µM). Its novel structure was determined as methyl (1-methoxy-1H-indol-3-yl)acetamidobenzoate by spectroscopic data and confirmed by single crystal X-ray diffraction. Through synthesis and structure-activity relationship (SAR) investigation of CI - 39 and 57 new derivatives (24 with EC50 values of 0.06-8.55 µM), two optimized derivatives 10f and 10i (EC50: 0.06 µM and 0.06 µM) having activity comparable to that of NVP (EC50 = 0.03 µM) were obtained. Further evaluation verified that 10f and 10i were RT DNA polymerase inhibitors and exhibited better activities and drug resistance folds compared to NVP against seven NNRTI-resistant strains carrying different mutations. Especially, 10i (EC50 = 0.43 µM) was more active to the L100I/K103N double-mutant strain as compared to both NVP (EC50 = 0.76 µM) and EFV (EC50 = 1.08 µM). The molecular docking demonstrated a possible binding pattern between 10i and RT and revealed activity mechanism of 10i against the NNRTI-resistant strains.

Two folate-derived analogues from an aqueous decoction of Uncaria rhynchophylla.

Two new folate-derived analogues, named uncarophyllofolic acids A (1) and B (2), respectively, were isolated from the Uncaria rhynchophylla hook bearing stem (Gouteng in Chinese). The distinct stereochemical structures of 1 and 2 were determined by spectroscopic data analysis in combination with acidic hydrolysis and Marfey's derivatization, along with comparison of their specific rotation and Cotton effect (CE) data with those of the biogenetically related known derivatives as well as theoretical calculations of electronic circular dichroism (ECD) spectra. A plausible biosynthetic pathway of 1 and 2, associating to folate metabolism and the previously reported orychophragines A-C from Orychophragmus violaceus, is discussed.

Gastrodin Derivatives from Gastrodia elata.

Nine new gastrodin derivatives, including seven p-hydroxybenzyl-modified gastrodin ethers (1-7), 6'-O-acetylgastrodin (8), and 4-[α-D-glucopyranosyl-(1 →6)-ß-D-glucopyranosyloxy]benzyl alcohol (9), together with seven known derivatives, were isolated from an aqueous extract of Gastrodia elata ("tian ma") rhizomes. Their structures were determined by spectroscopic and chemical methods as well as single crystal X-ray diffraction. Compounds 1-4, 7, 10, and 11 were also isolated from a reaction mixture by refluxing gastrodin and p-hydroxybenzyl alcohol in H2O. As both gastrodin and p-hydroxybenzyl alcohol exist in the plant, the reaction results provide evidence for the production and increase/decrease of potential effective/toxic components when "tian ma" is decocted solely or together with ingredients in Chinese traditional medicine formulations, though the isolates were inactive in the preliminarily cell-based assays at concentrations of 10 µM. Moreover, using ultra-performance liquid chromatography high-resolution electrospray ionization mass spectrometry (UPLC-HRESIMS), 4, 7, 10, and 11, as well as component variations, were detectable in the freshly prepared extracts of different types of samples, including the freeze-dried fresh G. elata rhizomes.

Whole-body spatially-resolved metabolomics method for profiling the metabolic differences of epimer drug candidates using ambient mass spectrometry imaging.

Investigation of the in vivo drug action and metabolic differences of epimer drugs is challenging. Whole-body MSI analysis can visually present the stereoscopic distribution of molecules related to the interaction of drugs and organisms, and can provide more comprehensive organ-specific profiling information. Herein, we developed a whole-body spatially-resolved imaging metabolomics method based on an air flow-assisted ionisation desorption electrospray ionisation (AFADESI)-MSI system coupled with a high-resolution mass spectrometer and highly discriminating imaging software. The epimeric sedative-hypnotic drug candidates YZG-331 and YZG-330 were selected as examples, and rats administered normal or high oral doses were used. By performing multivariate statistical data-mining on the combined MSI data, organ-specific differential ions were screened. By comparing the variations in the relative contents of the drugs, their metabolites, and endogenous neurotransmitters throughout whole-body tissue sections of the rats, rich information that could potentially explain the more significant sedative-hypnotic effects of YZG-330 compared to YZG-331 was obtained. Such as the increased ratio of gamma-aminobutyric acid in the brain and stomach of the rats (0.25, 0.47, 0.68, 0.30, and 0.89 for the control and YZG-331-H, YZG-330-H, YZG-331-L, and YZG-330-L, respectively) were interesting. This study provided a convenient and visual method to investigate in vivo molecular metabolic differences and provide insight towards a better understanding of the pharmacodynamic mechanisms of these sedative-hypnotic drug-candidates.

Sulfur-enriched alkaloids from the root of Isatis indigotica.

Five new sulfur-enriched alkaloids isatithioetherins A-E (1-5), and two pairs of scalemic enantiomers (+)- and (-)-isatithiopyrin B (6a and 6b) and isoepigoitrin and isogoitrin (7a and 7b), along with the known scalemic enantiomers epigoitrin and goitrin (8a and 8b), were isolated and characterized from an aqueous extract of the Isatis indigotica roots. Their structures were determined by extensive spectroscopic data analysis, including 2D NMR and theoretical calculations of electronic circular dichroism (ECD) spectra based on the quantum-mechanical time-dependent density functional theory (TDDFT). Compounds 1-5 represent a novel group of sulfur-enriched alkaloids, biogenetically originating from stereoselective assemblies of epigoitrin-derived units. Isolation and structure characterization of 6a and 6b support the postulated biosynthetic pathways for the diastereomers 9a and 9b via a rare thio-Diels-Alder reaction. Compounds 2 and 4 showed antiviral activity against the influenza virus A/Hanfang/359/95 (H3N2, IC50 0.60 and 1.92 µmol/L) and the herpes simplex virus 1 (HSV-1, IC50 3.70 and 2.87 µmol/L), and 2 also inhibited Coxsackie virus B3 (IC50 0.71 µmol/L).

C19-Diterpenoid alkaloid arabinosides from an aqueous extract of the lateral root of Aconitum carmichaelii and their analgesic activities.

Eight new C19-diterpenoid alkaloid arabinosides, named aconicarmichosides E-L (1-8), were isolated from an aqueous extract of the lateral roots of Aconitum carmichaelii (Fu Zi). Their structures were determined by spectroscopic and chemical methods including 2D NMR experiments and acid hydrolysis. Compounds 1-8, together with the previously reported four neoline 14-O-arabinosides from the same plant, represent the only examples of glycosidic diterpenoid alkaloids so far. At a dose of 1.0 mg/kg (i.p.), as compared with the black control, compounds 1, 2, and 4-6 exhibited analgesic effects with >65.6% inhibitions against acetic acid-induced writhing of mice. Structure-activity relationship was also discussed.

Anti-neuroinflammatory effects of 20C from Gastrodia elata via regulating autophagy in LPS-activated BV-2 cells through MAPKs and TLR4/Akt/mTOR signaling pathways.

20C, a novel bibenzyl compound, is isolated from Gastrodia elata. In our previous study, 20C showed protective effects on tunicamycin-induced endoplasmic reticulum stress, rotenone-induced apoptosis and rotenone-induced oxidative damage. However, the anti-neuroinflammatory effect of 20C is still with limited acquaintance. The objective of this study was to confirm the anti-neuroinflammatory effect of 20C on Lipopolysaccharide (LPS)-activated BV-2 cells and further elucidated the underlying molecular mechanisms. In this study, 20C significantly attenuated the protein levels of nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and interleukin (IL)-1ß, and secretion of nitric oxide (NO) and tumor necrosis factor (TNF)-α induced by Lipopolysaccharide (LPS) in BV-2 cells. Moreover, 20C up-regulated the levels of autophagy-related proteins in LPS-activated BV-2 cells. The requirement of mitogen-activated protein kinases (MAPKs) has been well documented for regulating the process of autophagy. Both 20C and rapamycin enhanced autophagy by suppressing the phosphorylation of MAPKs signaling pathway. Furthermore, 20C treatment significantly inhibited the levels of toll like receptor 4 (TLR4), phosphorylated-protein kinase B (Akt) and phosphorylated-mechanistic target of rapamycin (mTOR), indicating blocking TLR4/Akt/mTOR might be an underlying basis for the anti-inflammatory effect of 20C. These findings suggest that 20C has therapeutic potential for treating neurodegenerative diseases in the future.

Diglycosidic indole alkaloid derivatives from an aqueous extract of Isatis indigotica roots.

Six new indole alkaloid diglycosides named isatigotindolediosides A-F (1-6), along with three known analogs (7-9), were isolated from an aqueous extract of the Isatis indigotica roots (ban lan gen). Their structures including the absolute configurations were determined by comprehensive spectroscopic data analysis, combined with enzyme or acid hydrolysis, and comparison of experimental circular dichroism (CD) and calculated electronic circular dichroism (ECD) spectra. In the preliminary assays, compounds 3, 5, and 8 showed antiviral activity against Coxsackie virus B3.

DJ-1 regulating PI3K-Nrf2 signaling plays a significant role in bibenzyl compound 20C-mediated neuroprotection against rotenone-induced oxidative insult.

Oxidative stress is thought to be involved in the development of Parkinson's disease (PD). We previously reported that 20C, a bibenzyl compound isolated from Gastrodia elata, possesses antioxidative properties, but its in-depth molecular mechanisms against rotenone-induced neurotoxicity remains unknown. Recent studies indicate that without intact DJ-1, nuclear factor erythroid 2-related factor (Nrf2) protein becomes unstable, and the activity of Nrf2-mediated downstream antioxidant enzymes are thereby suppressed. In this study, we showed that 20C clearly protected PC12 and SH-SY5Y cells against rotenone-induced oxidative injury. Furthermore, 20C markedly up-regulated the levels of DJ-1, which in turn activated phosphoinositide-3-kinase (PI3K)/Akt signaling and inhibited glycogen synthase kinase 3ß (GSK3ß) activation, eventually promoted the nuclear translocation of Nrf2 and induced the expression of hemeoxygenase-1 (HO-1). The antioxidant effects of 20C could be partially blocked by ShRNA-mediated knockdown of DJ-1 and inhibition of the PI3K/Akt pathways with Akt1/2 kinase inhibitor, respectively. Conclusively, our findings confirm that DJ-1 is necessary for 20C-mediated protection against rotenone-induced oxidative damage, at least in part, by activating PI3K/Akt signaling, and subsequently enhancing the nuclear accumulation of Nrf2. The findings from our investigation suggest that 20C should be developed as a novel candidate for alleviating the consequences of PD in the future.

Bibenzyl compound 20c protects against endoplasmic reticulum stress in tunicamycin-treated PC12 cells in vitro.

AIM: Accumulation of α-synuclein (α-syn) in the brain is a characteristic of Parkinson's disease (PD). In this study, we investigated whether treatment with tunicamycin, an endoplasmic reticulum (ER) stress inducer, led to the accumulation of α-syn in PC12 cells, and where α-syn protein was accumulated, and finally, whether bibenzyl compound 20c, a novel compound isolated from Gastrodia elata (Tian ma), could alleviate the accumulation of α-syn and ER stress activation in tunicamycin-treated PC12 cells. METHODS: PC12 cells were treated with tunicamycin for different time (6 h, 12 h, 24 h, 48 h). Cell viability was determined by a MTT assay. Subcellular fractions of ER and mitochondria were extracted with the Tissue Endoplasmic reticulum Isolation Kit. The levels of α-syn protein and ER-stress-associated downstream chaperones were detected using Western blots and immunofluorescence. RESULTS: Treatment of PC12 cells with tunicamycin (0.5-10 µg/mL) dose-dependently increased the accumulation of α-syn monomer (19 kDa) and oligomer (55 kDa), and decreased the cell viability. Accumulation of the two forms of α-syn was observed in both the ER and mitochondria with increasing treatment time. Co-treatment with 20c (10-5 mol/L) significantly increased the viability of tunicamycin-treated cells, reduced the level of α-syn protein and suppressed ER stress activation in the cells, evidenced by the reductions in phosphorylation of eIF2α and expression of spliced ATF6 and XBP1. CONCLUSION: Tunicamycin treatment caused accumulation of α-syn monomer and oligomer in PC12 cells. Bibenzyl compound 20c reduces the accumulation of α-syn and inhibits the activation of ER stress, which protected PC12 cells against the toxicity induced by tunicamycin.

20C, a bibenzyl compound isolated from Gastrodia elata, protects PC12 cells against rotenone-induced apoptosis via activation of the Nrf2/ARE/HO-1 signaling pathway.

AIM: Our preliminary study shows that a bibenzyl compound isolated from Gastrodia elata, 2-[4-hydroxy-3-(4-hydroxybenzyl)benzyl]-4-(4-hydroxybenzyl)phenol (designated 20C), protects PC12 cells against H2O2-induced injury. In this study we investigated whether 20C exerted neuroprotective action in a cell model of Parkinson's disease. METHODS: A cell model of Parkinson's disease was established in PC12 cells by exposure to rotenone (4 µmol/L) for 48 h. Cell viability and apoptosis were assessed, and intracellular ROS level and the mitochondrial membrane potential (MMP) were detected. The expression of apoptosis-related proteins Bax, Bcl-2, cytochrome c, cleaved caspase-3, and oxidative stress-related proteins Nrf2, HO-1 and NQO1 were examined using Western blotting. The mRNA levels of HO-1 and NQO1 were determined with RT-PCR. The nuclear translocation of Nrf2 was observed with immunofluorescence staining. RESULTS: Treatment with rotenone significantly increased the number of apoptotic cells, accompanied by marked increases in the Bax/Bcl-2 ratio, cytochrome c release and caspase-3 activation. Rotenone also increased ROS accumulation, reduced MMP, and increased the nuclear translocation of Nrf2 as well as the mRNA and protein levels of the Nrf2 downstream target genes HO-1 and NQO1 in PC12 cells. Co-treatment with 20C (0.01-1 µmol/L) dose-dependently attenuated rotenone-induced apoptosis and oxidative stress in PC12 cells. Nrf2 knockdown by siRNA partially reversed the protective effects of 20C in rotenone-treated PC12 cells. CONCLUSION: The bibenzyl compound 20C protects PC12 cells from rotenone-induced apoptosis, at least in part, via activation of the Nrf2/ARE/HO-1 signaling pathway.