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.

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.

Hepatoprotective hemiterpene glycosides from the rhizome of Cibotium barometz (L.) J. Sm.

Five previously undescribed hemiterpene glycosides, cibotiumbarosides E-I, and two known hemiterpene glucosides, were isolated from the rhizome of Cibotium barometz (L.) J. Sm. The structures of cibotiumbarosides E-I were established by 1D and 2D NMR spectroscopic analyses and HRMS. The absolute configuration of the aglycone of cibotiumbaroside E was assigned by calculated ECD with the TDDFT method. Cibotiumbarosides F and I both exhibited remarkable hepatoprotective activity against APAP-induced acute liver damage in vitro, which were more effective than the positive control, bicyclol. On the other hand, seven hemiterpene glycosides were all inactive in assays of cytotoxicity, neuroprotection, antidiabetes and anti-inflammation.

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.

[Lignanoids from an aqueous extract of the roots of Codonopsis pilosula].

Sixteen lignanoids were isolated from an aqueous extract of the commonly used Chinese traditional medicine Dangshen, the dried roots of Codonopsis pilosula, by using a combination of various chromatographic techniques, including silica gel, macroporous adsorbent resin, MCI resin, sephadex LH-20, and reversed phase semi-preparative HPLC. On the basis of spectral data analysis, their structures were elucidated and identified as（-）-（7R,7'R,8R,8'S）-4,4'-dihydroxy-3,3',5,5',7-pentamethoxy-2,7'-cyclolignane（1）,（-）-（7R,8S）- dihydrodehydrodiconiferyl alcohol 4-O-ß-D-glucopyranosyl-（1'''→2''）-ß-D-glucopyranoside（2）,（-）-（7R,8S）- dihydrodehydrodiconiferyl alcohol（3）,（+）-（7S,8R）-dehydrodiconiferyl alcohol（4）,（+）-balanophonin（5）,（+）- demethoxypinoresinol（6）,（+）-pinoresinol（7）,（+）-epipinoresinol（8）,（-）-syringaresinol（9）,（-）-medioresinol（10）,（-）-lariciresinol（11）,（-）-secoisolariciresinol（12）,（-）-ent-isolariciresinol（13）,（+）-（7S,8S）-3-methoxy-3',7- expoxy-8,4'-neolignan-4,9,9'-triol（14）,（+）-（7S,8R）-3',4-dihydroxy-3-methoxy-8,4'-neolignan（15）, and（-）-（7R,8R）-3',4-dihydroxy-3-methoxy-8,4'-neolignan（16）. All these compounds were isolated from C. pilosula for the first time, while compound 1 is a new natural product of 2,7'-cyclolignan and 2 is a new 4',7-epoxy- 8,3'-neolignan diglucoside. Compound 12 showed activity against Fe(2+)-cysteine induced rat liver microsomal lipid peroxidation with an inhibition ratio of（63.4 ± 8.3） % at 1×10(-5) mol·L(-1).

[Central-adenosine A1 receptor involved in the thermal regulation effect of YZG-330, a N6-substituted adenosine derivative, in mice].

Adenosine receptors (AR) play an important role in the regulation processes for body temperature and vigilance states. During our previous studies, we noticed that aminophylline (a non-selective, blood-brain-barrier penetrably AR antagonist) could attenuate the effects of YZG-330 [(2R,3S,4R,5R)-2-(hydroxymethyl-5-(6-(((R)-1-phenylpropyl)amino)-9H-purin-9-yl)tetrahydrofuran-3, 4-diol] on lowering the body temperature. Hereby, we focused ourselves on the character of thermal regulation effect of YZG-330 in mice and tried to specify the receptor subtype via giving typical adenosine receptor antagonists. The results showed that both of the magnitude and lasting time of the effect that YZG-330 played on decreasing body temperature are in a dose-dependent manner: within the next 3 hour after intragastric administration (ig) of 0.25, 1 or 4 mg . kg-1 YZG-330, the extreme values on body temperature decreasing were (1.2 ± 0.3) °C, (3.6 ± 0.4) °C (P<0.001) and (7.4±0.5) °C (P<0.001), separately; whereas the duration that body temperature below 34 °C were 0, (10±5) and (153±4) min, separately. Adenosine A1 receptor (A1R) antagonist (DPCPX) could effectively reverse YZG-330's effect on decreasing body temperature, with intraperitoneal administration of DPCPX (5 mg . kg-1) 20 min prior than YZG-330 (4 mg.kg-1, ig), the extreme value on body temperature decreasing was (3.5 ± 0.7) °C (P<0.001), the duration that body temperature below 34 °C was (8±6) min (P<0.001). However, adenosine A2a receptor antagonist, SCH-58261, did not show any influence on the effects of YZG-330 at all. Combined with the fact that 8-SPT (a non-selective, blood-brain-barrier impenetrably AR antagonist) did not reverse the effect of YZG-330, we come to the conclusion that central-adenosine A, receptor plays a significant role on the thermal regulation effect of YZG-330.

EM-E-11-4 increases paclitaxel uptake by inhibiting P-glycoprotein-mediated transport in Caco-2 cells.

P-glycoprotein (P-gp) overexpression is the main mechanism involved in chemotherapy drug resistance such as paclitaxel resistance and therapy failure. The most widely studied P-gp inhibitors still have limited ability to reverse resistance in the clinic. In this study, EM-E-11-4, a lathyrane-type diterpenoid isolated from Euphorbia micractina, was found to significantly increase paclitaxel uptake in Caco-2 cells, which functionally overexpressed P-gp. In vitro transport experiments, carried out in the Caco-2 monolayer model, indicated that EM-E-11-4 significantly reduced the efflux ratio of paclitaxel transport by inhibiting P-gp function without affecting P-gp expression. We also found that EM-E-11-4 enhanced the intracellular accumulation of paclitaxel in a dose-dependent manner by LC-MS/MS and EM-E-11-4 showed low cytotoxicity. Hence, EM-E-11-4 is an effective potential agent to reverse P-gp-mediated paclitaxel resistance by inhibiting P-gp transport function and increasing the intracellular concentration of paclitaxel.

4-Hydroxybenzyl-substituted glutathione derivatives from Gastrodia elata.

Seven new 4-hydroxybenzyl-substituted glutathione derivatives (2-8), together with a known analogue (1), were isolated from the aqueous extract of Gastrodia elata Blume rhizomes. Their structures were determined by using spectroscopic and chemical methods. The absolute configurations of 1-8 were assigned by using Marfey's method, combined with comparing the NMR and CD spectroscopic data of sulfoxide moieties in 3-6 with those of S-(4-hydroxybenzyl)cysteine sulfoxide stereoisomers (9-12) synthesized as authentic samples. The configurations of 9-12 were confirmed by electronic CD calculations based on the quantum-mechanical time-dependent density functional theory. Furthermore, the structures of 1, 3, 5, 7, and 8 were verified by synthesis. Compound 3 was active against serum deprivation-induced PC12 cell damage and synthetic 9-14 exhibited activity against Fe(2+)-cysteine induced rat liver microsomal lipid peroxidation.

[Studies on chemical constituents of aqueous extract of Lonicera japonica flower buds].

From an aqueous extract of Lonicera japonica flower buds, sixteen compounds were isolated by a combination of various chromatographic techniques including column chromatography over macroporous resin, MCI gel, silica gel, and sephadex LH-20 and reversed-phase HPLC. Their structures were elucidated by spectroscopic data analysis as 6'-O-acetylvogeloside (1), 6'-O-acetylsecoxyloganin (2), dichlorogelignate (3), guanosinyl-(3' --> 5')-adenosine monophosphate(GpA,4) , 5'-O-methyladenosine (5), 2'-O-methyladenosine (6), adenosine (7), syringin (8), methyl 4-O-ß-D-glucopyranosyl caffeate (9), (-)-dihydrophaseic acid 4'-O-ß-D-glucopyranoside (10), ketologanin (11), 7α-morroniside (12), 7ß-morroniside (13), kingiside (14), cryptochlorogenic acid methyl ester (15), and 6-hydroxymethyl-3-pyridinol (16). All the compounds were obtained from this plant for the first time, compounds 1 and 2 are new compounds, 3 and 5 are new natural products, and 4 is the first example of dinucleoside monophosphate isolated from a plant extract.

NHBA isolated from Gastrodia elata exerts sedative and hypnotic effects in sodium pentobarbital-treated mice.

The rhizomes of Gastrodia elata have been used for the treatment of insomnia in oriental countries. N6-(4-hydroxybenzyl) adenine riboside (NHBA) was originally isolated from G. elata. For the first time we report a detailed study on the effects and mechanisms of NHBA on its sedative and hypnotic activity. Adenosine, an endogenous sleep factor, regulates sleep-wake cycle via interacting with adenosine A1/A(2A) receptors. Using radioligand binding studies and cAMP accumulation assays, our results show that NHBA may be a functional ligand for the adenosine A1 and A(2A) receptors. NHBA significantly decreases spontaneous locomotor activity and potentiates the hypnotic effect of sodium pentobarbital in mice. Sleep architecture analyses reveal that NHBA significantly decreases wakefulness time and increases NREM sleep times. However, NHBA does not affect the amount of REM sleep. Pretreatment with the adenosine A1 receptor antagonist DPCPX or the A(2A) receptor antagonist SCH 58261 significantly reverses the increase in sleeping time induced by NHBA in sodium pentobarbital treated mice. Immunohistochemical studies show that NHBA increases c-Fos expression in GABAergic neurons of the ventrolateral preoptic area (VLPO), which suggests that NHBA activates the sleep center in the anterior hypothalamus. Altogether, these results indicate that NHBA produces significant sedative and hypnotic effects. Such effects might be mediated by the activation of adenosine A1/A(2A) receptors and stimulation of the sleep center VLPO.

Butanolide derivatives from the bark of Machilus yaoshansis.

Six new butanolide derivatives with long aliphatic side chains (1-6), together with 23 known lipophilic constituents, were isolated from the bark of Machilus yaoshansis. Their structures were elucidated by spectroscopic and chemical methods. All the isolates were evaluated for cytotoxic and anti-inflammatory activities.

[Protective effect of new adenosine analog B2 against serum deprivation-induced PC12 cell injury].

This study is to investigate the effect of compound B2 on the damage of PC12 cells induced by serum deprivation and to explore its related mechanisms. The binding characteristics of B2 to rat striatum adenosine A2A receptor was studied by radioligand 3H-MSX-2 binding assay. Cell viability was detected by MTT assay. ROS formation was measured after DCFDA fluorescent staining. B2 has affinity to rat adenosine A2A receptor (K1 = 0.37 micromol x L(-1)). B2 remarkably increased PC12 cell survival rate in serum deprivation-induced PC12 cells. The percentage of serum deprivation-induced death of PC12 was 49.6%, and the treatment of B2 (0.1-100 micromol x L(-1)) increased the cell viability to 63.3%, 74.9%, 86.3% and 88.1%, respectively. Adenosine A2A receptor antagonist SCH 58261 could significantly block the protective effect of B2. The cell viability with 0.1 micromol x L(-1) SCH 58261 decreased by 16.1%, 24.0% and 19.8%, in the presence of B2 (0.1-10 micromol x L(-1)). Serum deprivation-induced ROS formation was 3.5 times more than that of control group, and treatment with B2 significantly and dose-dependently inhibited ROS over-formation. The protective effect of B2 may be related with adenosine A2A receptor. Decrease of serum-deprivation induced ROS formation may also be one of the mechanisms.

Minor constituents from the tubers of Gymnadenia conopsea.

Four new minor constituents including two cyclodipeptides (1 and 2) and two cyclopentene derivatives (3 and 4), together with four known cyclodipeptides, have been isolated from an ethanolic extract of the tubers of Gymnadenia conopsea. Their structures including absolute configurations were determined by spectroscopic data interpretation combined with chemical methods. Among them, compound 1 contains an abnormal S-(4''-hydroxybenzyl)cysteine residue, 3 and 4 possess [(4-methylcyclopentyl)methyl]benzene and (4-hydroxymethylcyclopentyl)benzene carbon skeletons, respectively, both of which are first found from the natural source.

Cytotoxic triterpenoid glycosides from the roots of Gordonia chrysandra.

Eight new oleanane triterpenoid glycosides, gordonosides A-H (1-8), were isolated from a 50% EtOH extract of the roots of Gordonia chrysandra. Their structures were determined by spectroscopic analysis, including 1D and 2D NMR and ESIMS, and by chemical methods. Among these substances, compounds 1, 3, 5, and 6 exhibited cytotoxic activity against several human cancer cell lines, with 3 being the most potent.

Triterpenoids from the roots of Pterospermum heterophyllum Hance.

Two new triterpenoids taraxer-14-ene-1alpha,3beta-diol (1) and 3beta-hydroxytaraxer-14-ene-1-one (2), together with the known triterpenes taraxerol (3), betulin (4), betulinic acid (5), sumaresinolic acid (6), and 5-hydroxy-2-methoxy-1,4-naphthoquinone (7), 5,7-dihydroxy-6,8-dimethylchromone (8), alpha-monpalmitin (9), palmitic acid (10), 6beta-hydroxystigmast-4-en-3-one (11), beta-sitostero1 (12), have been isolated from the petroleum ether fraction of the ethanolic extract of Pterospermum heterophyllum. Their structures were established by spectroscopic methods including IR, MS, 1D, and 2D NMR experiments. Compounds 1-8 were evaluated against several human cancer cell lines. Compound 1 showed in vitro selective cytotoxicity against human lung cancer cell lines (A549) with an IC(50) value of 1.22 microM. Compound 7 showed significant cytotoxicity against the A549, HCT-8, Bel7402, BGC-823, and A2780 cancer cell lines with IC(50) values of 0.21, 0.55, 0.40, 0.59, and 0.34 microM, respectively. However, the other compounds were inactive (IC(50)>10 microM).

[Coumarins from branch of Fraxinus sieboldiana and their antioxidative activity].

OBJECTIVE: To investigate the chemical constituents from the branch of Fraxinus sieboldiana, and evaluate their antioxidative activity. METHOD: The chemical constituents were isolated and purified by chromatographic techniques over silica gel, macroporous adsorbent resin, Sephadex LH-20, and preparative HPLC. Structures of the compounds were identified by spectroscopic methods. The antioxidant activity was evaluated by Fe(+2)-cystine induced rat liver microsomal lipid peroxidation. RESULT: Eight coumarins were obtained and their structures were elucidated as esculin (1) , esculetin (2), fraxin (3), fraxetin (4), 6, 7-di-O-beta-D-glucopyranosylesculetin (5), scopoletin (6), cleomiscosin D (7) and cleomiscosin B (8). At a concentration of 10(-6) mol x L(-1), compound 4 showed antioxidative activity inhibiting Fe(+2)-cystine induced rat liver microsomal lipid peroxidation with inhibitory rate of 60%. CONCLUSION: Compounds 5, 7 and 8 were obtained from the genus Fraxinus for the first time. Compound 4 showed remarkable antioxidative activity, which was higher than that of VE (35%).

Terpenoids from the tuber of Cremastra appendiculata.

Two new terpenoids including a cadinane sesquiterpene (1), and an ent-kaurane diterpene diglycoside (2), together with a known triterpene containing 32 carbons (3), have been isolated from the ethanolic extract of Cremastra appendiculata. Their structures were established by the spectroscopic methods including the IR, MS, 1D-, and 2D-NMR experiments as ( - )-cadin-4,10(15)-dien-11-oic acid (1), ( - )-ent-12beta-hydroxykaur-16-en-19-oic acid, 19-O-beta-D-xylopyranosyl-(1 --> 6)-O-beta-D-glucopyranoside (2), and (+)-24,24-dimethyl-25,32-cyclo-5alpha-lanosta-9(11)-en-3beta-ol (3). Compounds 1-3 were evaluated against several human cancer cell lines. Compound 3 showed in vitro-selective cytotoxicity against human breast cancer cell lines (MCF-7) with an IC50 of 3.18 microM, but 1 and 2 were inactive (IC50>10 microg/ml).