Integrating network pharmacology analysis and pharmacodynamic evaluation for exploring the active components and molecular mechanism of moutan seed coat extract to improve cognitive impairment.

Paeonia suffruticosa (Moutan) is a traditional medicinal plant in China. Its seed coat is rich in resveratrol oligomer, especially suffruticosol B (SB). Previous studies had shown that the seed coat extracts of Paeonia suffruticosa (PSCE) had good cholinesterase inhibitory activity and neuroprotective effect, but the effective dose range was unknown, and the pharmacodynamic components and molecular mechanism of PSCE had not been discussed. The current study aimed to screen the pharmacodynamic components in PSCE and investigate the improvement effect of PSCE and the selected SB on scopolamine-induced cognitive dysfunction in mice and its mechanism. The results of high-throughput sequencing and bioinformatics analysis showed that suffruticosol B (SB) and trans-gnetin H (GH) might be the main active components of PSCE; PSCE might improve cognitive dysfunction through p53, HIF-1, MAPK, and PI3K-Akt signaling pathways, while SB and GH might improve cognitive dysfunction through HIF-1 signaling pathway. SB and GH had good molecular docking activity with the target of HIF-1 signaling pathway. The pharmacodynamic activities of PSCE and SB were further verified by behavioral experiments. PSCE and SB could improve the recognition ability of familiar and new objects and shorten the escape latency in the Morris Water Maze test (PSCE 120 mg∙kg-1, p < 0.05; SB 60 mg∙kg-1, p < 0.01); PSCE and SB could increase Ach and GSH levels, enhance the activities of ChAT, SOD and CAT, decrease the levels of IL-1ß, IL-6, and TNF-α, and decrease the activity of AChE. In conclusion, the results indicated that PSCE might exert pharmacodynamic activity through multiple components, targets, and pathways, and SB and GH might be the main active components of PSCE. PSCE and SB might improve cognitive dysfunction by regulating cholinergic, antioxidant, and anti-inflammatory effects. These results indicated that PSCE and SB might be potential anti-AD drug candidates, providing a scientific basis for the development and utilization of Moutan bark.

Structural characterization, hypoglycemic effects and antidiabetic mechanism of a novel polysaccharides from Polygonatum kingianum Coll. et Hemsl.

The rhizome of Polygonatum kingianum has been used as a traditional medicine in China. In this study, a novel polysaccharides (PKPs-1) was isolated from P. kingianum and characterized by its molecular weight, primary structure. The hypoglycemic activity of PKPs-1was investigated by in vitro assay with the HepG2 cell line and in vivo test using STZ-induced diabetic mice. Results showed that the average molecular weight of PKPs-1 was 14.05 kDa and is composed mainly of glucose and mannose. Methylation analysis indicated that this polysaccharides fraction consisted mainly of ß1,2-link glucose. Besides, PKPs-1 exhibited significant anti-hyperglycemic activity on STZ-induced mice, improved insulin tolerance, and affected the metabolism of serum lipids. Results of real-time quantitative PCR (RT-PCR) showed that PKPs-1 significantly increased the expression of insulin receptor substrate-1 (IRS-1), phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT), indicating that PKPs-1 regulates glucose metabolism by activating the PI3K/AKT signaling pathway. This study provides new insights for investigating the hypoglycemic effects of PKPs-1 and suggests that PKPs-1 could be a promising functional food or medicine for treating T2DM.

Crepidtumines A and B, Two Novel Indolizidine Alkaloids from Dendrobium crepidatum.

Two new indolizidine alkaloids crepidatumines A (1) and B (2) together with the stereoisomer of dendrocrepidine B (3) and known analog dendrocrepine (4) were isolated from D. crepidatum. Their structures were determined by HR-ESI-MS, NMR, and Electronic Circular Dichroism (ECD) experiments together with comparison of analogues. Compound (1) possess a (5/6/6/5) tetra-hetero-cyclic ring, whereas compound (2) contains a tricyclic system with an unusual bridged ring, which are the first report in Nature. The biological evaluation revealed that dendrocrepine (4) displayed a potent hypoglycemic effect in vitro.

Stereochemical determination of four 10-membered ring resorcylic acid lactones from the desert plant endophytic fungus Chaetosphaeronema hispidulum.

Four 10-membered ring resorcylic acid lactones (RALs) including a new compound hispidulactone F (1) and three known analogs hispidulactone B (2), 2 R, 4R-sonnerlactone (3), and 2 R, 4S-sonnerlactone (4) were isolated from the special bioenvironmental desert plant endophytic fungus Chaetosphaeronema hispidulum. The structure of the new compound hispidulactone F (1) was determined by extensive spectra analysis including HR-ESI-MS, NMR (1H, 13C, 1H-1H COSY, HSQC, and HMBC). Hispidulactone F (1) and hispidulactone B (2) were a pair of stereoisomers at C-3, whereas 2 R, 4R-sonnerlactone (3) and 2 R, 4S-sonnerlactone (4) were another pair of stereoisomers at C-4. The stereochemistries of the hydroxyl groups at C-3 in 1 and 2, and at C-4 in 3 and 4 were first determined by modified Mosher's reactions. Thus, the absolute configuration C-3 in hispidulactone B (2) was not right in our previous report, and was rectified to be R. Compounds 1 and 4 were evaluated for their cytotoxic effects on the proliferation of HepG2. The possible biosynthetic pathway of compounds 1-4 was also presented.

Screening of acetylcholinesterase inhibitors and characterizing of phytochemical constituents from Dichocarpum auriculatum (Franch.) W.T. Wang & P. K. Hsiao through UPLC-MS combined with an acetylcholinesterase inhibition assay in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Dichocarpum is endemic to East Asia, and many of them are traditionally used folk medicine in China. Dichocarpum auriculatum (Franch.) W. T. Wang et P. K. Hsiao has the effect of clearing away heat, removing toxicity, and relieving swelling in southwestern China. Intriguingly, its root and whole herb also used as remedy for the neurological disease epilepsy. However, there are not any scientific reports on the phytochemistry and pharmacological activities of D. auriculatum. AIM OF STUDY: Traditional and folk medicinal knowledge would be useful for finding new pharmaceutical resources. There are many evidences over the years reported that an interaction probably exists between epilepsy and Alzheimer's disease (AD). The aim of the study was to investigate the potential AChE inhibitors and the phytochemical profiles of the specie D. auriculatum. MATERIALS AND METHODS: The AChE inhibitory activity of plant extracts of D. auriculatum and other 6 species from different regions of the genus Dichocarpum were evaluated in vitro assays and the UPLC-Q-TOF-MS technique was used to analyze the chemical constituents. Moreover, UPLC-ESI-MS/MS was used to determine the distribution of 12 standard compounds in samples. RESULTS: As a preferred source of potential acetylcholinesterase inhibitors of the genus Dichocarpum, D. auriculatum has been further investigated. The screening results show that the ability of root extracts from D. auriculatum (IC50 = 0.15 mg·mL-1) to inhibit AChE was better than other samples, it is consistent with traditional medicinal records. The phytochemical constituents of D. auriculatum was surveyed firstly by UPLC-Q-TOF-MS analysis, and 36 compounds, including 14 alkaloids, 16 flavonoids, 6 others, were identified tentatively. Further experiments showed that five compounds (columbamine, palmatine, dauricine, jatrorrhizine and berberine) from D. auriculatum were confirmed the potential inhibition of AChE activity in vitro (IC50: 0.24-6.37 µM) and UPLC-ESI-MS/MS results showed that the content of most active compounds in roots was much higher than in aerial parts. Palmatine (IC50 = 0.34 µM) and columbamine (IC50 = 0.24 µM) showed prominent AChE inhibitory activity among the tested compounds. CONCLUSIONS: This is the first report about the evaluation of AChE inhibitory activity and phytochemical profiles of D. auriculatum, led to the identification of 36 compounds including alkaloids and flavonoids, and five alkaloids exhibited a significant AChE inhibitory activity and had the potential as AChE inhibitors. This study provided scientific experimental basis for the traditional efficacy of neurological disease of the plant.

Crepidatumines C and D, Two New Indolizidine Alkaloids from Dendrobium crepidatum Lindl. ex Paxt.

Two new indolizidine alkaloids, crepidatumines C (1) and D (2), together with crepidine (3), isocrepidamine (4), and crepidamine (5) were isolated from the Dendrobium crepidatum Lindl. ex Paxt. X-ray diffraction experiments established the absolute configurations of known compounds 3 and 4. The planar structures and relative configurations of new compounds 1 and 2 were elucidated by extensive spectra analysis including HR-ESI-MS, NMR (1H, 13C, 1H-1H COSY, HSQC, HMBC, and NOESY spectra), and the absolute configurations of 1 and 2 were suggested on the basis of possible biosynthetic pathways. The biological results confirmed that isocrepidamine (4) displayed a potent hypoglycemic effect in vitro without cytotoxicity.

Ling-Yang-Gou-Teng-decoction prevents vascular dementia through inhibiting oxidative stress induced neurovascular coupling dysfunction.

ETHNOPHARMACOLOGICAL RELEVANCE: Vascular dementia (VaD) is the common cognitive disorder derived mainly from lacunar stroke (LS). The oxidative stress induced neurovascular coupling (NVC) dysfunction involves in the pathogenesis of VaD. Currently, there is no specific drug for VaD. Ling-Yang-Gou-Teng -Decoction (LG), a well-known traditional Chinese formula, has been used for preventing VaD in clinic. AIM OF THE STUDY: In this study, we aimed to investigate the underlying mechanism of LG on VaD in rats. MATERIALS AND METHOD: VaD was replicated with autologous micro-thrombi against the background of hypercholesterolemia induced with high fatty diet. PTX (68.90 mg/kg/day), LG with three dosages (2.58, 8.14, 25.80 g/kg/day) was orally administrated to VaD rats, respectively. The NVC sensitivity was defined as the ratio of the microcirculative cerebral blood velocity (CBV) to the electroencephalograph (EEG) before and after penicillin stimulation. Behavioral performance, pathological changes of brain and oxidation related molecules were detected to assess the effects of LG on VaD. RESULTS: LG exhibited beneficial effects on the VaD, which was demonstrated as improved exploratory, learning and memory abilities, relieved vascular or neural pathological changes in cerebral cortex or hippocampus. LG maintained NVC sensitivity, which was confirmed as significantly increased ΔCBV and the elevated ratio of ΔCBV/ΔqEEG. The underlying mechanisms of LG was associated with antioxidant effects, which was confirmed as significantly decreased nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) expression, and increased superoxide dismutase 3 (SOD3) expression. LG also reduced iNOS, increased nNOS and eNOS expression to restore NO bioavailability. CONCLUSIONS: The results suggested that LG prevented VaD may associate with inhibiting oxidative stress, protecting NO bioavailability, and then maintaining NVC sensitivity.

A novel pharmacodynamic model in rats for preventing vascular dementia from maintaining neurovascular coupling sensitivity.

Vascular dementia (VaD) is the common cognitive disorder derived mainly from lacunar stroke. The neurovascular coupling (NVC) dysfunction involves in its pathogenesis. VaD lacks suitable animal models for developing preventive therapies. This study aimed to confirm a model for preventing VaD via maintaining NVC sensitivity in rats. The model was replicated with autologous microthrombi against the background of hypercholesterolemia. A phosphodiesterase inhibitor (pentoxyfylline) was preventively administrated to confirm the role of NVC sensitivity. Cognitive function was evaluated as exploratory, learning and memorizing abilities. NVC sensitivity was defined as the ratio of microcirculative cerebral blood flow (∆CBF) to the quantitative electroencephalograph (∆qEEG) before and after penicillin stimulation. The pathogenesis of NVC dysfunction was explored as expressions of neuronal (nNOS), inducible (iNOS) and endothelial nitric oxide synthase (eNOS) in cerebral cortex. The model rats showed cognitive impairment, microvascular edema (2.54 ±â€¯0.30%, P < 0.01), neuronal edema (1.24 ±â€¯0.48%, P < 0.01) and nissl body loss (0.03 ±â€¯0.003%, P < 0.01) in cerebral cortex, and neuronal necrosis in hippocampal CA1 region (neuronal cell number 41.76 ±â€¯10.04 cells, P < 0.01) compared with sham group. The NVC dullness in model rats was confirmed as significantly decreased ratio of ∆CBF/∆qEEG (0.05 ±â€¯0.02%, P < 0.01) compared with sham group (0.20 ±â€¯0.06%). The underlying mechanism of NVC dysfunction was found as imbalanced NOS expressions (decreased nNOS and eNOS, while increased iNOS levels in cerebral cortex). The NVC dullness was significantly relieved in pentoxyfylline administrated rats (0.12 ±â€¯0.06%, P < 0.01). It indicated that this model was suitable to evaluate candidates for preventing VaD via maintaining NVC sensitivity.

Hypolipidemic activity and mechanisms of the total phenylpropanoid glycosides from Ligustrum robustum (Roxb.) Blume by AMPK-SREBP-1c pathway in hamsters fed a high-fat diet.

The aim of this study was to evaluate the hypolipidemic effect and mechanisms of total phenylpropanoid glycosides extracted from Ligustrum robustum (Roxb.) Blume (LRTPG) in hamsters fed a high-fat diet and to discover bioactive components in HepG2 cell model induced by oleic acid. LRTPG of high (1.2 g/kg), medium (0.6 g/kg), and low (0.3 g/kg) doses was administrated daily for 21 consecutive days in hamsters. We found that in hamsters fed a high-fat diet, LRTPG effectively reduced the concentrations of plasma triglycerides (TG), free fatty acid, total cholesterol, low-density lipoprotein cholesterol, and hepatic TG and total cholesterol. And the compounds acteoside, ligupurpuroside A, ligupurpuroside C, and ligupurpuroside D significantly inhibited lipid accumulation in HepG2 cell at the concentration of 50 µmol/L. Mechanism research demonstrated that LRTPG increased the levels of phospho-AMP-activated protein kinase and phospho-sterol regulatory element binding protein-1c in liver, further to suppress the downstream lipogenic genes as stearoyl-CoA desaturase 1, glycerol-3-phosphate acyltransferase, 1-acylglycerol-3-phosphate O-acyltransferase 2, and diacylglycerol acyltransferase 2. In addition, LRTPG increased the hydrolysis of circulating TG by up-regulating lipoprotein lipase activities. These results indicate that LRTPG prevents hyperlipidemia via activation of hepatic AMP-activated protein kinase-sterol regulatory element binding protein-1c pathway.

Crystal structure of the second fibronectin type III (FN3) domain from human collagen α1 type XX.

Collagen α1 type XX, which contains fibronectin type III (FN3) repeats involving six FN3 domains (referred to as the FN#1-FN#6 domains), is an unusual member of the fibril-associated collagens with interrupted triple helices (FACIT) subfamily of collagens. The results of standard protein BLAST suggest that the FN3 repeats might contribute to collagen α1 type XX acting as a cytokine receptor. To date, solution NMR structures of the FN#3, FN#4 and FN#6 domains have been determined. To obtain further structural evidence to understand the relationship between the structure and function of the FN3 repeats from collagen α1 type XX, the crystal structure of the FN#2 domain from human collagen α1 type XX (residues Pro386-Pro466; referred to as FN2-HCXX) was solved at 2.5 Šresolution. The crystal structure of FN2-HCXX shows an immunoglobulin-like fold containing a ß-sandwich structure, which is formed by a three-stranded ß-sheet (ß1, ß2 and ß5) packed onto a four-stranded ß-sheet (ß3, ß4, ß6 and ß7). Two consensus domains, tencon and fibcon, are structural analogues of FN2-HCXX. Fn8, an FN3 domain from human oncofoetal fibronectin, is the closest structural analogue of FN2-HCXX derived from a naturally occurring sequence. Based solely on the structural similarity of FN2-HCXX to other FN3 domains, the detailed functions of FN2-HCXX and the FN3 repeats in collagen α1 type XX cannot be identified.

Abrin P2 suppresses proliferation and induces apoptosis of colon cancer cells via mitochondrial membrane depolarization and caspase activation.

To explore the cytotoxic mechanism of abrin P2 on human colon cancer HCT-8 cells, abrin P2 was isolated from the seed of Abrus precatorius L. It was found that abrin P2 exhibited cytotoxicity toward 12 different human cancer cell lines. Our results demonstrated that abrin P2 suppressed the proliferation of human colon cancer cells (HCT-8 cells) and induced cell cycle arrest at the S and G2/M phases. The mechanism by which abrin P2 inhibited cell proliferation was via the down-regulation of cyclin B1, proliferating cell nuclear antigen and Ki67, as well as the up-regulation of P21. In addition, abrin P2 induced a dose- and time-dependent increase in the rate of HCT-8 cell apoptosis. Treatment with both Z-VAD-FMK, a broad-spectrum caspase inhibitor, and abrin P2 demonstrated that abrin P2 induced HCT-8 cell apoptosis via the activation of caspases. Together, our results revealed that abrin P2-induced apoptosis in HCT-8 cells was associated with the activation of caspases-3/-8/-9, the reduction in the Bcl-2/Bax ratio, the loss of mitochondrial membrane potential, and the increase in cytochrome c release. We further showed that abrin P2 administration effectively suppressed the growth of colon cancer xenografts in nude mice. This is the first report that abrin P2 effectively inhibits colon cancer cell growth in vivo and in vitro by suppressing proliferation and inducing apoptosis.

A rat model of vascular dementia for evaluating Chinese medicine prescriptions.

OBJECTIVE: To develop a new model of vascular dementia for evaluating Chinese medicine prescriptions. METHODS: Eighty-eight male Wistar rats were randomly divided into 4 groups. At d00, d42, d70, d98 (ni=20, 20, 24, 24) during fatty-feeding, rats in each group were further divided into 10 or 12 subgroups (ni=2), respectively. Lacunar stroke were replicated with the injection of thrombi which coagulated artificially from itself blood. The median lethal doses (LD50) were regressed from accumulative mortality in each geometric thrombus doses (k=0.75, 0.5, 0.85, 0.85), respectively. The degree of vascular dementia was evaluated as exploratory, learning and memorizing abilities. The median effective dose of thrombus for replicating rat model was regressed from dementia scores which were derived from the abilities. The linear correlation was regressed between the values of LD50 or effective dose (ED50) and the durations (days) of hypercholesterolemia. This model of vascular dementia was pathologically confirmed as the neural injuries from lacunar stroke in rats. RESULTS: The hypercholesterolemia was indicated as elevated total cholesterol, triglyeerides low-density lipoprotein cholesterol, and decreased high-density lipoprotein cholesterol. The values of LD50 with its 95% confidence intervals (CI) were 1525.0 (1361.0-1709.0), 584.3 (490.1-696.6), 168.7 (163.7-173.8), or 62.4 (59.5-65.4) mg/mL, at d00, d42, d70, and d98, respectively. There is a linear regression between the values of LD50 and the durations of hypercholesterolemia (y=-15.33x+1390.0, r=0.963, P<0.05). The values of ED50 with its 95% CI were 528.8 (340.5-821.4), 217.0 (20.84-2259.0), 96.3 (23.4-402.6), or 47.0 (43.7-50.6) mg/mL from dementia score, at d00, d42, d70, and d98, respectively. There is a linear regression between the values of ED50 and the durations of hypercholesterolemia (y=-4.992x+484.2, r=0.965, P<0.05). The neural injuries were demonstrated as neural degeneration and necrosis. CONCLUSIONS: For evaluating Chinese medicine, a model of vascular dementia in rats is set up with the lacunar stroke from self-thrombosis during hypercholesterolemia. This model from lacunar stroke is useful to investigate the pathogenesis and treatment of vascular dementia.

Anti-obesity effect of total phenylpropanoid glycosides from Ligustrum robustum Blume in fatty diet-fed mice via up-regulating leptin.

ETHNOPHARMACOLOGICAL RELEVANCE: In Chinese folk medicine, the leaves of Ligustrum robustum Blume (LR) were commonly used in the treatment of obesity and hyperlipidemia. This study aimed to evaluate the anti-obesity effect and mechanisms of total phenylpropanoid glycosides from Ligustrum robustum Blume (LRTPG) in fatty diet-fed C57BL/6J mice. MATERIALS AND METHODS: C57BL/6J mice were divided randomly into 6 groups, i.e., control, model, positive (Orlistat 0.12g/kg), and LRTPG at three dosages (0.3, 0.6 or 1.2g/kg), respectively. Control mice were fed with standard diet; the others were fed with fatty diet. After 4 weeks׳ modeling, therapy mice were intragastrically administrated with positive drug or LRTPG for 5 weeks, respectively. Pharmacodynamic effects including body weight, fat weight, Lee׳s index, serum lipid levels, morphological changes and adipocyte area ratio were evaluated. The mechanisms were explored as the factors related to lipids metabolism in gene expressions by real-time PCR, and assured as the protein level of differential gene by Western blotting. RESULTS: The anti-obesity effects of LRTPG in all treated mice were shown as decreased body weight, fat mass, Lee׳s index, total cholesterol (TC) level, and adipocyte area. The mechanisms were demonstrated as elevated mRNA and protein levels of adipose leptin, and consequently decreasing mRNA of adipose acyl coenzyme A: diacylglycerol acyltransferase (DGAT) with increasing mRNA of hepatic cholesterol 7α-hydroxylase (CYP7A1), which led to inhibition of triglyceride (TG) synthesis and promotion of cholesterol catabolism. CONCLUSIONS: The anti-obesity effect of LRTPG in fatty diet-fed mice was related to the up-regulation of leptin, which may provide scientific evidence supporting the traditional usage of LR on obesity in China.

Anticancer effect of total annonaceous acetogenins on hepatocarcinoma.

OBJECTIVE: To confirm the anticancer effect of total annonaceous acetogenins (TAAs) abstracted from Annona squamosa Linn. on human hepatocarcinoma. METHODS: The inhibitory effect of TAAs was demonstrated in H22-bearing mice. The potency of TAAs was confirmed as its 50% inhibiting concentration (IC50) on Bel-7402 cell under Sulfur Rhodamine B staining. Both underlying mechanisms were explored as cellular apoptosis and cell cycle under flow cytometry. Mitochondrial and recipient apoptotic pathways were differentiated as mitochondrial membrane potential under flow cytometry and caspases activities under fluorescence analysis. RESULTS: The inhibitory rate of TAAs in mice was 50.98% at 4 mg/kg dose. The IC50 of TAAs on Bel-7402 was 20.06 µg/mL (15.13-26.61µg/mL). Effective mechanisms of TAAs were confirmed as both of arresting cell cycle at G1 phase and inducing apoptosis dose- and time-dependently. Mitochondrial and recipient pathways involved in apoptotic actions of TAAs. CONCLUSION: TAAs is effective for hepatocarcinoma, via inhibiting proliferation and inducing apoptosis.

Hyperlipidemic guinea pig model: mechanisms of triglyceride metabolism disorder and comparison to rat.

Guinea pigs and rats are both common animal models for hyperlipidemia studies. However, many recent studies have suggested that rats do not develop hypertriglyceridemia in response to cholesterol feeding. In the present work, the differences in triglyceride metabolism between guinea pigs and rats were investigated. Feeding a high-fat diet containing 0.1% cholesterol and 10% lard for 4 weeks led to a significant increase in plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG) and free fatty acid (FFA) in guinea pigs but not in rats. By contrast, hepatic TG levels in rats were greatly increased in response to the high-fat diet, while it remained unchanged in guinea pigs. Furthermore, the hepatic acyl CoA:diacylglycerol acyltransferase (DGAT) activity and microsomal triglyceride transfer protein (MTTP) mRNA levels in guinea pigs fed a high-fat diet were significantly higher than those in the control group, which implies an increased very-low-density lipoprotein (VLDL)-TG secretion rate in guinea pigs in response to a high-fat diet. Hepatic carnitine palmitoyltransferase-1 (CPT-1) activity and peroxisome proliferator-activated receptor-α (PPARα) mRNA levels were upregulated in guinea pigs, but not rats, fed a high-fat diet. These findings may explain the differences in plasma and hepatic TG concentrations between guinea pigs and rats. These results suggest that there are differences in triglyceride metabolism between the two species when fed high-fat diets.