A network pharmacology approach to decipher the mechanism of total flavonoids from Dracocephalum Moldavica L. in the treatment of cardiovascular diseases.

AIM OF THE STUDY: Cardiovascular disease (CVD) seriously endangers human health and is characterized by high mortality and disability. The effectiveness of Dracocephalum moldavica L. in the treatment of CVD has been proven by clinical practice. However, the mechanism by which DML can treat CVD has not been systematically determined. MATERIALS AND METHODS: The active compounds in DML were screened by literature mining and pharmacokinetic analysis. Cytoscape software was used to construct the target-disease interaction network of DML in the treatment of CVD. Gene ontology and signalling pathway enrichment analyses were performed. The key target pathway network of DML compounds was constructed and verified by pharmacological experiments in vitro. A hydrogen glucose deprivation/reoxygenation model was established in H9c2 cells using hypoxia and glucose deprivation for 9 h combined with reoxygenation for 2 h. The model simulated myocardial ischaemic reperfusion injury to investigate the effects of total flavonoids of Cymbidium on cell viability, myocardial injury markers, oxidative stress levels, and reactive oxygen radical levels. Western blot analysis was used to examine NOX-4, Bcl-2/Bax, and PGC-1α protein expression. RESULTS: Twenty-seven active components were screened, and 59 potential drug targets for the treatment of CVD were obtained. Through the compound-target interaction network and the target-disease interaction network, the key targets and key signalling pathways, such as NOX-4, Bcl-2/Bax and PGC-1α, were obtained. TFDM significantly decreased LDH and MDA levels and the production of ROS and increased SOD activity levels in the context of OGD/R injury. Further studies indicated that NOX-4 and Bax protein levels and the p-P38 MAPK/P38 MAPK andp-Erk1/2/Erk1/2 ratios were suppressed by TFDM. The protein expression of Bcl-2 and PGC-1α was increased by TFDM. CONCLUSIONS: Our results showed that DML had multicomponent, multitarget and multichannel characteristics in the treatment of CVD. The mechanism may be associated with the following signalling pathways: 1) the NOX-4/ROS/p38 MAPK signalling pathway, which inhibits inflammation and reactive oxygen species (ROS) production, and 2) the Bcl-2/Bax and AMPK/SIRT1/PGC-1α signalling pathways, which inhibit apoptosis.

A network pharmacology approach to decipher the total flavonoid extract of Dracocephalum Moldavica L. in the treatment of cerebral ischemia- reperfusion injury.

BACKGROUND AND OBJECTIVE: Cerebral ischemia-reperfusion injury (CIRI) is a major injury that seriously endangers human health and is characterized by high mortality and high disability. The total flavonoid extract of Dracocephalum moldavica L.(TFDM) in the treatment of CIRI has been proved by clinical practice. But the mechanism for the treatment of CIRI by TFDM has not been systematically revealed. STUDY DESIGN AND METHODS: The active compounds contained in TFDM were screened by literature mining and pharmacokinetic parameters, and the targets related to CIRI were collected by searching Drugbank, Genecards and OMIM databases. Cytoscape software was used to construct the protein interaction network of TFDM for the prevention and treatment of CIRI. Geneontology and signal pathway enrichment were analyzed. The key target pathway network of TFDM compounds was constructed and verified by pharmacological experiment in vitro. RESULTS: 21 active components were screened, 158 potential drug targets for the prevention and treatment of CIRI were obtained, 53 main targets were further screened in the protein-protein interaction network, and 106 signal pathways, 76 biological processes, 26 cell components and 50 molecular functions were enriched (P<0.05). Through the compound-target-pathway network, the key compounds that play a role in the prevention and treatment of CIRI, such as acacetin, apigenin and other flavonoids, as well as the corresponding key targets and key signal pathways, such as AKT1, SRC and EGFR were obtained. TFDM significantly decreased LDH, MDA levels and increased the NO activity levels in CIRI. Further studies have shown that TFDM increases the number of SRC proteins, and TFDM also increases p-AKT/ AKT. Molecular docking results showed that acacetin-7-O (- 6''-acetyl) -glucopyranoside, acacetin7-O-ß-D-glucopyranoside, apigenin-7-O-ß-D-galactoside respectively had good affinity for SRC protein. Acacetin-7-O (- 6''-acetyl) -glucopyranoside,acacetin-7-O-ß-D-glucuronide, acacetin7-O-ß-D-glucopyranoside had good affinity for AKT1 protein, respectively. CONCLUSION: Our research showed that TFDM had the characteristics of multi-component, multi-target and multi-channel in the treatment of CIRI. The potential mechanism may be associated with the following signaling pathways:1) the signaling pathways of VEGF/SRC, which promote angiogenesis, 2) the signaling pathways of PI3K/AKT, which inhibit apoptosis, and 3) acacetin-7-O (- 6''-acetyl) -glucopyranoside is expected to be used as a candidate monomer component for natural drugs for further development.

Moldavica acid A, a new salicylic acid derivative from Dracocephalum moldavica.

In this paper, we present the discovery of a novel salicylic acid derivative, moldavica acid A (1), and a new natural dibenzo[b,f]oxepin, moldavica acid B (2), together with four known phenylpropionic acids (3-6) and protocatechuic acid (7) that were isolated from Dracocephalum moldavica L. Their structures were elucidated by comprehensive spectroscopic methods, including infrared and nuclear magnetic resonance. Compound 1 is the first example of salicylic acid linking a carboxylated α-pyrone via an ethyl bridge. Beyond expanding the knowledge of the chemical diversity of D. moldavica, both compounds 1 and 2 were shown to upregulate the expression of Kruppel-like factor 2, which could serve as a prospective therapeutic target for the treatment of atherosclerosis.

Total flavones of Dracocephalum moldavica L. protect astrocytes against H2O2-induced apoptosis through a mitochondria-dependent pathway.

BACKGROUND: The active components of Dracocephalum moldavica L. (TFDM) can inhibit myocardial ischemia by inhibiting oxidative stress. However, the effects of TFDM on astrocytes have not been investigated in vitro. The current study aimed to explore whether TFDM protects astrocytes against H2O2-induced apoptosis through a mitochondria-dependent pathway. METHODS: The human glioma cell line U87 was used to investigate the ability of TFDM to protect astrocytes against H2O2-induced apoptosis. The cell counting kit-8 assay and flow cytometry were used to detect cell viability, apoptosis, MMP, Ca2+ influx and reactive oxygen species (ROS). Lactate dehydrogenase (LDH) and malonic dialdehyde (MDA) levels were measured by ELISA. In addition, protein and mRNA expression changes were detected by Western blotting and qRT-PCR. RESULTS: TFDM (0.78~200 µg/ml) had limited cytotoxic effects on the viability of U87 cells. Compared with the model group (treated with H2O2 only), cells treated with medium- and high-dose TFDM exhibited reduced MDA concentrations (P < 0.05) and ROS production (P < 0.05) and decreased MMP (P < 0.05) and reduced apoptosis (P < 0.05). The percentage of annexin V-FITC-stained cells was markedly suppressed by TFDM, confirming its anti-apoptotic properties. WB results showed that protein expression of Bcl-2-associated X protein (BAX), Caspase-3, Caspase-9, Caspase-12, and B-cell leukemia/lymphoma 2 (Bcl2) was reduced in the TFDM group compared with that in the model group (P < 0.05) and that expression of these proteins was normalized by TFDM treatment in a dose-dependent manner. According to RT-qPCR results, TFDM pretreatment resulted in reduced mRNA expression of BAX, Caspase-9, Caspase-12, p38MAPK, and CaMKII and increased mRNA expression of mTOR compared with the model group. CONCLUSIONS: The current study revealed the protective effects of TFDM on U87 cells under oxidative stress conditions through the inhibition of a mitochondria-dependent pathway that is associated with the CaMKII/P38MAPK/ERK1/2 and PI3K/AKT/mTOR pathways.

Total flavonoid extract from Dracoephalum moldavica L. attenuates ß-amyloid-induced toxicity through anti-amyloidogenesic and neurotrophic pathways.

AIMS: Alzheimer's disease (AD) is an incurable neurodegenerative disorder characterized by global cognitive impairment that involves accumulation of amyloid-beta peptides (Aß) in the brain. Herbal approaches can be used as alternative medicines to slow the progression of AD. This study aimed to determine the beneficial effects and potential underlying mechanisms of total flavonoid extract from Dracoephalum moldavica L. (TFDM) for attenuating Alzheimer-related deficits induced by Aß. MAIN METHODS: We used amyloid precursor protein (APP) and presenilin 1 (PS1) double transgenic mice and copper-injured APP Swedish mutation overexpressing SH-SY5Y cells to evaluate the beneficial effects of TFDM. Further, identifying the mechanisms of action was conducted on anti-amyloidogenic and neurotrophic transductions. KEY FINDINGS: Our results indicated that TFDM treatment ameliorated cognitive impairments and neurodegeneration and improved the antioxidant defense system in APP/PS1 mice. TFDM also reduced Aß burden by relieving Aß deposition, decreasing insoluble Aß levels, and inhibiting ß-amyloidogenic processing pathway involving downregulation of ß-secretase and ß-C-terminal fragment in the brain. In the in vitro model of AD, TFDM treatment protected injured cells, and combined with the beneficial effects of decreasing APP levels, lowered Aß1-42 and regulated the redox imbalance. Moreover, TFDM preserved the extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway both in vitro and in vivo. SIGNIFICANCE: In conclusion, TFDM clearly demonstrated neuroprotective effects by restoring the anti-amyloidogenic and neurotrophic transductions in the context of AD-associated deficits. These findings indicate the potential use of herb-based substances as supplements or potential alternative supplements for attenuating the progression of AD.

Profiling microRNA from Brain by Microarray in a Transgenic Mouse Model of Alzheimer's Disease.

MicroRNAs (miRNAs) are small noncoding RNAs, which regulate numerous cell functions by targeting mRNA for cleavage or translational repression, and have been found to play an important role in Alzheimer's disease (AD). Our study aimed to identify differentially expressed miRNAs in AD brain as a reference of potential therapeutic miRNAs or biomarkers for this disease. We used amyloid precursor protein (APP) and presenilin 1 (PS1) double transgenic mice and age-matched wild-type (WT) littermates to determine the expression of miRNAs in the brain. MiRNAs were profiled by microarray, and differentially expressed miRNAs underwent target prediction and enrichment analysis. Microarray analysis revealed 56 differentially expressed miRNAs in AD mouse brain, which involved 39 miRNAs that were significantly upregulated and 19 that were downregulated at different ages. Among those miRNAs, a total of 11 miRNAs, including miR-342-3p, miR-342-5p, miR-376c-3p, and miR-301b-3p, were not only conserved in human but also predicted to have targets and signaling pathways closely related to the pathology of AD. In conclusion, in this study, differentially expressed miRNAs were identified in AD brain and proposed as biomarkers, which may have the potential to indicate AD progression. Despite being preliminary, these results may aid in investigating pathological hallmarks and identify effective therapeutic targets.

Development of solid lipid nanoparticles containing total flavonoid extract from Dracocephalum moldavica L. and their therapeutic effect against myocardial ischemia-reperfusion injury in rats.

Total flavonoid extract from Dracocephalum moldavica L. (TFDM) contains effective components of D. moldavica L. that have myocardial protective function. However, the cardioprotection function of TFDM is undesirable due to its poor solubility. In order to improve the solubility and efficacy of TFDM, we developed TFDM-loaded solid lipid nanoparticles (TFDM-SLNs) and optimized the formulation of TFDM-SLNs using central composite design and response surface methodology. The physicochemical properties of TFDM-SLNs were characterized, and the pharmacodynamics was investigated using the myocardial ischemia-reperfusion injury model in rats. The nanoparticles of optimal formulation for TFDM-SLNs were spherical in shape with the average particle size of 104.83 nm and had a uniform size distribution with the polydispersity index value of 0.201. TFDM-SLNs also had a negative zeta potential of -28.7 mV to ensure the stability of the TFDM-SLNs emulsion system. The results of pharmacodynamics demonstrated that both TFDM and TFDM-SLN groups afforded myocardial protection, and the protective effect of TFDM-SLNs was significantly superior to that of TFDM alone, based on the infarct area, histopathological examination, cardiac enzyme levels and inflammatory factors in serum. Due to the optimal quality and the better myocardial protective effect, TFDM-SLNs are expected to become a safe and effective nanocarrier for the oral delivery of TFDM.

Luteolin Inhibits Fibrillary ß-Amyloid1-40-Induced Inflammation in a Human Blood-Brain Barrier Model by Suppressing the p38 MAPK-Mediated NF-κB Signaling Pathways.

Amyloid-ß peptides (Aß) exist in several forms and are known as key modulators of Alzheimer's disease (AD). Fibrillary Aß (fAß) has been found to disrupt the blood-brain barrier (BBB) by triggering and promoting inflammation. In this study, luteolin, a naturally occurring flavonoid that has shown beneficial properties in the central nervous system, was evaluated as a potential agent to preserve barrier function and inhibit inflammatory responses at the BBB that was injured by fAß1-40. We established an in vitro BBB model by co-culturing human brain microvascular endothelial cells (hBMECs) and human astrocytes (hAs) under fAß1-40-damaged conditions and investigated the effect of luteolin by analyzing cellular toxicity, barrier function, cytokine production and inflammation-related intracellular signaling pathways. Our results demonstrated that, in cells injured by fAß1-40, luteolin increased cell viability of hBMECs and hAs. The cytoprotection of the co-culture against the damage induced by fAß1-40 was also increased at both the apical and basolateral sides. Luteolin protected the barrier function by preserving transendothelial electrical resistance and relieving aggravated permeability in the human BBB model after being exposed to fAß1-40. Moreover, in both the apical and basolateral sides of the co-culture, luteolin reduced fAß1-40-induced inflammatory mediator and cytokine production, including cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin 1 ß (IL-1ß), interleukin 6 (IL-6), and interleukin 8 (IL-8), however it did not show sufficient effects on scavenging intracellular reactive oxygen species (ROS) in hBMECs and hAs. The mechanism of BBB protection against fAß1-40-induced injury may be related to the regulation of inflammatory signal transduction, which involves inhibition of p38 mitogen-activated protein kinase (MAPK) activation, downregulation of phosphorylated inhibitory κB kinase (phosphor-IKK) levels, relief of inhibitory κB α (IκBα) degradation, blockage of nuclear factor κB (NF-κB) p65 nuclear translocation, and reduction of the release of inflammatory cytokines. Moreover, the employment of p38 MAPK and NF-κB inhibitors reversed luteolin-mediated barrier function and cytokine release. Taken together, luteolin may serve as a potential therapeutic agent for BBB protection by inhibiting inflammation following fAß1-40-induced injury.

Polyisoprenylated benzoylphloroglucinol derivatives from Hypericum scabrum.

Four new polyisoprenylated benzoylphloroglucinol derivatives, hyperscabrones J-M (1-4), were isolated from the air-dried aerial parts of Hypericum scabrum. Their structures were elucidated by spectroscopic methods and were subsequently confirmed by comparing with data of known compounds. The absolute configuration of the bicyclo[3.3.1]nonane-2,4,9-trione core was defined by the experimental and calculated electronic circular dichroism (ECD) spectra. The evaluation of their hepatoprotective activities against paracetamol-induced HepG2 cell damage showed that compounds 2 and 4 exhibited significant hepatoprotection at 10µM.

Polycyclic Polyprenylated Acylphloroglucinol Congeners from Hypericum scabrum.

Twenty polycyclic polyprenylated acylphloroglucinols (PPAPs), including the new compounds hyperscabrones A-I (1-9), were isolated from the air-dried aerial parts of Hypericum scabrum. These compounds comprise seven different structural types. All structures were determined by NMR spectroscopic methods and both experimental and calculated electronic circular dichroism (ECD) spectra. The evaluation of their neuroprotective effects on glutamate-induced toxicity in SK-N-SH cells showed that compounds 4-7 exhibited significant neuroprotection at 10 µM. Additionally, compounds 3, 4, 7, and 9 showed moderate hepatoprotective activities against paracetamol-induced HepG2 cell damage at 10 µM.

[Purification technology of Rhizoma et Radix Notopterygii and Radix Angelicae Pubescentis in Fufang Xuelian dropping pills by macroporous resin].

OBJECTIVE: To study the purification technology of Rhizoma et Radix Notopterygii and Radix Angelicae Pubescentis in Fufang Xuelian dropping pills by macroporous resin. METHOD: Taking osthole, isomperatorin as index ingredients, the type of resin sampling amount and elution solvent were decided, and the influence of sample concentration pH of sample and ratio of diameter to height of column to adsorption were studied. RESULT: HPD400A was chosen to purify, the suitable sampling ratio of resin volume to raw material was 1:2; pH 3.5 (crude drug) and ratio of diameter to height was 1:7; 95% ethanol of the elution solvent was satisfactory eluant for desorption. CONCLUSION: HPD400A macroporous resin can be used to purify Rhizoma et Radix Notopterygii and Radix Angelicae Pubescentis.

[Studies on chemical constituents in herbs of Dracocephalum moldavica II].

OBJECTIVE: To study the chemical constituents of Dracocephalum moldavica. METHOD: The compounds were isolated by using RA polystyrene resin, polyamide and silica gel column chromatography, The structures of the compounds were elucidated on the basis of physic-chemical properties and spectra data. RESULT: Six compounds were identified as syringaresinol4-O-beta-D-monoglucoside (I), sy-ringaresinol-4,4'-O-bis-beta-D-glucoside (II), kaempferol-3-O-beta-D-(6"-O-p-coumaroyl)-galactopyranoside (III), 2"-p-coumarylastragalin (IV), takakin-8-O-beta-D-glucopyranoside (V), beta-daucosterol (VI). CONCLUSION: Compounds I-V were obtained from genus Dracocephalum for the first time.