Salvianolic acid A relieves cognitive disorder after chronic cerebral ischemia: Involvement of Drd2/Cryab/NF-κB pathway.

Chronic cerebral ischemia (CCI) refers to long-term hypoperfusion of cerebral blood flow with the main clinical manifestations of progressive cognitive impairment. The pathological mechanism of CCI is complex, and there is a lack of effective treatments. Salvianolic acid A (SalA) is a neuroprotective extract of Salvia miltiorrhiza with the effects of anti-inflammation and anti-apoptosis. In this study, the effect of SalA on cognitive function and Drd2/Cryab/NF-κB signaling pathway in rats with CCI was investigated. Morris water maze and open field test were used to observe the effects of SalA on the cognitive function of CCI rats. The pathological changes in the brain were observed by HE, Nissl, and LFB staining. TUNEL staining, enzyme-linked immunosorbent assay, and western blot analysis were used to detect the inflammatory and apoptosis in the cortex and hippocampus. The expression of Drd2/Cryab/NF-κB pathway-related molecules and Drd2 localization were detected by western blotting and dual immunofluorescence, respectively. SH-SY5Y cells were exposed to chronic hypoglycemic and hypoxic injury in vitro, and Drd2 inhibitor haloperidol was used to verify the involved pathway. The results showed that SalA could improve the cognitive function of CCI rats, reduce pathological damage of cortex and hippocampus, inhibit neuroinflammation and apoptosis, and suppress the activation of NF-κB by regulating Drd2/Cryab pathway. And SalA inhibited NF-κB activation and nuclear translocation in SH-SY5Y cells by upregulating Drd2/Cryab pathway, which was reversed by haloperidol interference. In conclusion, SalA could relieve CCI-induced cognitive impairment in rats, at least partly through the Drd2/Cryab/NF-κB pathway.

The comprehensive study on the therapeutic effects of baicalein for the treatment of COVID-19 in vivo and in vitro.

Baicalein is the main active compound of Scutellaria baicalensis Georgi, a medicinal herb with multiple pharmacological activities, including the broad anti-virus effects. In this paper, the preclinical study of baicalein on the treatment of COVID-19 was performed. Results showed that baicalein inhibited cell damage induced by SARS-CoV-2 and improved the morphology of Vero E6 cells at a concentration of 0.1 µM and above. The effective concentration could be reached after oral administration of 200 mg/kg crystal form ß of baicalein in rats. Furthermore, baicalein significantly inhibited the body weight loss, the replication of the virus, and relieved the lesions of lung tissue in hACE2 transgenic mice infected with SARS-CoV-2. In LPS-induced acute lung injury of mice, baicalein improved the respiratory function, inhibited inflammatory cell infiltration in the lung, and decreased the levels of IL-1ß and TNF-α in serum. In conclusion, oral administration of crystal form ß of baicalein could reach its effective concentration against SARS-CoV-2. Baicalein could inhibit SARS-CoV-2-induced injury both in vitro and in vivo. Therefore, baicalein might be a promising therapeutic drug for the treatment of COVID-19.

The strategies and techniques of drug discovery from natural products.

Natural products have been the main sources of new drugs. The different strategies have been developed to find the new drugs based on natural products. The traditional and ethic medicines have provided information on the therapeutic effects and resulted in some notable drug discovery of natural products. The special activities of the medicine plants such as the side effects have inspired scientists to develop the novel small molecular. The microorganisms and the endogenous active substances from human or animal also become the important approaches to the drug discovery. The tremendous progress in technology led to the new strategies in drug discovery from natural products. The bioinformation and artificial intelligence have facilitated the research and development of natural products. We will provide a scene of strategies and technologies for drug discovery from natural products in this review.

Total flavonoids from Anchusa italica Retz. Improve cardiac function and attenuate cardiac remodeling post myocardial infarction in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant Anchusa italica Retz. (Anchusa azurea Mill.) has been traditionally used in Uygur medicine for the treatment of cardiovascular and cerebrovascular diseases in China. Our previous study showed that total flavonoids from Anchusa italica Retz. (TFAI) exhibited potent cardioprotection in acute ischemia/reperfusion injured rats. AIM OF THE STUDY: This study was undertaken to investigate the effects of TFAI on chronic myocardial infarction (MI) in mice and the underlying mechanism. MATERIALS AND METHODS: Total flavonoids were extracted from the whole herb of Anchusa italica Retz. and were characterized using HPLC-MS analysis. The left anterior descending branch of the coronary artery was ligated to simulate MI injury in mice. After surgery, mice were orally fed with TFAI at the doses of 10, 30 and 50 mg/kg body weight/day for a total of four weeks. Cardiac function and infarct size were measured, and inflammatory mediators were detected. Hematoxylin and eosin (H&E) staining and Masson's trichrome staining were performed on heart sections. The apoptotic factors, such as Bax, Bcl-2 and cleaved caspase 3, as well as the key proteins in the PI3K/Akt/mTOR signaling pathway were examined by Western blot. RESULTS: The content of total flavonoids in TFAI was 56.2%. Four weeks following the MI surgery, TFAI enhanced the survival rate in post-MI mice. TFAI treatment at the doses of 30 and 50 mg/kg remarkably reduced infarct size and improved cardiac function as indicated by elevated EF and FS. Assay of the inflammatory factors showed that sera levels of TNF-α, IL-1ß and IL-6 were markedly decreased by TFAI treatment compared to the MI group. H&E staining and Masson's trichrome staining demonstrated that TFAI suppressed myocyte hypertrophy and cardiac fibrosis as indicated by the decreased cross-section area and collagen volume. Western blot analysis showed that cleaved caspase 3 and Bax/Bcl-2 were significantly downregulated following TFAI treatment. Furthermore, TFAI treatment significantly suppressed the activation of the PI3K/Akt/mTOR signaling pathway. CONCLUSIONS: Our data suggest that TFAI exerts a potent protective effect against chronic MI injury, and its beneficial effects on cardiac function and cardiac remodeling might be attributable, at least in part, to anti-inflammation and inhibition of the PI3K/Akt/mTOR signaling pathway.

Chemical and pharmacological research on the polyphenol acids isolated from Danshen: A review of salvianolic acids.

Danshen, the dried root of Salvia miltiorrhiza Bge, is a common medicinal herb in Traditional Chinese Medicine, which has been used for the treatment of a number of diseases for thousands of years. More than 2000 years ago, the Chinese early pharmacy monograph "Shennong Materia Medica" recorded that Danshen could be used for the treatment of gastrointestinal diseases, cardiovascular diseases, certain gynecological diseases, etc. Since then, Danshen has been widely used clinically in many different prescriptions for many different diseases, especially for the treatment of cardiovascular diseases. Nowadays, many pharmacological studies about the water-soluble components from Danshen have been reported, especially salvianolic acids. It turned out that salvianolic acids showed strong anti-lipid peroxidation and anti-thrombic activities, and among them, SalAA and SalAB were the most potent. This review focused on the achievements in research of salvianolic acids regarding their bioactivities and pharmacological effects. These studies not only shed light on the water-soluble active components of Danshen and their mechanisms at the molecular level, but also provided theoretical information for the development of new medicines from Danshen for the treatment of cardiovascular and cerebrovascular diseases, inflammatory diseases, metabolic diseases, etc.

Inhibition of FOXO3a/BIM signaling pathway contributes to the protective effect of salvianolic acid A against cerebral ischemia/reperfusion injury.

Salvianolic acid A (SalA) is an effective compound extracted from traditional Chinese medicine Salvia miltiorrhiza Bunge. The Forkhead box O3a (FOXO3a) signaling pathway plays crucial roles in the modulation of ischemia-induced cell apoptosis. However, no information about the regulatory effect of SalA on FoxO3a is available. To explore the anti-cerebral ischemia effect and clarify the therapeutic mechanism of SalA, SH-SY5Y cells and Sprague-Dawley rats were applied, which were exposed to oxygen glucose deprivation/reoxygenation (OGD/R) and middle cerebral artery occlusion/reperfusion (MCAO/R) injuries, respectively. The involved pathway was identified using the specific inhibitor LY294002. Results showed that SalA concentration-dependently inhibited OGD/R injury triggered cell viability loss. SalA reduced cerebral infarction, lowered brain edema, improved neurological function, and inhibited neuron apoptosis in MCAO/R rats, which were attenuated by the treatment of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) specific inhibitor LY294002. SalA time- and concentration-dependently upregulated the phosphorylation levels of protein kinase B (AKT) and its downstream protein FOXO3a. Moreover, the nuclear translocation of FOXO3a was inhibited by SalA both in vivo and in vitro, which was also reversed by LY294002. The above results indicated that SalA fought against ischemia/reperfusion damage at least partially via the AKT/FOXO3a/BIM pathway.

Investigation of common chemical components and inhibitory effect on GES-type ß-lactamase (GES22) in methanolic extracts of Algerian seaweeds.

This study aimed to investigate the total phenolic content (TPC), the identification of the common compounds by HPLC-ESI-MS and HPLC-ESI-MS-TOF and the inhibitory effects against class A-type ß-lactamase (GES-22 variant, produced recombinantly) in methanolic extracts (MEs) of four Algerian seaweeds [Ulva intestinalis, Codium tomentosum, Dictyota dichotoma and Halopteris scoparia]. The TPC varied among the four species, ranging between 0.93 ±â€¯0.65 and 2.66 ±â€¯1.33 mg GAEs/g DW. C.tomentosum had higher total phenol content than other seaweeds while, all of them inhibited uncompetitively GES-22 activity in a dose-dependent manner. Nitrocefin was used as chromogenic substrate to evaluate the inhibitory effect on GES-22. The methanolic extract of D.dichotoma exhibited significant inhibitory effect on GES-22 (IC50 = 13.01 ±â€¯0.046 µg/mL) more than clavulanate, sulbactam and tazobactam (classical ß-lactam inhibitors) (IC50 = 68.38 ±â€¯0.17 µg/mL, 52.68 ±â€¯0.64 µg/mL, and 29.94 ±â€¯0.01 µg/mL, respectively). IC50 of the other ME of U.intestinalis, C.tomentosum, and H.scoparia were 16.87 ±â€¯0.10 µg/mL, 16.54 ±â€¯0.048 µg/mL, and 25.72 ±â€¯0.15 µg/mL, respectively. Except H. scoparia, other three seaweed extracts showed almost two times or more inhibition on GES-22. Furthermore, four common compounds in these MEs were identified, α-linolenic acid (C18:3ω3), linoleic acid (C18:2ω6), oleic acid (C18:1ω9), the eicosanoid precursors ''arachidonic acid'' (C20:4ω6). Baicalein (C15H10O5) was identified in U.intestinalis and D.dichotoma seaweeds. The fact that all seaweed extracts inhibited the GES-22 better than commercial samples makes these seaweeds candidate for discovering new inhibitors against ß-lactamases. Besides that, they contain important components with potential health benefits.

Salvianolic acid A attenuates ischemia reperfusion induced rat brain damage by protecting the blood brain barrier through MMP-9 inhibition and anti-inflammation.

Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia Miltiorrhiza Bge, a traditional Chinese medicine, which has been used for the treatment of cerebrovascular diseases for centuries. The present study aimed to determine the brain protective effects of SAA against cerebral ischemia reperfusion injury in rats, and to figure out whether SAA could protect the blood brain barrier (BBB) through matrix metallopeptidase 9 (MMP-9) inhibition. A focal cerebral ischemia reperfusion model was induced by middle cerebral artery occlusion (MCAO) for 1.5-h followed by 24-h reperfusion. SAA was administered intravenously at doses of 5, 10, and 20 mg·kg-1. SAA significantly reduced the infarct volumes and neurological deficit scores. Immunohistochemical analyses showed that SAA treatments could also improve the morphology of neurons in hippocampus CA1 and CA3 regions and increase the number of neurons. Western blotting analyses showed that SAA downregulated the levels of MMP-9 and upregulated the levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) to attenuate BBB injury. SAA treatment significantly prevented MMP-9-induced degradation of ZO-1, claudin-5 and occludin proteins. SAA also prevented cerebral NF-κB p65 activation and reduced inflammation response. Our results suggested that SAA could be a promising agent to attenuate cerebral ischemia reperfusion injury through MMP-9 inhibition and anti-inflammation activities.

Pharmacokinetic study of salvianolic acid D after oral and intravenous administration in rats.

A sensitive, specific and rapid LC-MS method was developed and validated for the determination of salvianolic acid D (SalD) in rat plasma. This method used a single quadrupole mass spectrometer with an electrospray ionization (ESI) source. A single ion monitoring scanning (SIM) mode was employed. It showed good linearity over the concentration range from 3.3 to 666.7 ng/mL for the determination of SalD. The R.S.D.% of intra-day and inter-day precision values were no more than 7.69%, and the accuracy was within 91%-104% at all quality control levels. This LC-MS method was applied to the pharmacokinetic study of SalD in rats. A two-compartmental model analysis was employed. The plasma concentrations at 2 min (C 2min) were 5756.06±719.61, 11,073.01±1783.46 and 21,077.58±5581.97 µg/L for 0.25, 0.5 and 1 mg/kg intravenous injection, respectively. The peak plasma concentration (C max) was 333.08±61.21 µg/L for 4 mg/kg oral administration. The area under curve (AUC0-t ) was 14,384.379±8443.184, 22,813.369±11,860.823, 46,406.122±27,592.645 and 8201.740±4711.961 µg/L·h for intravenous injection (0.25, 0.5 and 1 mg/kg) and oral administration (4 mg/kg), respectively. The bioavailability of SalD was calculated to be 4.159%±0.517%.

Lack of dose dependent kinetics of methyl salicylate-2-O-ß-D-lactoside in rhesus monkeys after oral administration.

ETHNOPHARMACOLOGICAL RELEVANCE: Methyl salicylate-2-O-ß-d-lactoside (MSL) is one of the main active components isolated from Gaultheria yunnanensis, which is a traditional Chinese medicine used to treat arthritis and various aches and pains. Pharmacological researches showed that MSL had various effective activities in both in vivo and in vitro experiments. However, the pharmacokinetics features and oral bioavailability of MSL in primates were not studied up to now. AIM: To study the pharmacokinetics of different doses of MSL in rhesus monkeys and investigate the absolute bioavailability of MSL after oral administration. MATERIALS AND METHODS: Male and female rhesus monkeys were either orally administrated with MSL 200, 400 and 800 mg/kg or received an intravenous dose of 20mg/kg randomly. The levels of MSL and salicylic acid (SA) in plasma were simultaneous measured by a simple, sensitive and reproducible high performance liquid chromatography method. RESULTS: Mean peak plasma concentration values for groups treated with 200, 400 and 800 mg/kg doses ranged from 48.79 to 171.83 µg/mL after single-dose oral administration of MSL, and mean area under the concentration-time curve values ranged from 195.16 to 1107.76 µg/mL h. Poor linearity of the kinetics of SA after oral administration of MSL was observed in the regression analysis of the Cmax-dose plot (r(2)=0.812), CL-dose plot (r(2)=0.225) and AUC(0-t)-dose plot (r(2)=0.938). Absolute bioavailability of MSL was assessed to be 118.89 ± 57.50, 213.54 ± 58.98 and 168.72 ± 76.58%, respectively. CONCLUSIONS: Bioavailability of MSL after oral administration in rhesus monkeys was measured for the first time. Pharmacokinetics parameters did not appear to be dose proportional among the three oral doses of treatments, and MSL showed an apparent absolute bioavailability in excess of 100% in rhesus monkeys based on the present study. In addition, a rapid, sensitive and reliable HPLC method was established and demonstrated for the research of traditional Chinese medicine in this study.

[Pharmacokinetics of salvianolic acid A after single intravenous administration in Rhesus monkey].

Salvianolic acid A (Sal A) is one of the most effective compounds isolated from the root of Salvia miltiorrhiza. Up to now, several studies regarding the pharmacokinetic profiles of Sal A have been reported, however there is no such study reported in monkeys, the species which is more similar to human. The aim of this study is to develop a LC-MS method for the determination of Sal A in monkey plasma and apply it to the pharmacokinetic studies of monkeys. After single intravenous administration of Sal A, the plasma concentration-time curves were observed and the main pharmacokinetic parameters were calculated. The plasma concentration at 2 min (C2 (min)) values were (28.343 ± 6.426), (45.679 ± 12.301) and (113.293 ± 24.360) mg x L(-1) for Rhesus monkeys treated with Sal A at 2.5, 5 and 10 mg x kg(-1). The area under the concentration-time curve (AUC(0-∞)) values were (3.316 ± 0.871), (5.754 ± 2.150) and (13.761 ± 2.825) µg x L(-1) x h, respectively. Furthermore, this method was improved and applied to the simultaneous determination of Sal A, Sal B and Sal C, which provided useful information for preclinical studies and clinical trials of Sal A, Sal B and Sal C.

Pharmacokinetic study of salvianolic acid A in beagle dog after oral administration by a liquid chromatography-mass spectrometry method: a study on bioavailability and dose proportionality.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvianolic acid A (SAA) is one of the main water-soluble components isolated from Salvia miltiorrhiza Bunge. Pharmacological researches revealed that it had various curative activities after oral and intravenous administration, including beneficial effects on diabetes and its complications, cardioprotective effect, anti-platelet aggregation, and so on. However, there is no report regarding the pharmacokinetics of SAA in beagle dogs after oral administration up to now. AIM OF THE STUDY: To study the pharmacokinetics of different doses of SAA in beagle dogs and figure out the absolute bioavailability and dose proportionality of SAA after oral administration. MATERIALS AND METHODS: Male and female beagle dogs were orally administered SAA 5, 10 and 20mg/kg randomly. The plasma drug concentration was detected by a rapid, sensitive and reproducible liquid chromatography-mass spectrometry (LC-MS) method. The pharmacokinetic parameters were calculated from plasma concentration-time data using the DAS pharmacokinetic software Data Analysis System Version 3.0 program. RESULTS: After single-dose oral administration of SAA, the mean peak plasma concentration (Cmax) values for groups treated with 5, 10 and 20 mg/kg doses ranged from 14.38 to 38.18 µg/L, and the mean area under the concentration-time curve (AUC(0-t)) values ranged from 38.77 to 130.33 (µg/L·h). SAA showed lack of dose proportionality over the dose range 5-20mg/kg, based on the power model. However, the increase in systemic exposure with dose appeared linear. The absolute bioavailability was calculated to range from 1.47% to 1.84%. CONCLUSION: The pharmacokinetic properties of SAA in beagle dogs after oral administration were characterized as rapid oral absorption, quick clearance, and poor absolute bioavailability. Systemic exposure exhibited lack of dose proportionality over the dose range 5-20mg/kg. Furthermore, a readily preparative LC-MS method was demonstrated in this study for the research of traditional Chinese medicine.

Quercetin protects against the Aß(25-35)-induced amnesic injury: involvement of inactivation of rage-mediated pathway and conservation of the NVU.

Quercetin has demonstrated protective effects against Aß-induced toxicity on both neurons and endothelial cells. However, whether or not quercetin has an effect on the neurovascular coupling is unclear. In the present study, we aim to investigate the anti-amnesic effects of quercetin and to explore the underlying mechanisms. Aß(25-35) (10 nmol) was administrated to mice i.c.v. Quercetin was administrated orally for 8 days after injection. Learning and memory behaviors were evaluated by measuring spontaneous alternation in Morris Water Maze test and the step-through positive avoidance test. The regional cerebral blood flow was monitored before the Aß(25-35) injection and on seven consecutive days after injection. Mice were sacrificed and cerebral cortices were isolated on the last day. The effects of quercetin on the neurovascular unit (NVU) integrity, microvascular function and cholinergic neuronal changes, and the modification of signaling pathways were tested. Our results demonstrate that quercetin treatment for Aß(25-35)-induced amnesic mice improved the learning and memory capabilities and conferred robust neurovascular coupling protection, involving maintenance of the NVU integrity, reduction of neurovascular oxidation, modulation of microvascular function, improvement of cholinergic system, and regulation of neurovascular RAGE signaling pathway and ERK/CREB/BDNF pathway. In conclusion, in Aß(25-35)-induced amnesic mice, optimal doses of quercetin administration were beneficial. Quercetin protected the NVU likely through reduction of oxidative damage, inactivation of RAGE-mediated pathway and preservation of cholinergic neurons, offering an alternative medication for Alzheimer's disease.

Pharmacokinetic study of baicalein after oral administration in monkeys.

Baicalein, a flavonoid originally isolated from the root of Scutellaria baicalensis Georgi, has numerous pharmacological activities. Up to now, several studies regarding the pharmacokinetic profiles of baicalein have been described, while there is no such study reported in monkey, the species which is more similar to human. The purpose of this study was to investigate the pharmacokinetic profiles of baicalein after oral administration in monkeys. After orally administrating three doses of baicalein in monkeys, multi-peaks of the plasma concentration-time curves were observed and the non-linear pharmacokinetics for baicalein and its metabolite baicalin were found at doses of 50-500mg/kg. In order to calculate the absolute bioavailability, the intravenous pharmacokinetic study was also carried out after intravenous administration of 10mg/kg baicalein. The absolute bioavailability of baicalein in different doses was ranged from 13.1% to 23.0%. In this study, baicalein and baicalin were determined by LC-MS method. The chromatographic separation was performed on Agilent Poroshell 120 SB-C18 column (2.7µm, 2.1×50mm). Baicalein and baicalin were detected by single quadrupole mass spectrometer equipment with electrospray ionization interface with the selected ion monitoring mode. The assay was linear for both baicalein and baicalin with the correlation coefficients>0.99. The intra- and inter-day precisions for baicalein and baicalin were all less than 15% by relative standard deviation. The analytes were stable during samples storage and handling, and no matrix effects were observed. The method we developed in this study was sensitive, precise, stable and producible.