Chinese herbal medicines for mild cognitive impairment: A protocol for meta-analysis and trial sequential analysis.

BACKGROUND: Mild cognitive impairment (MCI), as a common neurodegenerative aging disease representing an intermediate stage between normal cognitive functioning and dementia, poses an excessive burden on health care. The clinical benefit of Chinese herbal medicines (CHMs) for MCI remains inconclusive. This study is aimed at evaluating the efficacy and acceptability of CHMs through meta-analysis and trial sequential analysis (TSA). METHODS: We applied extensive strategies on preliminary literature screening to identify relevant randomized controlled trials which meticulously compare any of CHMs interventions with placebo groups as monotherapy for MCI. The primary outcome of this study is the change of global cognitive function, and the secondary outcomes include assessments of activities of daily living, mood, and adverse events. Data synthesis, risk of bias assessment, sensitivity and subgroup analyses, and TSA will be conducted with application of Review Manager, Stata, and TSA software. The quality of the evidence will be evaluated using the Grading of Recommendations Assessment, Development and Evaluation instrument. INPLASY registration number: INPLASY202190006 (https://inplasy.com/inplasy-2021-9-0006/). RESULTS: This study will confirm the clinical efficacy and safety of CHMs when used in the treatment of patients with MCI. CONCLUSION: This study will provide reliable evidence and references for the selection of CHMs in therapy and future clinical research of MCI.

Neuroprotection of Cilostazol against ischemia/reperfusion-induced cognitive deficits through inhibiting JNK3/caspase-3 by enhancing Akt1.

Cilostazol(CTL) is a phosphodiesterase inhibitor, which has been widely used as anti-platelet agent. It also has preventive effects on various central nervous system (CNS) diseases, including ischemic stroke, Parkinson's disease and Alzheimer disease. However, the molecular mechanism underlying the protective effects of CTL is still unclear, and whether CTL can prevent I/R induced cognitive deficit has not been reported. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The open field tasks and Morris water maze were used to assess the effect of CTL on anxiety-like behavioral and cognitive impairment after I/R. Western blotting were performed to examine the expression of related proteins, and HE-staining was used to detect the percentage of neuronal death in the hippocampal CA1 region. Here we found that CTL significantly improved cognitive deficits and the behavior of rats in Morris water maze and open field tasks (P<0.05). HE staining results showed that CTL could significantly protect CA1 neurons against cerebral I/R (P<0.05). Additionally, Akt1 phosphorylation levels were evidently up-regulated (P<0.05), while the activation of JNK3, which is an important contributor to I/R-induced neuron apoptosis, was reduced by CTL after I/R (P<0.05), and caspase-3 levels were also decreased by CTL treatment. Furthermore, all of CTL's protective effects were reversed by LY294002, which is a PI3K/Akt1 inhibitor. Taken together, our results suggest that CTL could protect hippocampal neurons and ameliorate the impairment of learning/memory abilities and locomotor/ exploratory activities in ischemic stroke via a PI3K-Akt1/JNK3/caspase-3 dependent mechanism.

Biotransformation and in vitro metabolic profile of bioactive extracts from a traditional Miao-nationality herbal medicine, Polygonum capitatum.

Polygonum capitatum Buch.-Ham.ex D. Don, a traditional Miao-nationality herbal medicine, has been widely used in the treatment of various urologic disorders. Recent pharmacological studies demonstrated that a pure compound, FR429, isolated from the ethanol extracts of P. capitatum could selectively inhibit the growth of four hepatocellular carcinoma (HCC) cell lines in a dose-dependent manner. Thus, P. capitatum probably exhibits potential antitumor activity. However, there is very little information on the metabolism of substances present in P. capitatum extracts. In this study, gallic acid, quercetrin, ethanol extracts and ethyl acetate fraction of ethnolic extract (EtOAc fraction) of P. capitatum were cultured anaerobically with rat intestinal bacteria. A highly sensitive and selective liquid chromatography electrospray ionization-ion trap-time of fight mass spectrometry (LC/MSn-IT-TOF) technique was employed to identify and characterize the resulting metabolites. A total of 22 metabolites (M1-M22), including tannins, phenolic acids and flavonoids, were detected and characterized. The overall results demonstrated that the intestinal bacteria played an important role in the metabolism of P. capitatum, and the main metabolic pathways were hydrolysis, reduction and oxidation reactions. Our results provided a basis for the estimation of the metabolic transformation of P. capitatum in vivo.

Metabolite profiling analysis of FR429, an ellagitannin purified from Polygonum capitatum, in rat and human liver microsomes, cytosol and rat primary hepatocytes in vitro.

FR429, an ellagitannin (a type of polyphenol), is isolated and purified from Polygonum capitatum Buch.-Ham.ex D. Don which is the original herbal medicine of the "Re-Lin-Qing" formula used clinically to treat urinary tract infection in China. FR429 has been investigated for its antitumor potential in tumor-bearing nude mice in vivo, but its in vitro anti-tumor effect in hepatoma cell lines was low. Thus, it was of our interest to investigate its metabolism pathways for supporting its in vivo antitumor potential. The metabolic profiles of FR429 were studied in vitro by liquid chromatography coupled to ion trap time-of-flight mass spectrometry. Total eight metabolites were identified in rat and human liver microsomes, cytosol, and rat primary hepatocytes in vitro. Ellagic acid, a reported anti-angiogenic agent, was one of the main metabolites in these biological matrices. Methylated metabolites catalyzed by catechol-O-methyl transferase (COMT) were observed mainly in the in vitro incubation with rat liver cytosol, which was verified by using a COMT specific inhibitor entacapone and supported by molecular docking analysis. Methylated and sulfated metabolites were also found in rat primary hepatocytes in a time-dependent manner. In conclusion, the in vitro metabolism pathways of FR429 were hydrolysis, methylation and sulfation. The anti-tumor effects of its major metabolites should be further studied.

In vitro identification of cytochrome P450 isoforms responsible for the metabolism of 1-hydroxyl-2,3,5-trimethoxy-xanthone purified from Halenia elliptica D. Don.

1-Hydroxyl-2,3,5-trimethoxyxanthone (HM-1) is one of the main constituents extracted from Halenia elliptica D. Don, which is a traditionally used Tibetan medicinal plant. The aim of this study was to illustrate the proposed metabolic pathways of HM-1 and identify which cytochrome P450 (CYP450) isoforms involved in its metabolism by using pooled human liver microsomes (HLMs) and recombinant CYP450 isoforms with selective chemical inhibitors. Metabolites were identified by high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LCMS(n)-ESI-IT-TOF) and nuclear magnetic resonance spectroscopy (hydrogen-1 NMR and carbon-13 NMR). Three metabolites (M1-M3) were identified, which demonstrated that demethylation and hydroxylation were the major Phase I metabolic reactions for HM-1 in HLMs. The structure of another metabolite (M4) was still unclear. The enzymatic kinetics of M1 (K(m)=23.19±14.20 µM) and M2 (Km=32.06±17.09 µM) exhibited substrate inhibition; whereas, the formation of M3 (K(m)=5.73±0.70 µM) and M4 (K(m)=16.43±5.12 µM) displayed Michaelis-Menten kinetics. The intrinsic clearance (V(max)/K(m)) of M3 was highest among these metabolites, suggesting that M3 was the major metabolite of HM-1. Moreover, CYP3A4 and CYP2C8 were the primary CYP450 isoform responsible for the metabolism of HM-1. CYP1A2, CYP2A6, CYP2B6, CYP2C9 and CYP2C19 were also involved in HM-1 metabolism, especially in the formation of M3. This study finally provides evidence of substrate inhibition and metabolism-based drug-drug interaction for the medicinal preparations containing HM-1 used in clinic.

Identification of metabolites of FR429, a potential antitumor ellagitannin, transformed by rat intestinal bacteria in vitro, based on liquid chromatography-ion trap-time of flight mass spectrometry analysis.

FR429 is an ellagitannin with a potential antitumor activity, isolated and purified from Polygonum capitatum Buch.-Ham.ex D.Don, which is a traditional Miao-nationality herbal medicine in Guizhou and Yunnan of China. Our preliminary result of pharmacology study has indicated that the antitumor activity of FR429. However, the metabolism of FR429 has not been reported yet. In this study, LC-ion trap-time of flight mass spectrometry (LC-IT-TOF/MS) was used to characterize unpredictable metabolites of FR429 biotransformed by intestinal bacteria in vitro. Total thirteen metabolites were detected and characterized via comparisons of their accurate molecular masses and fragment ions of each MS(n) stage with those of the parent drug, and four of them were also elucidated by NMR. The results demonstrated that FR429 could be transformed by intestinal bacteria in vitro, mainly via hydrolysis and reduction reaction. This work provided a basis for the further study on the biotansformation of FR429 in vivo.

Enzyme kinetic and molecular docking studies on the metabolic interactions of 1-hydroxy-2,3,5-trimethoxy-xanthone, isolated from Halenia elliptica D. Don, with model probe substrates of human cytochrome P450 enzymes.

Halenia elliptica D. Don is a Tibetan herb and medicinal preparations containing Halenia elliptica have been commonly used for the treatment of hepatitis B virus infection in China. The metabolism of 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) to its metabolites is mediated through cytochrome P450 enzymes. This study aimed to investigate the herb-drug interaction potential of HM-1 by studying its effects on the metabolism of model probe substrates of five major CYP450 isoforms in human liver microsomes. HM-1 showed moderate inhibitory effects on CYP1A2 (IC50 = 1.06 µM) and CYP2C9 (IC50 = 3.89 µM), minimal inhibition on CYP3A4 (IC20 = 11.94 µM), but no inhibition on model CYP2D6 (dextromethorphan) and CYP2E1 (chlorzoxazone) probe substrates. Inhibition kinetic studies showed that the K(i) values of HM-1 on CYP1A2, CYP2C9 and CYP3A4 were 5.12 µM, 2.00 µM and 95.03 µM, respectively. HM-1 competitively inhibited testosterone 6ß-hydroxylation (CYP3A4) but displayed mixed type inhibitions for phenacetin O-deethylation (CYP1A2) and tolbutamide 4-hydroxylation (CYP2C9). Molecular docking study confirmed the inhibition modes of HM-1 on these human CYP isoforms.

In vitro study on metabolite profiles of bioactive xanthones isolated from Halenia elliptica D. Don by high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry.

The metabolisms of five xanthones isolated from a Tibetan medicinal herb Halenia elliptica D. Don, including 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1), 1-hydroxy-2,3,4,7-tetramethoxy-xanthone (HM-2), 1-hydroxy-2,3,4,5-tetramethoxy-xanthone (HM-3), 1,7-dihydroxy-2,3,4,5-tetramethoxy-xanthone (HM-4) and 1,5-dihydroxy-2,3-dimethoxy-xanthone (HM-5), were studied in rat liver microsomes in vitro. High performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC-ESI-IT-TOF) was applied for identification of metabolites of five xanthones mentioned above and (1)H NMR was used to elucidate the major metabolites. The structures of thirteen metabolites were identified and seven of them had not been reported before. Moreover, xanthone isomers herein could be distinguished by difference of fragmentation behaviors with increase of stages or relative abundances. The results indicated that in vitro metabolic transformation of HM-1, HM-2, HM-3, HM-4 and HM-5 occurred mainly at 2-, 4-, 5-, 7-carbonic positions on their structures of parent drugs. The metabolites could be new vasoactive substances. This work will provide a basis for study on the structure-activity relationships of these xanthones and their derivatives from Tibetan herbal in the next work.