Integrated pharmacokinetics and pharmacometabolomics to reveal the synergistic mechanism of a multicomponent Chinese patent medicine, Mailuo Shutong pills against thromboangiitis obliterans.

BACKGROUND: Mailuo Shutong Pills (MLST) have displayed pharmacological activity against thromboangiitis obliterans (TAO). However, the active ingredients and therapeutic mechanism of MLST against TAO remained to be further clarified. PURPOSE: The aim of this study was to explore the active components of MLST and their synergistic mechanism against TAO by integrating pharmacokinetics (PK) and pharmacometabolomics (PM). METHODS: TAO model rats were established by sodium laurate solution. Firstly, the efficacy of MLST was evaluated by gangrene score, blood flow velocity, and hematoxylin-eosin (H&E) staining. Secondly, PK research was conducted on bioavailable components to characterize their dynamic behaviors under TAO. Thirdly, multiple plasma and urine metabolic biomarkers for sodium laurate-induced TAO rats were found by untargeted metabolomics, and then variations in TAO-altered metabolites following MLST treatment were analyzed utilizing multivariate and bioinformatic analysis. Additionally, metabolic pathway analysis was performed using MetaboAnalyst. Finally, the dynamic link between absorbed MLST-compounds and TAO-associated endogenous metabolites was established by correlation analysis. RESULTS: MLST significantly alleviated gangrene symptoms by improving the infiltration of inflammatory cells and blood supply in TAO rats. Significant differences in metabolic profiles were found in 17 differential metabolites in plasma and 24 in urine between Sham and TAO rats. The 10 bioavailable MLST-compounds, such as chlorogenic acid and paeoniflorin, showed positive or negative correlations with various TAO-altered metabolites related to glutamate metabolism, histidine metabolism, arachidonic acid metabolism and so on. CONCLUSION: This study originally investigated the dynamic interaction between MLST and the biosystem, providing unique insight for disclosing the active components of MLST and their synergistic mechanisms against TAO, which also shed light on new therapeutic targets for TAO and treatment.

[Comprehensive evaluation of Pinellia ternata germplasm resources based on phenotypic trait classification].

The evaluation of germplasm resources is the prerequisite for the development, utilization, and conservation of Chinese medicinal resources. The selection of excellent germplasm is the key to the breeding and orderly production of Pinellia ternata. In this study, 21 germplasm materials of P. ternata from major production areas in China were collected and analyzed for population diversity after phenotypic preliminary screening. The results have revealed that the P. ternata population has abundant phenotypic variation, and the phenotypic changes could be divided into five phenotypes in terms of organ trait variation. Further analysis of variation in 20 quantitative traits of the population revealed that the coefficient of variation for adenosine content(339.05%) was the largest, while the coefficient of variation for the underground plant height(16.35%) was the smallest. Correlation analysis showed that there was a strong correlation among various traits, with 52 pairs of traits showing highly significant correlation(P<0.01) and 19 pairs of traits showing a significant correlation(P<0.05). The 21 germplasms in the test could be classified into three major clusters by cluster analysis, with Cluster Ⅱ having the highest number and content of nucleosides, making it suitable for the selection and breeding of P. ternata varieties with high content of nucleosides. The yield in Cluster Ⅲ was higher than that in other groups, making it suitable for the selection and breeding of P. ternata varieties with a high yield. All trait indicators could be simplified into five principal component factors through principal component analysis, and the cumulative contribution rate was up to 86.04%. Further, comprehensive analysis using membership function and stepwise regression analysis identified nine traits, such as plant height, main leaf length, and underground plant height as characteristic indicators for the comprehensive evaluation of germplasm resources of P. ternata. BX007, BX008, and BX005 were identified as germplasms with both high yield and high uridine content, with BX007 having the highest uridine content of 479.51 µg·g~(-1). It belonged to the germplasm of P. ternata with double bulbils and could be cultivated as a potential good variety. Based on the phenotypic classification of P. ternata, systematic resource evaluation was carried out in this study, which could lay a foundation for the excavation of genetic resources and the breeding of new varieties of P. ternata.

Identifying quality markers of Mailuoshutong pill against thromboangiitis obliterans based on chinmedomics strategy.

BACKGROUND: Mailuoshutong pill (MLSTP) is a traditional Chinese medicine (TCM) for the treatment of Thromboangiitis obliterans (TAO, Buerger's disease) which is a segmental non-atherosclerotic inflammatory occlusive disorder. However, the mechanism and quality standards of MLSTP have not been sufficiently studied. PURPOSE: This work aims to investigate the potential mechanisms and quality markers (Q-markers) of MLSTP treating TAO based on the chinmedomics strategy. METHODS: The therapeutical effect of MLSTP on TAO rats was evaluated by changes in body weight and clinical score, regional blood flow velocity and perfused blood vessel distribution, hematoxylin-eosin (H&E) staining, serum metabolic profile. Moreover, both endogenous metabolites and exogenous components were simultaneously detected in serum based on ultra-high performance liquid chromatography coupled with a Q Exactive hybrid quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS), and multivariate analysis was applied to identify the biomarkers, as well as the dynamic changes of metabolites were observed to explore the mechanism of action of MLSTP. In addition, the pharmacodynamic material basis were identified by correlation analysis between biomarkers and absorbed constituents. Finally, the Q-markers of MLSTP were determined according to the screening principles of Q-marker and validated the measurability. RESULTS: MLSTP treatment alleviated disease severity of TAO, reduced inflammatory infiltration, and ameliorated vascular function. 26 potential biomarkers associated with glutamate metabolism, linoleic acid metabolism, arachidonic acid metabolism and so on were identified. Besides, 27 prototypical components were identified in serum, 16 of which were highly correlated with efficacy and could serve as the pharmacodynamic material basis of MLSTP against TAO. In addition, 7 compounds, namely, sweroside, chlorogenic acid, calycosin-7-glucoside, formononetin, paeoniflorin, liquiritigenin and 3-butylidenephthalide, were considered as potential Q-markers of MLSTP. Ultimately, the measurability of the seven Q-markers was validated by rapid identifcation and quantifcation. CONCLUSION: This study successfully clarified the therapeutic effect and Q-markers of MLSTP by chinmedomics strategy, which is of great significance for the establishment of quality standards. Furthermore, it provides a certain reference for the screening of Q-markers in TCM prescriptions.

Effects of paired associated stimulation with different stimulation position on motor cortex excitability and upper limb motor function in patients with cerebral infarction.

OBJECTIVE: To investigate the effects of paired associated stimulation (PAS) with different stimulation position on motor cortex excitability and upper limb motor function in patients with cerebral infarction. METHOD: A total of 120 volunteers with cerebral infarction were randomly divided into four groups. Based on conventional rehabilitation treatment, the PAS stimulation group was given the corresponding position of PAS treatment once a day for 28 consecutive days. The MEP amplitude and RMT of both hemispheres were assessed before and after treatment, and a simple upper limb Function Examination Scale (STEF) score, simplified upper limb Fugl-Meyer score (FMA), and improved Barthel Index (MBI) were used to assess upper limb motor function in the four groups. RESULTS: Following PAS, the MEP amplitude decreased, and the RMT of abductor pollicis brevis (APB) increased on the contralesional side, while the MEP amplitude increased and the RMT of APB decreased on the ipsilesional side. After 28 consecutive days the scores of STEF, FMA, and MBI in the bilateral stimulation group were significantly better than those in the ipsilesional stimulation group and the contralesional stimulation group, but there was no significant difference in the scores of STEF, FMA, and MBI between the ipsilesional stimulation group and the contralesional stimulation group. CONCLUSION: The excitability of the motor cortex can be changed when the contralesional side or the ipsilesional side was given the corresponding PAS stimulation, while the bilateral PAS stimulation can more easily cause a change of excitability of the motor cortex, resulting in better recovery of the upper limb function.

Protein tyrosine phosphatase 1B inhibitory activities of ursane-type triterpenes from Chinese raspberry, fruits of Rubus chingii.

Protein tyrosine phosphatase 1B (PTP1B) has led to an intense interest in developing its inhibitors as anti-diabetes, anti-obesity and anti-cancer agents. The fruits of Rubus chingii (Chinese raspberry) were used as a kind of dietary traditional Chinese medicine. The methanolic extract of R. chingii fruits exhibited significant PTP1B inhibitory activity. Further bioactivity-guided fractionation resulted in the isolation of three PTP1B inhibitory ursane-type triterpenes: ursolic acid (1), 2-oxopomolic acid (2), and 2α, 19α-dihydroxy-3-oxo-urs-12-en-28-oic acid (3). Kinetics analyses revealed that 1 was a non-competitive PTP1B inhibitor, and 2 and 3 were mixed type PTP1B inhibitors. Compounds 1-3 and structurally related triterpenes (4-8) were further analyzed the structure-activity relationship, and were evaluated the inhibitory selectivity against four homologous protein tyrosine phosphatases (TCPTP, VHR, SHP-1 and SHP-2). Molecular docking simulations were also carried out, and the result indicated that 1, 3-acetoxy-urs-12-ene-28-oic acid (5), and pomolic acid-3ß-acetate (6) bound at the allosteric site including α3, α6, and α7 helix of PTP1B.

Bioactive terpenoids from Silybum marianum and their suppression on NO release in LPS-induced BV-2 cells and interaction with iNOS.

Three new (1-3) and one known (4) bioactive terpenoids were isolated from the seeds of Silybum marianum based on the investigation to get new NO inhibitors. Their structures were determined by extensive NMR (1D and 2D NMR) and MS spectroscopic data, and the absolute configurations were identified by experimental and calculated ECD spectra. The NO inhibitory activities in murine microglial BV-2 cells and interactions with iNOS protein by molecular docking were evaluated for all compounds. The results showed that these compounds had potent NO inhibitory effects.

Icetexane diterpenoids from Perovskia atriplicifolia.

Five new icetexane diterpenoids, namely, perovskatones B-D (1, 3, 4), 1α-hydroxybrussonol (2), and 1α-hydroxypisiferanol (5), were isolated from Perovskia atriplicifolia, together with a new natural product (6) and two known compounds, przewalskin E (7) and brussonol (8). The structures of the new compounds were elucidated by detailed analyses of their MS, IR, 1D, and 2D NMR data. Compounds 1-8 were assayed for their inhibitory hepatitis B virus activities in the HepG 2.2.15 cell line. The results suggested that compounds 1 and 2 possessed noticeable anti-hepatitis B virus activity in vitro, suppressing the replication of hepatitis B virus DNA with selectivity index values of 154.3 and 137.7, respectively.

[Inhibitory effect of losartan on prostatic hyperplasia in spontaneous hypertension rats and its pathophysiological mechanism].

OBJECTIVE: To investigate the effect of losartan on prostatic hyperplasia in spontaneous hypertension rats (SHRs) and its pathophysiological mechanism. METHODS: We randomly divided 36 male SHRs into three groups of equal number to be treated intragastrically with high-dose losartan (30 mg per kg per d), low-dose losartan (15 mg per kg per d) and distilled water (control group). After 6 weeks of intervention, we measured the body weight and tail artery blood pressure of the rats and compared them with the baseline data. We collected blood from the heart for determination of the levels of serum angiotensin II (Ang II), insulin-like growth factor-1 (IGF-1) and interleukin-6 (IL-6) by enzyme-linked immunosorbent assay (ELISA), and harvested their prostates for measurement of their weight, observation of the tissue ultrastructures under the electron microscope and detection of the expression of endothelial nitric oxide synthase (eNOS) in the prostate tissue by immunohistochemistry. RESULTS: Compared with the control group, the low- and high-dose losartan groups showed significant decreases in systolic blood pressure ([203.75 +/- 10.28] vs [184.54 +/- 16.90] mmHg, P = 0.013; [203.75 +/- 10.28] vs [166.88 +/- 14.74] mmHg, P = 0.001) and diastolic blood pressure ([151.58 +/- 9.96] vs [136.71 +/- 14.28] mmHg, P = 0.022; [151.58 +/- 9.96] vs [122.71 +/- 11.56] mmHg, P < 0.001) of the lower tail artery after treatment, as well as in the prostate weight ([0.73 +/- 0.08] vs [0.64 +/- 0.10] mg, P = 0.011; [0.73 +/- 0.08 ] vs [0.50 +/- 0.17] mg, P < 0.001). Electron microscopy revealed edema of the basal and columnar epithelial cells, concentrated and marginated heterochromatin and widened nuclear gap of interstitial fibroblast nuclei, and reduced mitochondria and endoplasmic reticula in the low-dose losartan group, and even more obvious in the high-dose group. The level of serum Ang II was remarkably higher in the low- and high-dose losartan groups than in the control ([61.32 +/- 2.49] vs [54.85 +/- 7.20] pg/ml, P = 0.021; [65.49 +/- 6.78] vs [54.85 +/- 7.20] pg/ml, P < 0.001]) , that of serum IGF-1 was lower in high-dose losartan than in the control group ([1.50 +/- 0.11] vs [1.60 +/- 0.10] ng/ml, P = 0.03), but the serum IL-6 levels exhibited no significant differences among the three groups. The expression of eNOS in the prostate tissue was significantly higher in the losartan groups than in the controls (P = 0.022), even higher in the high-dose than in the low-dose group. CONCLUSION: Losartan can suppress the progression of prostate hyperplasia in spontaneous hypertension rats by inhibiting RAS, IGF-1 and angiogenesis.

[Effect of lemon essential oil on caries factors of Streptococcus sobrinus].

OBJECTIVE: To investigate the mechanism of lemon peel essential oil (LPE) on the cariogenicity of Streptococcus sobrinus (Ss). METHODS: LPE was extracted by the authors, and the minimum inhibition concentration (MIC) was measured by disc diffusion method. The LPE was used as the experimental group with concentrations ranging from 2.250 g/L to 0.281 g/L prepared with trypticase peptone yeast (TPY) culture medium, and TPY culture medium was used as the control group. Ss at the concentration of 10(8) CFU/ml was added to each group, and cultured for 6, 18, 24, 48 hours. Neson-Somogyi method was used to measure the content of reducing sugar, and glucosyltransferase (GTF) activity. The activity of lactate dehydrogenase (LDH) was measured by lactic acid and pyruvic acid continuous monitoring method. The content of water insoluble glucan (WIG) was measured by anthrone method, and the pH value of the culture solution was detected. The value of pH before the experiment and the time difference was alculated as ΔpH. RESULTS: At the same time point, the activity of GTF and LDH and the concentration of WIG and the value ΔpH decreased gradually with the increase of concentration of LPE. There were significant differences between each experimental group and control group (P < 0.01). The control group had the maximum value, GTF: (6.71 ± 0.61) mIU, LDH: (135.8 ± 1.7) U/L, WIG: (47.15 ± 5.12) mg/L, ΔpH: (2.67 ± 0.01). The highest drug concentration group had the minimum value: GTF: (0.39 ± 0.07) mIU, LDH: (95.0 ± 5.4) U/L, WIG: (2.44 ± 0.38) mg/L, ΔpH: (0.61 ± 0.01). CONCLUSIONS: The LPE below the MIC could still inhibit the GTF, LDH activity and lead to the decrease of WIG and the acid production.

[Effects of lemon peel extracts on lactate dehydrogenase and sucrase activity of Streptococcus mutans].

OBJECTIVE: To investigate the effect of lemon peel extracts (LPE) on the activity of lactate dehydrogenase and sucrase of Streptococcus mutans (Sm). METHODS: After serial dilution with trypticase soy broth (TSB) medium containing 2% glucose, LPE was used as the experimental group, and TSB without LPE as the control group. Sm was added to each group, which was then cultured for 6, 18, 24 and 48 hours in the anaerobic tank. The activity of lactate dehydrogenase(LDH) was measured with the method of oxidation of reduction coenzymeIand the pH value of the culture solution was also detected. The activity of the sucrose was determined with the method of coloration of 3,5-dinitrosalicylic acid. RESULTS: The activity of LDH, sucrase and the changes of solution pH were decreased with the increase of the concentration of LPE (P < 0.01). The activity of LDH were declined from (0.8025 ± 0.0913) × 10(3) U/L to (0.2099 ± 0.0283) × 10(3) U/L; the activity of sucrase were declined from (-0.0107 ± 0.0003) × 10(3) U/L to (-0.0078 ± 0.0002) × 10(3) U/L; the ΔpH were declined from (2.8067 ± 0.0404) to (2.5033 ± 0.0416) (24 h results). The differences were significant between experimental groups and the control group (P < 0.01), and there were also significant differences among experimental groups with different LPE concentration (P < 0.01). The inhibitory effect of acid generation and lactate dehydrogenas' activity of Sm were positively correlated (P < 0.01). CONCLUSIONS: LPE can inhibit the activity of lactate dehydrogenase, sucrase and the acid production capacity of the Sm in a dose dependent manner. The inhibitory effects in logarithmic phase is stronger than that in other phases of growth cycle.

[PET study of effects of combination of different points on glucose metabolism in the patient of cerebral infarction].

OBJECTIVE: To observe effects of combination of different points on glucose metabolism in the patient of cerebral infarction. METHODS: Neiguan (PC 6), Shuigou (GV 26), Fengchi (GB 20), Sanyinjiao (SP 6), Yinlingquan (SP 9), Taichong (LR 3) were selected for the Xingnao Kaiqiao group (n =7), and Hegu (LI 4), Quchi (LI 11), Zusanli (ST 36), Yanglingquan (GB 34) and Xuanzhong (GB 39) for the routine acupuncture group (n = 5). PET-CT was used to observe the local cerebral glucose metabolism. RESULTS: The cerebral regions activated in the the Xingnao Kaiqiao group included infarction center, gyrus temporalis superior, thalamus, gyrus temporalis inferior, gyrus rectus, insula, lateral occipital lobe, parietal lobe and cerebellum, and in the routine acupuncture group included gyrus frontalis inferior, caudate nucleus, cingulated gyrus, hippocampus, precuneus and parietal lobe. CONCLUSION: PET is very suitable for exploring acupuncture mechanisms in treatment of cerebral infarction, and further shows the specificity of acupoints-brain functional region.