Alleviation of endothelial dysfunction of Pheretima guillemi (Michaelsen)-derived protein DPf3 in ponatinib-induced thrombotic zebrafish and mechanisms explored through ox-LDL-induced HUVECs and TMT-based proteomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Thrombus generation is one of the leading causes of death in human, and vascular endothelial dysfunction is a major contributor to thrombosis. Pheretima guillemi (Michaelsen), a traditional medicinal animal known as "Dilong", has been utilized to cure thrombotic disorders for many years. DPf3, a group of functional proteins extracted from P. guillemi, has been characterized and identified to possess antithrombotic bioactivity via in vitro and ex vivo experiments. AIM OF THE STUDY: This study is aimed to investigate the vascular-protection activity and related mechanism of antithrombotic protein DPf3 purified from Pheretima guillelmi systematically. MATERIALS AND METHODS: The antithrombotic activity and vascular endothelium protection effect of DPf3 was explored in vivo using ponatinib-induced vascular endothelial injury zebrafish thrombus model. Then, (hi) ox-LDL-induced HUVECs was applied to investigate the protection mechanism of DPf3 against the injury of vascular endothelium. In addition, TMT-based proteomics analysis was used to study the biomarkers, biological processes and signal pathways involved in the antithrombotic and vascular protective effects of DPf3 holistically. RESULTS: DPf3 exerted robust in vivo antithrombosis and vascular endothelial protection ability. DPf3 was identified to prevent HUVECs from damage by reducing ROS production, and to reduce monocyte adhesion by decreasing the protein content of adhesion factor VCAM 1. DPf3 was also observed to weaken the migration ability of injured cells and inhibit abnormal angiogenesis. The mechanism of DPf3's antithrombotic and vascular protective activity was mainly related to the regulation of lipid metabolism, energy metabolism, complement and coagulation system, ECM receptor interaction, MAPK signal pathway, etc. CONCLUSIONS: This study demonstrates that DPf3 has strong antithrombotic and endothelial protective effects. The endothelial protective ability and related mechanisms of DPf3 provide a scientific reference for the traditional use of earthworms in the treatment of thrombosis.

Protective Effect of Trillium tschonoskii Maxim Components Against Glutamate-Induced SH-SY5Y Cells Damage Through Regulating Apoptosis.

Context: Among the Tujia people, the root or rhizome of Trillium tschonoskii Maxim.in Bull.Acad (TTM) is considered a miraculous herb for headaches. Previous studies have shown ethyl acetate extract (TTM1) can protect SH-SY5Y cells against glutamate injury. Objective: This study clarified TTM1's mechanism against glutamate-induced cell damage, focusing on the regulation of apoptosis. The compounds were separated, identified, and performed molecular docking with pro-apoptotic proteins. Materials and Methods: SH-SY5Y cells were treated with glutamate (2 mM) for 12 hour, and the effect of TTM1 (2.5, 5, 10, and 20 µg/mL) was evaluated with MTT and LDH release assays, taking EGb761(40 µg/mL) as a control. Cell apoptosis was detected with Hoechst 33258 and Annexin V-FITC and measurements of intracellular calcium and caspase-3. The major components were separated and identified by LCMS-IT-TOF and NMR, then the proapoptotic activity of TTM1 was confirmed by molecular docking method. Results: TTM1 protected SH-SY5Y cells by resisting apoptosis, TTM1 (10 and 20 µg/mL) decreased apoptotic bodies and nuclear fragments, increased the proportion of normal cells to 68.38 ± 5.63% and 92.80 ± .88%, decreased VA cells to 4.30 ± .76% and 3.58 ± .45% and caspase-3 to .365 ± .034 and .344 ± .047 ng/mL.TTM1 (10 µg/mL) decreased intracellular free calcium to 2.77 ± .40. Polyphyllin VI and pennogenin 3-O-ß-chacotrioside were identified in TTM1 at 15.04% and 2.84%, and had potential anti-apoptosis activities. Discussion and Conclusions: Folk records of TTM for headache may be related to its anti-apoptosis of nerve cells. Identification and content determination of index components based on effective extract provides research paradigms for rare and endangered ethnic plants.

[Effect of nanoparticles of different stiffness combined with menthol/curcumol on mechanical properties of bEnd.3 cells].

This study aimed to investigate the effects of nanoparticles PLGA-NPs and mesoporous silicon nanoparticles(MSNs) of different stiffness before and after combination with menthol or curcumol on the mechanical properties of bEnd.3 cells. The particle size distributions of PLGA-NPs and MSNs were measured by Malvern particle size analyzer, and the stiffness of the two nanoparticles was quantified by atomic force microscopy(AFM). The bEnd.3 cells were cultured in vitro, and the cell surface morphology, roughness, and Young's modulus were examined to characterize the roughness and stiffness of the cell surface. The changes in the mechanical properties of the cells were observed by AFM, and the structure and expression of cytoskeletal F-actin were observed by a laser-scanning confocal microscope. The results showed that both nanoparticles had good dispersion. The particle size of PLGA-NPs was(98.77±2.04) nm, the PDI was(0.140±0.030), and Young's modulus value was(104.717±8.475) MPa. The particle size of MSNs was(97.47±3.92) nm, the PDI was(0.380±0.016), and Young's modulus value was(306.019±8.822) MPa. The stiffness of PLGA-NPs was significantly lower than that of MSNs. After bEnd.3 cells were treated by PLGA-NPs and MSNs separately, the cells showed fine pores on the cell surface, increased roughness, decreased Young's modulus, blurred and broken F-actin bands, and reduced mean gray value. Compared with PLGA-NPs alone, PLGA-NPs combined with menthol or curcumol could allow deepened and densely distributed surface pores of bEnd.3 cells, increase roughness, reduce Young's modulus, aggravate F-actin band breakage, and diminish mean gray value. Compared with MSNs alone, MSNs combined with menthol could allow deepened and densely distributed surface pores of bEnd.3 cells, increase roughness, reduce Young's modulus, aggravate F-actin band breakage, and diminish mean gray value, while no significant difference was observed in combination with curcumol. Therefore, it is inferred that the aromatic components can increase the intracellular uptake and transport of nanoparticles by altering the biomechanical properties of bEnd.3 cells.

Differences in in vivo absorption of flavone glycosides, flavone aglycones and terpene lactones under different dosage forms and physiological conditions.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba L. extract (GBE) oral preparations have been used for many years in the prevention and treatment of cardiovascular and cerebrovascular diseases, and the main active ingredients are flavonoids and terpene lactones. Among them, the oral absorption of the prototype components of flavonoid glycosides into the blood needs to be further clarified, and the differences in the oral absorption of different components in GBE by different dosage forms and physiological conditions are not clear yet. AIM OF THE STUDY: To clarify the oral absorption of the prototype flavonoid glycosides in vivo, and to further explore the differences in the oral absorption of various active compounds under different oral dosage forms and dietary conditions. MATERIALS AND METHODS: Firstly, the target compounds were selected based on the characteristic chromatogram of GBE and literature. Then, the content differences of three different oral GBE preparations were studied, and their pharmacokinetics (PK) were compared. Finally, the PK differences of the preparations with better oral absorption under different dietary conditions were studied. RESULTS: Five flavonoid glycosides, three aglycones and four terpene lactones were selected as the research objects. The content determination results of GBE tablets, guttate pills and tinctures showed that the content of several components especially flavonoid glycosides in the tincture was higher than that of the other two preparations. After oral administration of these three preparations, the PK study showed different results from previous studies. The PK behavior of flavonoid glycosides was also determined at the same time as flavonoid glycosides and terpene lactones. and the bioavailability of flavonoid glycosides in the tincture was higher than that of the other two preparations. PK results of fasting and non-fasting showed that taking GBE tincture on an empty stomach increased the absorption of various compounds, especially flavonoid glycosides. However, due to the existence of food residues in the gastrointestinal tract, the oral bioavailability of flavonoid glycosides was significantly improved. CONCLUSIONS: This study discussed the differences in the content and oral absorption of active compounds in different oral preparations of GBE, clarified the in vivo absorption of flavonoid glycosides prototype, as well as the influence of diet on the PK of active compounds, which has certain guiding significance for the clinical application of GBE oral preparations.

Chemical and pharmacological difference between honey-fried licorice and fried licorice.

ETHNOPHARMACOLOGICAL RELEVANCE: According to textual research of books from ancient times till now, there are three main preparation methods of "fried licorice", including frying licorice without excipients (F), frying licorice after dipping with water (W), and frying licorice with honey (H). However, with the development over many successive generations, honey frying has gradually become the main processing form of licorice, whereas the fried licorice is nowadays rarely used. AIM OF THE STUDY: The objectives of this study were to clarify the differences of the three forms of "fried licorice" in chemical composition and pharmacological activities, and to screen quality markers for differently processed licorice. It is expected to provide a scientific basis for the rational choice of "fried licorice" as medicine. MATERIALS AND METHODS: Non-target metabolomic analysis based on UHPLC-QE-Orbitrap-MS was conducted to compare the chemical differences between the differently processed licorice material. Pharmacodynamically, the differences in immunomodulatory activity (including intestinal flora experiment), anti-inflammatory activity, and hepatoprotective activity of the differently processed licorice were evaluated. Furthermore, multivariate statistical analysis was performed to screen potential quality markers of honey-fried licorice. The serum concentration of selected markers was determined by UHPLC-QqQ-MS. RESULTS: Metabolomic analysis showed no difference in the chemical composition of F and W, whereas the chemical composition of H was significantly different from that of F and R. The immunomodulatory activity, anti-acute inflammatory effect, and hepatoprotective effect of licorice were significantly improved after frying with honey; No significant differences were observed between F and H in term of immunomodulatory activity and anti-acute inflammatory effect, whereas, H is better than F in terms of liver-protective activity. The intestinal flora experiment confirmed that H does have immunomodulatory activity, while F may induce an increased abundance of certain pathogenic bacteria in the intestine. Multivariate statistical analysis suggests that the content of liquiritin (2), liquiritigenin (3), isoliquiritin (5), isoliquiritigenin (6) and glycyrrhizic acid (7) plusing glycyrrhetinic acid (8) in H group is closely correlated with its improved effects. CONCLUSIONS: This study provides a scientific rational for the selection of "fried licorice" processing methods. In addition, it provides a scientific basis for the selection of quality markers of differently processed licorice.

Differentiation of three Asparagus species by UHPLC-MS/MS based molecular networking identification and chemical profile analysis.

Asparagi Radix (AR), a traditional Chinese medicine, is the dried roots of Asparagus cochinchinensis (Lour.) Merr. Modern pharmacological studies have shown that AR has various excellent bioactivities, such as antioxidative, antitumor, antibacterial, anti-inflammatory, and hypoglycemic effects. However, the quality control method of AR is incomplete and there are various AR adulterants in markets due to their similar morphological characters. Here, holistic and practical quality evaluation methods were developed to chemically distinguish three common Asparagus species in markets, including Asparagus cochinchinensis (Lour.) Merr., Asparagus officinalis L., and Asparagus lycopodineus (Baker) F.T.Wang & Tang. The chemical constituents of three species were rapidly tentatively annotated using a combination of ultra-high pressure liquid chromatography-linear ion trap-orbitrap high resolution mass spectrometry (UHPLC-LTQ-Orbitrap-MS) and molecular networking (MN). Fifty-six steroidal saponins were annotated, including common and characteristic chemical constituents of the three Asparagus species. Besides, to establish holistic and practical methods to differentiate three Asparagus species, an HPLC-ELSD (evaporative light scattering detector) was applied for fingerprint analysis and content determination of the sum of protoneodioscin and protodioscin of twenty samples. Each Asparagus species showed characteristic chemical profile and AR showed much higher level of the sum of protoneodioscin and protodioscin than that in the others. The above analyses showed that the three Asparagus species mainly contain steroidal saponins and the developed HPLC-ELSD profile of saponin can be used to differentiate them. In conclusion, this study reveals the different chemical constituents of three Asparagus species and provides relatively feasible quality evaluation methods for them which are essential for the rational utilization of these Asparagus species.

Synergistic mechanism for the bioactivity fortification of licorice by honey.

ETHNOPHARMACOLOGICAL RELEVANCE: Honey-processed licorice has been used since ancient times. It was recorded that honey-processing has the effect of improving the immunomodulatory efficacy of licorice, which has been confirmed by modern pharmacological studies. However, it is still unknown why honey-processing can enhance the immunomodulatory activity of licorice. Our previous research demonstrated that honey has natural deep eutectic solvent (NADES) characteristics. In this study, we investigated the synergistic effect of honey on licorice to elucidate the possible potentiation of honey-frying on licorice. MATERIALS AND METHODS: Immunological experiments were conducted to investigate whether the honey-processing could enhance the immunomodulatory efficacy of licorice in vivo. Then, the synergistic mechanism of honey and licorice was explored based on cell bioactivity tests, metabolomics analysis, bioavailability test, and Fourier transform-infrared (FT-IR) spectra. RESULTS: Pharmacological experiment verified that honey-processing enhanced the immunomodulatory efficacy of licorice. Moreover, honey increased the total flavonoid and polysaccharide contents in licorice decoction, improved the thermal stability and oral bioavailability of certain pharmacologically active constituents, and augmented their overall immunostimulatory functions. Similar effects of honey were also observed with a honey analogue GFSH, a NADES made of glucose, fructose, and sucrose with certain amount of water. The above effects might be due to multiple molecular interactions between active compounds and sugar molecules of honey. CONCLUSION: These findings indicate that the biological activities of medicinal plants might be fortified by honey due to the synergism between licorice and honey. At the meantime, these findings provide theoretical and empirical basis for potential novel applications of honey or other NADESs at augmenting the health-promoting effects of medicinal plants.

[Puerarin-Chuanxiong oil submicron emulsion combining medicine and adjuvant].

This study was mainly based on the compatibility of Puerariae Lobatae Radix and Chuanxiong Rhizoma to prepare submicron emulsion and evaluated its physical and pharmaceutical properties. Firstly, pseudo-ternary phase diagrams were drawn by dripping method which took Chuanxiong oil as the oil phase and the area of microemulsion region as the index. On this basis, suitable emulsifier and co-emulsifier were screened for the preparation of Chuanxiong oil submicron emulsion. Then, the formula realizing the largest oil loading was selected. Finally, puerarin substituted part of emulsifier and co-emulsifier to lower their content, so as to form puerarin-Chuanxiong oil submicron emulsion featuring the combination of medicine and adjuvant. Its particle size, zeta potential, centrifugal stability and storage stability were determined, and the in vitro drug release behavior was investigated by dialysis bag method, based on which the quality of the as-prepared submicron emulsion was evaluated comprehensively. The proposed method was proved feasible for the preparation of Chuanxiong oil submicron emulsion, which adopted polyoxyethylene castor oil(EL-40) as the emulsifier and was free from co-emulsifier. The formula of the maximum oil loading was found as Chuanxiong oil∶EL-40∶water 3∶7∶90. Further, puera-rin successfully replaced up to 10% of the emulsifier in submicron emulsion. Eventually, the optimal drug-loading formula was determined as puerarin∶Chuanxiong oil∶EL-40∶water 7∶30∶63∶900. The quality evaluation results of the as-prepared submicron emulsion demonstrated that the average emulsion droplet size was 333.9 nm, the PDI 0.26, and the zeta potential-10.12 mV. The submicron emulsion had a good centrifugal stability and did not present any instable phenomena such as delamination and precipitation during its standing still for 50 days. The evaluation of in vitro drug release behavior indicated that the submicron emulsion was capable of releasing the drug completely. The puerarin-chuanxiong oil submicron emulsion prepared in this study possessed a stable quality and to some extent increased the solubility of puerarin along with a sustained-release effect. This study provided ideas for the clinical application of puerarin.

[HPLC fingerprint of famous traditional formula Sanpian Decoction and quality value transmitting of Chuanxiong Rhizoma].

Famous traditional formula Sanpian Decoction(SPD)comes from Dialectical Records of Chen Shiduo of the Qing Dynasty,and ranks among 100 classic prescriptions of Classic Famous Traditional Formula catalogue(the First Batch). SPD was prepared according to Management Standards for Traditional Chinese Medicine Decoction Room in Medical Institutions. According to the polarity of different components in SPD,two HPLC fingerprints were established, in which six herbs, namely Chuanxiong Rhizoma, Paeoniae Randix Alba, Sinapis Semen, Glycyrrhizae Radix et Rhizoma, Pruni Semen, Angelicae Dahuricae Radix,are all reflected in the fingerprints; The dry extract rate, transfer rate and similarities of fingerprints were used as indicators to study the relationship between the quality value transmitting of medicinal herbs-decoction pieces-whole decoction of Chuanxiong Rhizoma. Experiment result shows that,the transfer rate of ferulic acid from medicinal herbs to decoction pieces is between 72.00% and 108.36%; the transfer rate of ferulic acid from decoction pieces to SPD is between 31.76% and 64.09%; the dry extract rate of the whole decoction is between 14.69% and 20.16%;The similarity range of fingerprint 1 of 15 batches of SPD is between 0.971 and 0.998, and the similarity range of fingerprint 2 is between 0.980 and 0.996. The established fingerprint has rich information,and the established quality evaluation method is suitable for the quality control of medicinal herbs-decoction pieces-whole decoction of Chuanxiong Rhizoma, which can provide a certain reference for developing the quality control evaluation method for formulated granules, famous formulae and other terminal products derived from traditional Chinese medicine decoction.

Quality Markers for Astragali Radix and Its Products Based on Process Analysis.

Due to the complex nature of traditional medicines, quality control methods need to cover two aspects: compliance of raw materials with quality standards and process control. Astragali radix (AR), the roots of Astragalus mongholicus Bunge, was selected in this study as an example of a widely used traditional medicine in various formulations. Astragaloside IV (AG IV) and calycosin 7-O-ß-D-glucoside (CG) are used as the markers for the quality control of AR and its products in the Chinese Pharmacopoeia. However, in the raw materials, malic acid esters of the CG and acetate esters of the astragaloside are easily decomposed into CG and AG IV during storage and processing of AR to make extracts for various preparations. The thermal stability of the isoflavonoids and astragalosides in decoction was studied. The level of CG and astragalosides (AG I/AG II/AG IV) was strongly affected by prolonged heat during processing, while calycosin was stable in the conditions. Also the major astragalosides in AR could fully converted into AG IV which eventually reaches a stable level under certain conditions. With calycosin and AG IV as marker components, practical, reproducible, and precise methods were established and applied to the quality analysis of AR from its raw materials to its intermediates and products. This study demonstrates that a full chemical profiles analysis of the whole manufacturing process (from "raw materials-intermediates/extracts-final product") is important to identify quality markers (Q-markers) and even to establish proper analysis methods for traditional Chinese medicine products.

Aging Changes Effective Connectivity of Motor Networks During Motor Execution and Motor Imagery.

Age-related neurodegenerative and neurochemical changes are considered to be the basis for the decline of motor function; however, the change of effective connections in cortical motor networks that come with aging remains unclear. Here, we investigated the age-related changes of the dynamic interaction between cortical motor regions. Twenty young subjects and 20 older subjects underwent both right hand motor execution (ME) and right hand motor imagery (MI) tasks by using functional magnetic resonance imaging. Conditional Granger causality analysis (CGCA) was used to compare young and older adults' effective connectivity among regions of the motor network during the tasks. The more effective connections among motor regions in older adults were found during ME; however, effective within-domain hemisphere connections were reduced, and the blood oxygenation level dependent (BOLD) signal was significantly delayed in older adults during MI. Supplementary motor area (SMA) had a significantly higher In+Out degree within the network during ME and MI in older adults. Our results revealed a dynamic interaction within the motor network altered with aging during ME and MI, which suggested that the interaction with cortical motor neurons caused by the mental task was more difficult with aging. The age-related effects on the motor cortical network provide a new insight into our understanding of neurodegeneration in older individuals.

Panax notoginseng saponins suppress lipopolysaccharide-induced barrier disruption and monocyte adhesion on bEnd.3 cells via the opposite modulation of Nrf2 antioxidant and NF-κB inflammatory pathways.

Dysfunction of the blood-brain barrier (BBB) is a prerequisite for the pathogenesis of many cerebral diseases. Oxidative stress and inflammation are well-known factors accounting for BBB injury. Panax notoginseng saponins (PNS), a clinical commonly used drug against cerebrovascular disease, possess efficient antioxidant and anti-inflammatory activity. In the present study, the protective effects of PNS on lipopolysaccharide (LPS)-insulted cerebral microvascular endothelial cells (bEnd.3) were assessed and the underlying mechanisms were investigated. The results showed that PNS mitigated the decrease of Trans-Endothelial Electrical Resistance, increase of paracellular permeability, and loss of tight junction proteins in bEnd.3 BBB model. Meanwhile, PNS suppressed the THP-1 monocytes adhesion on bEnd.3 monolayer. Moreover, PNS prevented the pro-inflammatory cytokines secretion and reactive oxygen species generation in bEnd.3 cells stimulated with LPS. Mechanism investigations suggested that PNS promoted the Akt phosphorylation, activated Nrf2 antioxidant signaling, and inhibited the NF-κB activation. All the effects of PNS could be abolished by PI3K inhibition at different levels. Taken together, these observations suggest that PNS may act as an extrinsic regulator that activates Nrf2 antioxidant defense system depending on PI3K/Akt and inhibits NF-κB inflammatory signaling to attenuate LPS-induced BBB disruption and monocytes adhesion on cerebral endothelial cells in vitro.

Transcriptomic-proteomics-anticoagulant bioactivity integrated study of Pheretima guillemi.

ETHNOPHARMACOLOGICAL RELEVANCE: Earthworms, a type of animal drugs from traditional Chinese medicine, have been used to treat coagulation for many years with less adverse effects and similar anticoagulant effects compared to the commonly used anticoagulants. There are four species of earthworms recorded in Chinese Pharmacopoeia, while few of them were studied and deficient information were involved in the NCBI and UniProt earthworm protein database. We have adopted a transcriptomic-proteomics-anticoagulant bioactivity integrated approach to investigate a seldom-studied Chinese Pharmacopoeia recorded species, Pheretima guillelmi. AIM OF THE STUDY: In the present study, we aimed to reveal the anticoagulant bioactivity of Pheretima guillelmi, and identify its functional proteins via LC-MS/MS-transcriptome cross identification. METHODS AND RESULTS: With the aid of fibrinogen-thrombin time assay, Pheretima guillelmi was found to possess strong anticoagulant activity, and the bioactivity was quite stable under 30-50 °C and near-neutral conditions. A comprehensive non-reference transcriptome assembly of P. guillelmi was first established to supplement the currently inadequate earthworm protein database and to illustrate the active proteins. Illumina RNA sequencing generated 25,931,175 of clean reads with over 97% high-quality clean reads (Q20) and assembled an average of 133,228 of transcript and 106,717 of unigenes. A total of 11,259 coding sequences were predicted via ESTScan (3.0.3). The P. guillelmi unigenes were searched and annotated against public database. The bioactive proteins in P. guillelmi were with broad distribution of molecular weight. With bottom-up proteomics analysis, ten proteins were identified against UniProt and NCBI earthworm database; and 31 proteins with high-confidence were matched against transcriptomic established P. guillelmi database. CONCLUSION: This study illuminated the therapeutic potency of P. guillelmi for antithrombus and provide a new strategy to investigate animal drugs of Chinese materia medica.

Study on the Material Basis of Houpo Wenzhong Decoction by HPLC Fingerprint, UHPLC-ESI-LTQ-Orbitrap-MS, and Network Pharmacology.

Houpo Wenzhong Decoction (HWD) as a classical prescription has been widely used for hundreds of years. However, the quality of HWDs is difficult to control because of its herb materials from different regions. It is a new idea to use HPLC fingerprints, LTQ-ESI-Orbitrap-MS, and network pharmacology in combination to screen common components (CCs) as potential quality control indicators. In this paper, the fingerprints of HWDs were established, which were used to determine HWDs compounded from different sources of traditional Chinese medicines (TCMs). Through the similarity analysis, 45 CCs were selected. UHPLC-LTQ-ESI-Orbitrap-MS was used to carry out the chemical composition analysis of HWD. Seventy-three chemical constituents were distinguished, and 30 CCs were identified. Through network pharmacology, networks of candidate CCs, diseases, and candidate targets were constructed. Finally, four CCs were screened as potential active ingredient markers of HWD, and a method for content determination of these four components was established. A rapid, reasonable, and effective method for quality evaluation and control of HWDs was established. It provides a reference for the further development and research of HWDs and a new way of thinking for the research of other Chinese medicine prescriptions.

Effects of Panax Notoginseng Saponins on Esterases Responsible for Aspirin Hydrolysis In Vitro.

Herb⁻drug interactions strongly challenge the clinical combined application of herbs and drugs. Herbal products consist of complex pharmacological-active ingredients and perturb the activity of drug-metabolizing enzymes. Panax notoginseng saponins (PNS)-based drugs are often combined with aspirin in vascular disease treatment in China. PNS was found to exhibit inhibitory effects on aspirin hydrolysis using Caco-2 cell monolayers. In the present study, a total of 22 components of PNS were separated and identified by UPLC-MS/MS. Using highly selective probe substrate analysis, PNS exerted robust inhibitory potency on human carboxylesterase 2 (hCE2), while had a minor influence on hCE1, butyrylcholinesterase (BChE) and paraoxonase (PON). These effects were also verified through molecular docking analysis. PNS showed a concentration-dependent inhibitory effect on hydrolytic activity of aspirin in HepaRG cells. The protein level of hCE2 in HepaRG cells was suppressed after PNS treatment, while the level of BChE or PON1 in the extracellular matrix were elevated after PNS treatment. Insignificant effect was observed on the mRNA expression of the esterases. These findings are important to understand the underlying efficacy and safety of co-administration of PNS and aspirin in clinical practice.

Network pharmacology-based identification of protective mechanism of Panax Notoginseng Saponins on aspirin induced gastrointestinal injury.

BACKGROUND & AIMS: Aspirin is the first line therapy for cardiovascular and cerebrovascular diseases and is widely used. However aspirin-induced gastrointestinal injury is one of its most common side effect which limits long-term use. Panax Notoginseng Saponins(PNS) which is also used to prevent thrombus may alleviate this side effect according to previous clinical evidences. Owing to the complexity of drug combination, the protective mechanism of PNS on aspirin-induced gastrointestinal injury remains unclear. Therefore, a network pharmacology-based strategy was proposed in this study to address this problem. METHODS: A network pharmacology approach comprising multiple components, candidate targets of each component, known therapeutic targets, network analysis has been used in this study. Also, we establish aspirin-induced gastrointestinal injury model by the oral administration of aspirin (0.5 g/kg body weight) to verify the predicted targets from network pharmacology. All rats was randomly allocated to control groups (n = 6),aspirin groups (n = 6)and aspirin + PNS groups (n = 6) and conducted H&E staining and ELISA for VEGFA. RESULTS: The comprehensive systematic approach was successfully to identify 5 compounds and 154 candidate targets in PNS and 479 candidate targets in aspirin. After network establishment and analysis, 27 potential targets hit by PNS, aspirin and 6 kind of gastrointestinal diseases were found. The experiments results indicated that aspirin group has visible inflammation and lesions while aspirin + PNS group have not. The higher expression of VEGFA in aspirin + PNS group verified the predicted potential protective targets of PNS. CONCLUSIONS: PNS may have protective function for aspirin-induced gastrointestinal injury through increasing VEGFA expression. Network pharmacology strategy may provide a forceful tool for exploring the mechanism of herb medicine and discovering novel bioactive ingredients.

Rapid Characterization of Components in Bolbostemma paniculatum by UPLC/LTQ-Orbitrap MSn Analysis and Multivariate Statistical Analysis for Herb Discrimination.

Bolbostemma paniculatum is a traditional Chinese medicine (TCM) showed various therapeutic effects. Owing to its complex chemical composition, few investigations have acquired a comprehensive cognition for the chemical profiles of this herb and explicated the differences between samples collected from different places. In this study, a strategy based on UPLC tandem LTQ-Orbitrap MSn was established for characterizing chemical components of B. paniculatum. Through a systematic identification strategy, a total of 60 components in B. paniculatum were rapidly separated in 30 min and identified. Then based on peak intensities of all the characterized components, principle component analysis (PCA) and hierarchical cluster analysis (HCA) were employed to classify 18 batches of B. paniculatum into four groups, which were highly consistent with the four climate types of their original places. And five compounds were finally screened out as chemical markers to discriminate the internal quality of B. paniculatum. As the first study to systematically characterize the chemical components of B. paniculatum by UPLC-MSn, the above results could offer essential data for its pharmacological research. And the current strategy could provide useful reference for future investigations on discovery of important chemical constituents in TCM, as well as establishment of quality control and evaluation method.

Effect of aspirin on the pharmacokinetics and absorption of panax notoginseng saponins.

BACKGROUND: Panax notoginseng saponins, a traditional Chinese medicine extraction, and aspirin are both widely used to treat cerebral infarction in China. Good results in clinical practice have been achieved, when Panax notoginseng saponins was taken together with aspirin. METHODS: To investigate the interaction of the two drugs in vivo, the concentration of notoginsenoside R1, ginsenoside Rg1, Rb1, Re and Rd. in blood were simultaneously measured by UPLC/MS/MS. Sample preparation was carried out by the protein precipitation technique with an internal standard saikosaponin A standard. The separation of six components was achieved by using an ACQUITY UPLC ®BEH C18 column (1.7µm 2.1×100mm) by gradient elution using water (containing 0.2% formic acid) and acetonitrile (containing 0.2% formic acid) as the mobile phase at a flow rate of 0.2mL/min. The pharmacokinetic parameters were determined using non-compartmental analysis. The transport of notoginsenoside R1, ginsenoside Rg1, Rb1, Re and Rd. in MDCK -MDR1 cell monolayer was also used to verify the conclusion of pharmacokinetic drug-drug interaction and study the mechanism of drug interaction. RESULTS: The concentrations of the five components increased in a certain extent when the two drugs administered together in rats. The values of apparent permeability coefficients were significantly increased when the two drugs were used together. Aspirin and salicylic acid could destroy the tight junction protein and open the intercellular space to increase the absorption of Panax notoginseng saponins. CONCLUSION: Pharmacokinetic drug-drug interaction in vivo existed between Panax notoginseng saponins and aspirin. The drug-drug interaction mainly occurred in the process of absorption.

A Practical Quality Control Method for Saponins Without UV Absorption by UPLC-QDA.

Saponins are a class of important active ingredients. Analysis of saponin-containing herbal medicines is a major challenge for the quality control of medicinal herbs in companies. Taking the medicine Astragali radix (AR) as an example, it has been shown that the existing evaporative light scattering detection (ELSD) methods of astragaloside IV (AG IV) has the disadvantages of time-consuming sample preparation and low sensitivity. The universality of ELSD results in an inapplicable fingerprint with huge signals from primary compounds and smaller signals from saponins. The purpose of this study was to provide a practical and comprehensive method for the quality control of the astragalosides in AR. A simple sample preparation method with sonication extraction and ammonia hydrolyzation was established, which shortens the preparation time from around 2 days to less than 2 h. A UPLC-QDA method with the SIM mode was established for the quantification of AG IV in AR. Methanol extract was subjected to UPLC-QDA for fingerprinting analysis, and the common peaks were assigned simultaneously with the QDA. The results showed that with the newly established method, the preparation time for a set of samples was less than 90 min. The fingerprints can simultaneously detect both saponins and flavonoids in AR. This simple, rapid, and comprehensive UPLC-QDA method is suitable for quality assessment of RA and its products in companies, and also provides references for the quality control of other saponin ingredients without UV absorption.

Pharmacokinetics of Panax notoginseng Saponins in Adhesive and Normal Preparation of Fufang Danshen.

BACKGROUND AND OBJECTIVE: Fufang Danshen formula, a famous Chinese patent medicine containing Salvia miltiorrhiza, Panax notoginseng and borneol, has been widely used in the treatment of coronary heart disease. The application is restricted by low bioavailability partly due to Panax notoginseng saponins (PNS) instability and low in vivo absorption. Thus, adhesive pellets were developed to improve bioavailability. The objectives of the present study were to evaluate the adhesive preparation by describing PNS's plasma pharmacokinetics in vivo and compare adhesive micro pills with normal preparation. METHOD: LC-MS/MS method was established to analyze five ingredients, notoginsenoside R1 (R1), ginsenoside Rg1 (Rg1), ginsenoside Rb1 (Rb1), ginsenoside Re (Re), and ginsenoside Rd (Rd), in rats' plasma to describe the pharmacokinetic parameters of PNS. RESULTS: The pharmacokinetic parameters were significantly different after oral administration three formulations. The results show adhesive formulations are superior to Fufang Danshen tablet (FDT); there are differences between the two adhesive, but not obvious. CONCLUSIONS: It was found that the modification with adhesive materials improved PNS bioavailability in Fufang Danshen formula. These findings provide a way for further in vivo evaluation of different formulations.