[Acupuncture of revised acupoint combination around the skull base for post-stroke mild cognitive impairment: a randomized controlled trial].

OBJECTIVE: To observe the clinical efficacy of acupuncture of revised acupoint combination around the skull base in treating post-stroke mild cognitive impairment (PSMCI), and preliminary explore its action mechanism. METHODS: A total of 76 PSMCI patients were randomly divided into an observation group (38 cases, 4 cases dropped off) and a control group (38 cases, 3 cases dropped off, 1 case was removed). In the observation group, acupuncture of revised acupoint combination around the skull base (bilateral Fengchi [GB 20], Wangu [GB 12], Tianzhu [BL 10] and Yamen [GV 15], Baihui [GV 20]) was used for treatment. In the control group, 8 non-meridian and non-acupoint points at the distal end were selected for shallow puncture treatment. Retaining the needles of 30 min, once every other day,3 times a week for 4 weeks in both groups. The scores of Montreal cognitive assessment (MoCA), mini-mental state examination (MMSE), Barthel index (BI) and serum levels of cystatin C (Cys-C) and homocysteine (Hcy) were compared in the two groups before and after treatment, and the clinical efficacy was evaluated. RESULTS: After treatment, the scores of MoCA were increased compared with those before treatment in the two groups (P<0.05), and the score in the observation group was higher than that in the control group (P<0.05). The scores of MMSE and BI were increased compared with those before treatment in the observation group (P<0.05), and the score of MMSE in the observation group was higher than that in the control group (P<0.05). After treatment, the serum levels of Cys-C and Hcy were decreased compared with those before treatment in the observation group (P<0.05), and lower than those in the control group (P<0.05). After treatment, the serum level of Cys-C was increased compared with that before treatment in the control group (P<0.05). The total effective rate of the observation group was 88.2% (30/34), which was higher than 32.4% (11/34) of the control group (P<0.05). CONCLUSION: Acupuncture of revised acupoint combination around the skull base can improve cognitive function and daily living ability of PSMCI patients, which may be related to the down regulation of serum levels of Cys-C and Hcy.

An integrated strategy by absorbed component characterization, pharmacokinetics, and activity evaluation for identification of potential nephroprotective substances in Zhu-Ling decoction.

An efficient strategy for the identification of potential nephroprotective substances in Zhu-Ling decoction has been established with the integration of absorbed components characterization, pharmacokinetics, and activity evaluation. A qualitative method was developed to characterize the chemical constituents absorbed components in vivo of Zhu-Ling decoction by using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. A quantitative method was established and validated for the simultaneous determination of eight compounds in rat plasma by using ultra-performance liquid chromatography-triple quadruple tandem mass spectrometry. Finally, the nephroprotective activities of absorbed components with high exposure were assessed by cell survival rate, superoxide dismutase, and malondialdehyde activities in hydrogen peroxide-induced Vero cells. As a result, 111 compounds in Zhu-Ling decoction and 36 absorbed components were identified in rat plasma and urine, and poricoic acid A, poricoic acid B, alisol A, 16-oxo-alisol A, and dehydro-tumulosic acid had high exposure levels in rat plasma. Finally, poricoic acid B, poricoic acid A, 16-oxo-alisol A, and dehydro-tumulosic acid showed remarkable nephroprotective activity against Vero cells damage induced by hydrogen peroxide. Besides, superoxide dismutase and malondialdehyde activities were obviously regulated in hydrogen peroxide-induced Vero cells by treatment with the four compounds mentioned above. Therefore, these four compounds were considered to be effective substances of Zhu-Ling decoction due to their relatively high exposure in vivo and biological activity. This study provided a chemical basis for the action mechanism of Zhu-Ling decoction in the treatment of chronic kidney diseases.

Simultaneous determination of multiple constituents of Qi-Lin pill by UPLC-MS/MS: Applications to pharmacokinetics and testicular tissue distribution in rats.

Qi-Lin pill (QLP) is an effective traditional Chinese medicine prescription (TCMP) that has been used for the treatment of the oligoasthenozoospermia in China. Recently, some articles described the pharmacological effects of QLP and multiple ingredients in QLP contribute to its effects. However, the pharmacokinetic and target tissue distribution data of QLP are still unknown. In the present study, according to the Bioanalytical Method Validation Guidance of FDA, a sensitive and selective UPLC-MS/MS method was developed and validated for simultaneous determination of multiple constituents in rat plasma and testicular tissue, including morusimic acid A, codonopyrridium B, magnoflorine, emodin, 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (THSG), ecliptasaponin A, paeoniflorin, albiflorin, gallic acid, danshensu, salvianolic acid A, catechin, isosinensetin, nobiletin, formononetin, calycosin, icariside II, icariin and epimedin C. For 19 analytes, the LLOQs reached 0.01-4 ng/mL. And all calibration curves showed favorable linearity (r ≥ 0.9903) in linear ranges. The intra-day and inter-day precision (relative standard deviation) for all analytes was less than 14.92 %, and the accuracies (as relative error) were in the range of - 6.44 % to 6.22 %. Extraction recoveries and matrix effects of analytes and IS were acceptable. All analytes were stable during the assay and storage in plasma samples. The method was successfully applied for the pharmacokinetics and testis distribution of multiple chemical constituents in QLP after a single oral dose. As a result, high exposure of danshensu, gallic acid, paeoniflorin and albiflorin were observed in rat plasma and testicular tissue. Among the flavonoids, isosinensetin and nobiletin had high exposure in testicular tissue. Moreover, alleviation of progesterone reduction was evaluated in H2O2-induced R2C leydig cells, and danshensu, gallic acid, paeoniflorin, albiflorin and nobiletin showed potent activity. Therefore, these five components were considered to be the effective components of QLP due to their relatively high exposure in vivo and biological activity. This finding also provided relevant information on action mechanism of QLP in the treatment of oligoasthenozoospermia.

Pharmacokinetics, hepatic disposition, and heart tissue distribution of 14 compounds in rat after oral administration of Qi-Li-Qiang-Xin capsule via ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry.

In the present study, a specific and sensitive approach using ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry was developed and validated for the quantitative analysis of 14 constituents in rat plasma, liver, and heart. The method was fully validated and successfully applied to pharmacokinetic, hepatic disposition, and heart tissue distribution studies of 14 compounds after the oral administration of Qi-Li-Qiang-Xin capsule. Ginsenoside Rb1, alisol A, astragaloside IV, and periplocymarin were found to be highly exposed in rat plasma, while toxic components such as hypaconitine, mesaconitine, and periplocin had low circulation levels in vivo. Moreover, sinapine thiocyanate, neoline, formononetin, calycosin, and alisol A exhibited significant liver first-pass effects. Notably, high levels of alisol A, periplocymarin, benzoylmesaconine, and benzoylhypaconine were observed in the heart. Based on high exposure and appropriate pharmacokinetic features in the systemic plasma and heart, astragaloside IV, ginsenoside Rb1, periplocymarin, benzoylmesaconine, benzoylhypaconine, and alisol A can be considered as the main potentially effective components. Ultimately, the results provide relevant information for discovery of effective substances, as well as further anti-heart failure action mechanism investigations of Qi-Li-Qiang-Xin capsule.

Discovery of potential Q-marker of traditional Chinese medicine based on plant metabolomics and network pharmacology: Periplocae Cortex as an example.

BACKGROUND: Quality control exerted great importance on the clinical application of drugs for ensuring effectiveness and safety. Due to chemical complexity, diversity among different producing areas and harvest seasons, as well as unintentionally mixed with non-medicinal parts, the current quality standards of traditional Chinese medicine (TCM) still faced challenges in evaluating the overall chemical consistency. PURPOSE: We aimed to develop a new strategy to discover potential quality marker (Q-marker) of TCM by integrating plant metabolomics and network pharmacology, using Periplocae Cortex (GP, the dried root bark of Periploca sepium Bge.) as an example. METHODS: First, plant metabolomics analysis was performed by UPLC/Q-TOF MS in 89 batches of samples to discover chemical markers to distinguish medicinal parts (GP) and non-medicinal parts (the dried stem bark of Periploca sepium Bge. (JP)), harvest seasons and producing region of Periplocae Cortex. Second, network pharmacology was applied to explore the initial linkages among chemical constituents, targets and diseases. Last, potential Q-marker were selected by integrating analysis of plant metabolomics and network pharmacology, and the quantification method of Q-marker was developed by using UPLC-TQ-MS. RESULTS: The chemical profiling of GP and JP was investigated. Fifteen distinguishing features were designated as core chemical markers to distinguish GP and JP. Besides, the content of 4-methoxybenzaldehyde-2-O-ß-d-xylopyranosyl-(1→6)-ß-d-glucopyranoside could be used to identify Periplocae Cortex harvested in spring-autumn or summer. Meanwhile, a total of 15 components targeted rheumatoid arthritis were screened out based on network pharmacology. Taking absorbed constituents into consideration, 23 constituents were selected as potential Q-marker. A simultaneous quantification method (together with 11 semi-quantitative analysis) was developed and applied to the analysis of 20 batches of commercial Periplocae Cortex on the market. The PLS-DA model was successfully developed to distinguish GP and JP samples. In addition, the artificially mixed GP sample, which contained no less than 10% of the adulterant (JP), could also be correctly identified. CONCLUSION: Our results indicated that 9 ingredients could be considered as Q-marker of Periplocae Cortex. This study has also demonstrated that the plant metabolomics and network pharmacology could be used as an effective approach for discovering Q-marker of TCM to fulfill the evaluation of overall chemical consistency among samples from different producing areas, harvest seasons, and even those commercial crude drugs, which might be mixed with a small amount of non-medicinal parts.

Dissection of the potential anti-influenza materials and mechanism of Lonicerae japonicae flos based on in vivo substances profiling and network pharmacology.

Lonicerae japonicae flos.(LJF) was widely used as a drug to treat upper respiratory tract infection or a tea to clear heat in Asian countries for thousands of years. Despite of its curative effects confirmed by modern pharmacological methods, its functional materials and mechanism against influenza were still unclear and needed further investigation. In this study, an integrated strategy based on in vivo substances profiling and network pharmacology was proposed and applied to screen out the potential anti-influenza substances and mechanism of LJF. An UHPLC/Q-TOF MS method was utilized to profile the chemical components in LJF and their metabolites in rats. The targets of absorbed prototypes were predicted by Swiss Target Prediction, and they were further analyzed by String and Kyoto Encyclopedia of Genes and Genomes (KEGG). As a result, a total of 126 chemical components mainly featuring three chemical structure types were characterized, including 70 iridoid glycosides, 17 caffeoylquinic acids, 24 flavonoids, and 15 other types compounds. Among them, ten N-contained iridoid glycosides were characterized as potential novel compounds. Moreover, 141 xenobiotics (74 prototypes and 67 metabolites) were clearly screened out in rat plasma and urine after ingestion of LJF. Phase II reactions (sulfation, glucuronidation, methylation) and phase I reactions (dehydroxylation, hydrogenation, hydrolysis, N-heterocyclization) were the main metabolic reactions of LJF in rats. Further, a total of 338 targets were predicted and TNF, PTGS2 and EGFR were the three main targets involved in the pathology of influenza. In addition to normal NF-κB pathway, T cell signal pathway and mTOR signal pathway were the other patterns for LJF to achieve its anti-flu effects. Our work provided the meaningful data for further pharmacological validation of LJF against influenza, and a new strategy was also proposed for minimizing the process to reveal the mechanism and functional basis of TCMs.

Discovery of anti-flu substances and mechanism of Shuang-Huang-Lian water extract based on serum pharmaco-chemistry and network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuang-Huang-Lian preparation has captured wide attention since its clinical applications for the successful treatment of upper respiratory tract infection. However, its functional basis under actual therapeutic dose in vivo was still unrevealed. AIM OF THE STUDY: This study aimed to reveal the anti-flu substances and mechanism of Shuang-Huang-Lian water extract (SHL) on H1N1 infected mouse model by a strategy based on serum pharmaco-chemistry under actual therapeutic dose and network pharmacology. MATERIALS AND METHODS: H1N1 infected mouse model was employed for evaluation of the anti-flu effects of SHL. A simultaneous quantification method was developed by UPLC-TQ-XS MS coupled switch-ions mode and applied to characterize the pharmacokinetics of the multiple components of SHL under actual therapeutic dose. The potential active ingredients were screened out based on their pharmacokinetic parameters. And then, a compound mixture of these active candidates was re-evaluated for the anti-flu activity on H1N1 infected mouse model. Furthermore, the anti-flu mechanism of SHL was also predicted by network pharmacology coupled with the experimental result. RESULTS: SHL significantly increased the survival rate and prolonged survival days on H1N1 infected mice at a dosage of 20 g crude drug/kg/day by reversing the increased lung index, down-regulating the inflammatory cytokines (TNF-α, IL-1ß, IL-6) and inhibiting the release of IFN-ß in bronchoalveolar lavage fluids (BALF). Concomitantly, the pharmacokinetic parameters of fourteen quantified and twenty-one semi-quantified constituents of SHL were characterized. And then, five compounds (baicalin, sweroside, chlorogenic acid, forsythoside A and phillyrin), which displayed satisfactory pharmacokinetic features, were considered as potential active ingredients. Thus, a mixture of these five ingredients was administered to H1N1-infected mice at a dose of 4.24 mg/kg/day. As a result, the therapeutical effects of the mixture were similar to SHL in terms of survival rate, lung index and the release of cytokines (TNF-α, IL-1ß and IL-6) in BALF. Moreover, network pharmacology analysis indicated that the TNF-signal pathways might play a role in the anti-flu mechanism of SHL. CONCLUSIONS: A mixture of five compounds (baicalin, sweroside, chlorogenic acid, forsythoside A and phillyrin) were the anti-flu substances of SHL. The strategy based on serum pharmaco-chemistry under actual therapeutic dose provided a new sight on exploring in vivo effective substances of TCM.

Systematically characterization of in vivo substances of Ziziphi Spinosae Semen in rats by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry analysis.

Ziziphi Spinosae Semen (ZSS), the seeds of Ziziphus jujuba var. spinosa, is widely used in China or other Asian countries for the treatment of insomnia and palpitation. In our previous work, chemical constituents in ZSS were profiled by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC/Q-TOF MS). Notably, characterization of substances in vivo was of great importance to reveal the therapy basis or mechanism in further work. Till now, there were few reports about in vivo substances' investigation of ZSS. In the present study, an integrated strategy contained represented compounds and diagnostic ions extraction was applied to characterize metabolism feature of ZSS in rats based on UHPLC/Q-TOF MS method. First, the metabolic information of four compounds (spinosin, isovitexin, jujuboside B, betulinic acid) featuring three representative chemical structures (flavonoids, saponins, terpenes) in ZSS was conducted, and their metabolism features were summarized, especially for flavonoid C-glycosides. Second, the absorbed compounds and representative compounds-related metabolites were quickly screened out; during this time, the diagnostic ions were sorted out. Last, with the help of diagnostic ions and summarized metabolic reactions, other metabolites were characterized. As a result, a total of 151 xenobiotics (58 prototypes and 93 metabolites) were identified or tentatively characterized in rats after ingestion of ZSS. Among them, 16 substances were presented in plasma, 114 in urine, 51 in bile, and 120 in feces, respectively. Hydrogenation, hydrolysis, and glucuronidation were the major metabolic reactions of ZSS in rats. The present study provided meaningful data for further pharmacological mechanism research or pharmacokinetics evaluation of ZSS.

An integrated strategy for revealing the pharmacological changes based on metabolites profiling and network pharmacology: Arctiin as an example.

Traditional Chinese medicine was widely used in China since its definite effects and therapy. The components of TCM were absorbed into the circle system as the format of prototypes or metabolites, which contributed to the therapy or side effects. Declaring the functional changes in this process was of great importance to the clinical applications. In this work, an integrated strategy based on metabolites' profiling and network pharmacology was proposed for exploring the pharmacological changes of compounds in vivo. Arctiin, the main component in Fructus Arctii with various kinds of bioactivities, was used as an example. An ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry and metabolynx™software was applied to characterize the metabolites of arctiin in rats at a dosage of 100 mg/kg; network pharmacology was applied to characterize the functional changes. As a result, fifty-three metabolites (32 in plasma, 40 in urine, 19 in bile, 20 in feces, 1 in brain, 12 in liver and 4 in lungs) were screened out and characterized, and 3 of them were unambitiously identified by comparison with standard substances. Among them, 38 metabolites were reported for the first time. It was found the major metabolic pathways of arctiin in rats were demethylation, lactone-opening and phase II conjugations with sulfate and glucuronide.It also confirmed that M14, M15, M18, M23, M22, M43 and M45 were the major circulating forms of arctiin in rats following oral administration. In addition to the above metabolic reactions, phase I reactions of hydrolysis, demethylation, dehydroxylation were also observed, and dehydrogenation were first revealed metabolic patterns of arctiin in rats. Meanwhile, in addition to the main targets of arctiin (MTOR, EGFR and MAPK14), its metabolites targeted additional 392 targets with additional functions of anti-hepatitis B or viral carcinogenesis (SRC, CAPS3, PIK3CA, CDK4, ESR1, MMP9 and ERBB2). The above results provided very important information for understanding the metabolism and functional changes of arctiinin vivo, and supporting data for further pharmacological evaluation. Our work also provided a newsight for elucidation of functional changes of TCMs in vivo.

Characterization of lignans in Forsythiae Fructus and their metabolites in rats by ultra-performance liquid chromatography coupled time-of-flight mass spectrometry.

OBJECTIVES: This study was designed to profile the chemical information of Forsythiae Fructus (FF) and investigate the in-vivo FF-related xenobiotics, especially for lignans. METHODS: Rats were oral administrated of FF and pinoresinol-4-O-glucoside, respectively. Blood and urine samples were collected after ingestion, and xenobiotics was profiled by an UPLC/Qtof MS method. KEY FINDINGS: A total of 19 lignans were identified or tentatively characterized in FF, and 63 lignan-related xenobiotics were found in rat plasma and urine after ingestion of FF. It was found that lignans could be transformed into metabolites by furan ring opening, hydrogenation, demethylation, dehydration and phase II reactions (sulfation and glucuronidation). The whole metabolic behaviour of bisepoxylignan was revealed by evaluating the metabolism of pinoresinol-4-O-glucoside in vivo. It was found that the configuration of C-8/C-8' was retained after furan ring opening and metabolic reactions always occurred at position of C-3/C-4/C-5 or C-3'/C-4'/C-5'. Additionally, other types components in FF and in vivo were also characterized. CONCLUSIONS: This work revealed the in-vivo metabolism of FF, and reported the characteristic metabolic reactions of lignans for the first time. It was also provided the foundation for the further investigation on pharmacodynamic components of FF or TCMs containing FF.

Characterization of chemical components of Periplocae Cortex and their metabolites in rats using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Periplocae Cortex, named Xiang-Jia-Pi in China, has been widely used to treat autoimmune diseases, especially rheumatoid arthritis. However, the in vivo substances of Periplocae Cortex remain unknown yet. In this study, an ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used for profiling the chemical components and related metabolites of Periplocae Cortex. A total of 98 constituents were identified or tentatively characterized in Periplocae Cortex: 42 C21 steroidal glycosides, 10 cardiac glycosides, 23 organic acids, 4 aldehydes, 7 triterpenes, and 12 other types. Among them, 18 components were unambiguously identified by comparison with reference standards. In addition, 176 related xenobiotics (34 prototypes and 142 metabolites) were screened out and characterized in rats' biosamples (plasma, urine, bile, and feces) after the oral administration of Periplocae Cortex. Moreover, the metabolic fate of periplocoside S-4a, a C21 steroidal glycoside, was proposed for the first time. In summary, phase II reactions (methylation, glucuronidation, and sulfation), phase I reactions (hydrolysis reactions, oxygenation, and reduction), and their combinations were the predominant metabolic reactions of Periplocae Cortex in rat. It is the first report to reveal the in vivo substances and metabolism feature of Periplocae Cortex. This study also provided meaningful information for further pharmacodynamics study of Periplocae Cortex, as well as its quality control research.