Characterisation and critical processes identification for production of herbal preparations using 1H-NMR and chemometrics: A case study of Trichosanthis Pericarpium injection.

INTRODUCTION: Herbal preparations are extensively utilised for the treatment of diseases in Asian countries. However, the variations in origin, climate, and production processes can lead to inconsistencies in the quality of herbal preparations. Existing quality control methods only target a few components in the finished product but ignore the control in the pharmaceutical process. Therefore, this study intends to develop a comprehensive component analysis method for intermediates in the pharmaceutical process to reveal the change patterns of substances and deepen the process understanding. OBJECTIVE: This study aims to develop a rapid and comprehensive process characterisation and critical process identification method for herbal preparations. METHODS: Six batches of Trichosanthis Pericarpium injection (TPI) intermediates were collected from the production process. Proton nuclear magnetic resonance (1H-NMR) spectra were acquired for qualitative and quantitative analysis of the se intermediates. Subsequently, chemometrics were used to identify critical processes and potential chemical markers. RESULTS: A total of 39 components in intermediates were identified, and the transfer of 25 components during the production process was investigated. Column chromatography was determined as the critical process. Nine components were identified as chemical markers. CONCLUSION: The application of 1H-NMR facilitated a comprehensive reflection of the chemical composition information of process intermediates, enabling investigations into the transfer of multi-component substances and accurate identification of critical processes and chemical markers.

A multivariate curve resolution-alternating least squares (MCR-ALS) technology assisted 1 H-NMR methodology for multi-component quantitation of Trichosanthis Pericarpium injection.

INTRODUCTION: Trichosanthis Pericarpium injection (TPI) is a traditional Chinese medicine preparation obtained from Trichosanthis Pericarpium by extraction, purification and sterilisation. It contains amino acids, alkaloids, nucleotides and other components. Existing quantitative methods only analyse a few components in injections, so this study intends to develop a method for comprehensive analysis of TPI components. OBJECTIVE: To develop a method for quantification of components in TPI by multivariate curve resolution-alternating least squares (MCR-ALS) assisted proton nuclear magnetic resonance (1 H-NMR). METHODS: A 1 H-NMR method was developed for the quantification of components in TPI. For components with independent signals, 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt (TSP) was used as an internal standard to calculate the component contents. For components with overlapping signals, the method of MCR-ALS was used. RESULTS: A total of 36 components were identified in TPI, of which 33 were quantified. Methodological validation results showed that the developed 1 H-NMR method has good linearity, accuracy, precision, robustness and specificity. CONCLUSION: The use of 1 H-NMR provides a reliable and universal method for the TPI components identification and quantification. Also, it can be used as a powerful tool for analysing the contents in a complex mixture as a quality control measure.

Sodium Tanshinone IIA Sulfonate Improves Adverse Ventricular Remodeling Post-MI by Reducing Myocardial Necrosis, Modulating Inflammation, and Promoting Angiogenesis.

BACKGROUND AND OBJECTIVE: Myocardial infarction (MI) leads to pathological cardiac remodeling and heart failure. Sodium tanshinone IIA sulfonate (STS) shows to possess therapeutic potential. The present study aimed to explore the potential role of STS in ventricular remodeling post-MI. METHODS: Mice were randomly divided into sham, MI + normal saline (NS) and MI + STS (20.8 mg/kg/day intraperitoneally) groups. MI was established following left anterior descending artery ligation. Cardiac function was evaluated using echocardiography. Scar size and myocardial fibrosis-associated markers were detected using Masson's trichrome staining and western blot analysis (WB). Necrosis and inflammation were assessed using H&E staining, lactate dehydrogenase (LDH) detection, ELISA, immunohistochemical staining, and WB. Furthermore, angiogenesis markers and associated proteins were detected using immunohistochemical staining and WB. RESULTS: Mice treated with STS exhibited significant improvements in cardiac function, smaller scar size, and low expression levels of α-smooth muscle actin and collagen I and III at 28 days following surgery, compared with the NS-treated group. Moreover, treatment with STS reduced eosinophil necrosis, the infiltration of inflammatory cells, plasma levels of LDH, high mobility group protein B1, interleukin-1ß and tumor necrosis factor- α, and protein expression of these cytokines at 3 days. Macrophage infiltration was also decreased in the STS group in the early phase. Additionally, CD31+ vascular density, protein levels of hypoxia-inducible factor- 1α, and vascular endothelial growth factor were elevated in the STS-treated mice at 28 days. CONCLUSION: STS improved pathological remodeling post-MI, and the associated therapeutic effects may be a result of a decrease in myocardial necrosis, modulation of inflammation, and an increase in angiogenesis.

Novel TIPP (H-Tyr-Tic-Phe-Phe-OH) analogues displaying a wide range of efficacies at the δ opioid receptor. Discovery of two highly potent and selective δ opioid agonists.

Analogues of the δ opioid antagonist peptide TIPP (H-Tyr-Tic-Phe-Phe-OH; Tic=1,2,3,4-tetrahydroisoquinoline3-carboxylic acid) containing various 4'-[N-(alkyl or aralkyl)carboxamido]phenylalanine analogues in place of Tyr(1) were synthesized. The compounds showed subnanomolar or low nanomolar δ opioid receptor binding affinity and various efficacy at the δ receptor (antagonism, partial agonism, full agonism) in the [(35)S]GTPγS binding assay. Two analogues, [1-Ncp(1)]TIPP (1-Ncp=4'-[N-(2-(naphthalene-1-yl)ethyl)carboxamido]phenylalanine) and [2-Ncp(1)]TIPP (2-Ncp=4'-[N-(2-(naphthalene-2-yl)ethyl)carboxamido]phenylalanine), were identified as potent and selective δ opioid agonists.

Bifunctional µ/δ opioid peptides: variation of the type and length of the linker connecting the two components.

On the basis of evidence that opioid compounds with a mixed µ agonist/δ antagonist profile may produce an antinociceptive effect with low propensity to induce side effects, bifunctional opioid peptides containing the µ agonist H-Dmt-d-Arg-Phe-Lys-NH(2) ([Dmt(1) ]DALDA; Dmt = 2',6'-dimethyltyrosine) connected tail-to-tail via various α,ω-diaminoalkyl- or diaminocyclohexane linkers to the δ antagonists H-Tyr-TicΨ[CH(2) -NH]Cha-Phe-OH (TICP[Ψ]; Cha = cyclohexylalanine, Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), H-Dmt-Tic-OH or H-Bcp-Tic-OH (Bcp = 4'-[N-((4'-phenyl)phenethyl)carboxamido]phenylalanine) were synthesized and pharmacologically characterized in vitro. Bifunctional [Dmt(1) ]DALDA→NH-(CH(2) )(n) -NH←TICP[Ψ] compounds (n = -12) showed decreasing µ and δ receptor binding affinities with increasing linker length. As expected, several of the bifunctional peptides were µ agonist/δ antagonists with low nanomolar µ and δ receptor binding affinities. However, compounds with unexpected opioid activity profiles, including a µ partial agonist/δ partial agonist, µ antagonist/δ antagonists and µ agonist/δ agonists, were also identified. These results indicate that the binding affinities and intrinsic efficacies of these bifunctional compounds at both receptors depend on the length and type of the linker connecting the µ and δ components. An important recommendation emerging from this study is that the in vitro activity profiles of bifunctional compounds containing an agonist and an antagonist component connected via a linker need to be determined prior to their pharmacological evaluation in vivo.

Development of peptidic dopamine transporter inhibitors via aromatic modification-mediated conformational restriction.

The dopamine transporter plays an important role in the molecular mechanism of cocaine dependence. It is suggested that inhibitors of the dopamine transporter would have strong therapeutic potential. Here we report that aromatic modification can constrain a linear peptide into the beta-turn conformation, which is preferred by the dopamine transporter. On the basis of this finding, a novel selective and competitive peptidic inhibitor of the dopamine transporter was developed. The peptide binds to the dopamine- and cocaine-binding site of the dopamine transporter and has behavioral effects different from those of cocaine in mice.

Expression of monomeric insulin precursor in Pichia pastoris and its conversion into monomeric B27 Lys destripeptide insulin by tryptic hydrolysis.

Monomeric B27 Lys destripeptide insulin (B27 Lys DTrI) was designed and produced from its precursor expressed in Pichia pastoris through tryptic hydrolysis instead of the less efficient tryptic transpeptidation. The monomeric B27 Lys DTrI precursor (MIP) was purified from a cultured medium of P. pastoris by a combination of hydrophobic, size-exclusion, and ion-exchange chromatography. The purified MIP was converted, by tryptic hydrolysis, to B27 Lys DTrI, which was then purified by ion-exchange chromatography to homogeneity as assessed by native gel electrophoresis, HPLC, amino acid composition, and electrospray mass-spectrometric analysis. B27 Lys DTrI exhibited superior monomeric properties in size-exclusion chromatography. The yield of MIP was 200 mg per liter of culture, and the overall yield of purified B27 Lys DTrI from the crude MIP was 70%. The in vivo biological activity of B27 Lys DTrI as determined by the mouse convulsion assay was 21 U/mg, identical to that obtained by semisynthesis.

Monomeric B27 Lys destripeptide insulin: semisynthesis, characterization and biological activity.

In this paper, we report the semisynthesis of B27 Lys destripeptide insulin (B27 Lys DTrI), i.e. destetrapeptide insulin with an additional Lys residue at the C-terminus of B-chain. B27 Lys DTrI is also monomeric as shown by gel filtration. Its in vivo biological activity is 80% in comparison with that of native insulin. The addition of a Lys residue at the C-terminus of B-chain makes it possible to obtain monomeric B27 Lys DTrI from a precursor expressed in Saccharomyces cerevesiae by tryptic hydrolysis instead of the less efficient tryptic transpeptidation.