A Metabolomic Strategy to Screen the Prototype Components and Metabolites of Shuang-Huang-Lian Injection in Human Serum by Ultra Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry

Shuang-huang-lian injection (SHLI) is a famous Chinese patent medicine, which has been wildly used in clinic to treat acute respiratory tract infection, pneumonia, influenza, and so forth. Despite the widespread clinical application, the prototype components and metabolites of SHLI have not been fully elucidated, especially in human body. To discover and screen the constituents or metabolites of Chinese medicine in biofluids tends to be more and more difficult due to the complexity of chemical compositions, metabolic reactions and matrix effects. In this work, a metabolomic strategy to comprehensively elucidate the prototype components and metabolites of SHLI in human serum conducted by UPLC-Q-TOF/MS was developed. Orthogonal partial least squared discriminant analysis (OPLS-DA) was applied to distinguish the exogenous, namely, drug-induced constituents, from endogenous in human serum. In the S-plot, 35 drug-induced constituents were found, including 23 prototype compounds and 12 metabolites which indicated that SHLI in human body mainly caused phase II metabolite reactions. It was concluded that the metabolomic strategy for identification of herbal constituents and metabolites in biological samples was successfully developed. This identification and structural elucidation of the chemical compounds provided essential data for further pharmacological and pharmacokinetics study of SHLI

A Metabolomic Strategy to Screen the Prototype Components and Metabolites of Shuang-Huang-Lian Injection in Human Serum by Ultra Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry

Shuang-huang-lian injection (SHLI) is a famous Chinese patent medicine, which has been wildly used in clinic to treat acute respiratory tract infection, pneumonia, influenza, and so forth. Despite the widespread clinical application, the prototype components and metabolites of SHLI have not been fully elucidated, especially in human body. To discover and screen the constituents or metabolites of Chinese medicine in biofluids tends to be more and more difficult due to the complexity of chemical compositions, metabolic reactions and matrix effects. In this work, a metabolomic strategy to comprehensively elucidate the prototype components and metabolites of SHLI in human serum conducted by UPLC-Q-TOF/MS was developed. Orthogonal partial least squared discriminant analysis (OPLS-DA) was applied to distinguish the exogenous, namely, druginduced constituents, from endogenous in human serum. In the S-plot, 35 drug-induced constituents were found, including 23 prototype compounds and 12 metabolites which indicated that SHLI in human body mainly caused phase II metabolite reactions. It was concluded that the metabolomic strategy for identification of herbal constituents and metabolites in biological samples was successfully developed. This identification and structural elucidation of the chemical compounds provided essential data for further pharmacological and pharmacokinetics study of SHLI

Plasma Metabolomic Profiling to Reveal Antipyretic Mechanism of <i>Shuang-Huang-Lian</i> Injection on Yeast-Induced Pyrexia Rats

<div><p><i>Shuang-huang-lian</i> injection (<i>SHLI</i>) is a famous Chinese patent medicine, which has been wildly used in clinic for the treatment of acute respiratory tract infection, pneumonia, influenza, etc. The existing randomized controlled trial (RCT) studies suggested that <i>SHLI</i> could afford a certain anti-febrile action. However, seldom does research concern the pharmacological mechanisms of <i>SHLI</i>. In the current study, we explored plasma metabolomic profiling technique and selected potential metabolic markers to reveal the antipyretic mechanism of <i>SHLI</i> on yeast-induced pyrexia rat model using UPLC-Q-TOF/MS coupled with multivariate statistical analysis and pattern recognition techniques. We discovered a significant perturbance of metabolic profile in the plasma of fever rats and obvious reversion in <i>SHLI</i>-administered rats. Eight potential biomarkers, i.e. 1) 3-hydeoxybutyric acid, 2) leucine, 3) 16∶0 LPC, 4) allocholic acid, 5) vitamin B₂, 6) Cys-Lys-His, 7) 18∶2 LPC, and 8) 3-hydroxychola-7, 22-dien-24-oic acid, were screened out by OPLS-DA approach. Five potential perturbed metabolic pathways, i.e. 1) valine, leucine, and isoleucine biosynthesis, 2) glycerophospholipid metabolism, 3) ketone bodies synthesis and degradation, 4) bile acid biosynthesis, and 5) riboflavin metabolism, were revealed to relate to the antipyretic mechanisms of <i>SHLI</i>. Overall, we investigated antipyretic mechanisms of <i>SHLI</i> at metabolomic level for the first time, and the obtained results highlights the necessity of adopting metabolomics as a reliable tool for understanding the holism and synergism of Chinese patent drug.</p></div

The results of S-plots of OPLS-DA models.

<p>(A) At positive ion mode. (B) At negative ion mode. Note: NC (▴), M (•) and SHLI (▪).</p

The changing trend of rectal temperatures in NC, M and <i>SHLI</i> treatment group.

<p>Data was expressed as mean ± SD. ^###<i>P</i><0.001 vs., NC group; **<i>P</i><0.01 vs., M group.</p

Metabolites selected by OPLS-DA with VIP >1 and significant test <i>P</i><0.05 between the pyretic model group and the normal control group.

<p>Note: ^aMetabolites were identified based on database information in METLIN, Lipid MAPs or HMDB; ↑showed up-regulated metabolites and ↓showed down-regulated metabolites; ^#<i>p<0.05</i>, ^{# #}<i>p<0.01</i>, ^{# # #}<i>p<0.001</i> Model vs. normal control; *<i>p<0.05</i>, **<i>p<0.01</i>, ***<i>p<0.001</i> SHLI vs. Model.</p

The results of multiple pattern recognition of plasma metabolites (PCA scores).

<p>(A) At positive ion mode. (B) At negative ion mode. Note: NC (▴), M (•) and SHLI (▪).</p

The results of relative integral levels of metabolites among NC, M and <i>SHLI</i> treatment groups.

<p>(A) At positive ion mode. (B) At negative ion mode. ^a<i>P</i><0.05 or <i>P</i><0.01 among NC, M, and <i>SHLI</i> treatment group.</p

Typical base peak intensity (BPI) chromatograms ofplasma samples from each groups.

<p>(A) NC at positive ion mode. (B) M at positive ion mode (C) <i>SHLI</i> treatment at positive mode (Blue arrows show drug induced components).</p