Abstract

   In this study, a fingerprint-activity relationship between chemical
   fingerprints and hepatoprotective activity was established to evaluate
   the quality of salt-treated Apocyni Veneti Folium (AVF). Characteristic
   fingerprints of AVF samples exposed to different concentrations of salt
   were generated by ultrafast liquid chromatography tandem triple
   time-of-flight mass/mass spectrometry (UFLC-Triple TOF-MS/MS), and a
   similarity analysis was performed based on common characteristic peaks
   by hierarchical clustering analysis (HCA). Then, the hepatoprotective
   activity of AVF against CCl[4]-induced acute liver damage in mice was
   investigated by assessing biochemical markers and histopathology, which
   showed that a high dose of AVF exposed to low levels of salt stress
   produced a marked amelioration of hepatic damage compared with the
   other salt-treated AVF. Finally, fingerprint-activity relationship
   modeling, which was capable of discovering the bioactive markers used
   in the quality evaluation, was investigated by the chemical
   fingerprints and the hepatoprotective activities utilizing multivariate
   statistical analysis, gray correlation analysis (GCA) and bivariate
   correlation analysis (BCA). The results showed that the accumulation of
   polyphenols, such as flavonoids and phenolic acids, in AVF subjected to
   low levels of salt stress could result in the effective scavenging of
   free radicals. Therefore, the present study may provide a powerful
   strategy to holistically evaluate the quality of salt-treated AVF in
   combination with chemical fingerprint and bioactivity evaluation.

   Subject terms: Salt, Secondary metabolism, Mass spectrometry

Introduction

   Apocynum venetum L., as one of the known medicinal halophytes, has
   attracted much attention in terms of its antioxidant properties
   associated with human well-being. This plant grows widely in China,
   especially in a variety of saline habitats, and therefore, it is
   characterized by a high physiological plasticity for salt tolerance.
   Salinity has adverse effects on all aspects of plant health by
   activating salinity-induced molecular networks associated with salt
   stress perception, ion and osmotic homeostasis, and regulation of
   stress-related genes, proteins and metabolic pathways^[42]1,[43]2.
   However, halophytes develop a robust and sophisticated protective
   system and accumulate secondary metabolites under appropriate levels of
   salt stress, which may be responsible for maintaining homeostasis and
   protecting against excessive reactive oxygen species-induced oxidative
   stress^[44]3.

   Apocyni Veneti Folium (AVF) contains abundant bioactive compounds,
   including the main and prominent antioxidative constituents of phenolic
   acid and flavonoids^[45]4. Modern pharmacological studies have
   demonstrated that AVF has anti-hypertension, anti-depressant,
   hepatoprotection, anti-anxiety, antioxidation and diuretic
   functions^[46]5,[47]6. In the Chinese Pharmacopoeia (2015 edition),
   hyperoside, a stable active ingredient of AVF occurring at a high
   concentration, was selected as the marker for the quality control
   analysis of AVF^[48]7. In fact, most published reports quantify one or
   a limited number of components to achieve quality
   evaluation^[49]8,[50]9. However, considering the fluctuations in
   chemical compositions and contents on the basis of many factors, such
   as abiotic stress, cultivation region and harvest time, it is difficult
   to achieve consistent quality in medicinal herbs^[51]3. Furthermore, it
   is generally recognized that Chinese medicines exert therapeutic
   efficacies holistically through a ‘multicomponent, multitargeted, and
   multipathway’ mode^[52]10. In addition, as mentioned above, AVF is
   composed of many bioactive components, and its therapeutic effects are
   not confined to the individual or simple effects of a single bioactive
   component. Therefore, it is necessary to establish a truly meaningful
   protocol to effectively and systematically achieve quality control of
   this plant.

   The fingerprint test proved to be a useful tool in assessing the
   chemical consistency of traditional Chinese medicine (TCM) and has been
   widely accepted by the World Health Organization (WHO), China Food and
   Drug Administration (CFDA), the United States Food and Drug
   Administration (FDA), and European Medicines Agency (EMEA). Several
   chromatographic techniques, including liquid chromatography
   (LC)^[53]11, gas chromatography (GC)^[54]12, thin-layer chromatography
   (TLC)^[55]13, and high-performance capillary electrophoresis
   (HPCE)^[56]14, have been used to construct chemical fingerprints of
   AVF, which has allowed researchers to visualize and identify as many
   components of the plant as possible. Among these techniques, a
   high-performance liquid chromatography (HPLC) system is typically
   employed to establish the chemical fingerprints and quality assessment
   of AVF because of its ease of operation, high selectivity, and
   accuracy^[57]9,[58]11,[59]15. However, limits of this HPLC system
   include the identification of unknown components. Accurate masses and
   molecular formulae of untargeted compounds acquired from LC coupled
   with mass spectrometry/mass spectrometry (LC-MS/MS) have been used to
   predict and find such components^[60]5,[61]16,[62]17. In particular,
   triple time-of-flight mass spectrometry/mass spectrometry (Triple
   TOF-MS/MS) is considered to be the first accurate, high-throughput and
   high-resolution system of its kind and operates by means of
   information-dependent acquisition^[63]18,[64]19. Therefore, the
   ultrafast liquid chromatography (UFLC)-Triple TOF-MS/MS system could be
   introduced to obtain AVF chemical fingerprints. Although similarity
   analysis of chromatographic fingerprints can directly reflect whether
   an analyzed sample is chemically similar to others, it alone cannot
   assess quality consistency. Many studies have proven that samples with
   high similarity values do not always exhibit the expected equivalent
   efficacies^[65]20,[66]21. Hence, fingerprint-activity relationship
   modeling correlating major components to bioactivity is meaningful to
   achieve quality consistency and discover bioactive
   markers^[67]22–[68]25.

   Liver injury induced by carbon tetrachloride (CCl[4]) is one of the
   most widely used experimental models^[69]26. Increased serum alanine
   aminotransferase (ALT), aspartate aminotransferase (AST), and hepatic
   malondialdehyde (MDA) contents along with decreased superoxide
   dismutase (SOD), catalase (CAT) and peroxidase (POD) activities
   demonstrate hepatotoxicity induced by CCl[4] in mice^[70]27. Many
   published studies have indicated that natural substances, such as
   phenolic acids and flavonoids from plants, exhibit strong antioxidative
   activity that could act against CCl[4]-induced liver
   damage^[71]28,[72]29. References have shown that AVF has protective