Abstract

   Nonsteroidal anti-inflammatory drugs (NSAIDs), such as cyclooxygenase
   (Cox)-1/2 inhibitor, have emerged as potent antipyretics and
   analgesics. However, few herbs with Cox-1/2 inhibitory activity are
   commonly used for heat-clearing in China. Although these are known to
   have antipyretic activity, there is a lack of molecular data supporting
   their activity. Using the traditional Chinese medicine herb honeysuckle
   (Hon) as an example, we explored key antipyretic active compounds and
   their mechanisms of action by assessing their metabolites and
   metabolomics. Mitogen-activated protein kinase (MAPK) 3 and protein
   kinase B (AKT) 1 were suggested as key targets regulated primarily by
   chlorogenic acid (CA) and swertiamarin (SWE). CA and SWE
   synergistically inhibited the production of interleukin (IL)-1 and
   IL-6, alleviated generation of prostaglandin E2, and played an
   antipyretic role equivalent to honeysuckle extract at the same dose
   contents within 3 h. Collectively, these findings indicated that
   lipopolysaccharide-induced fever can be countered by CA with SWE
   synergistically, allowing the substitution of a crude extract of
   complex composition with active compounds. Our findings demonstrated
   that, unlike the traditional NSAIDs, the Hon extract showed a remote
   and indirect mechanism for alleviating fever that depended on the
   phosphatidylinositol-3-kinase–AKT and MAPK pathways by regulating the
   principal mediator of inflammation.

   Keywords: metabolites, metabolomics, chlorogenic acid, swertiamarin,
   antipyretic, anti-inflammatory

1. Introduction

   Starting with aspirin, nonsteroidal anti-inflammatory drugs (NSAIDs),
   which target cyclooxygenase (COX)-1/2, have emerged as potent
   antipyretics and analgesics [[34]1]. However, almost no heat-clearing
   herbal medicines containing COX-1/2 inhibitors based on traditional
   Chinese medicine are being used for alleviating fever. One of the few
   examples of plants that are being used is honeysuckle (Hon), which
   refers to the flower buds of Lonicera japonica Thunb., possessing as it
   does potential anti-pyretic and anti-inflammatory activities, and which
   is widely used clinically after extraction [[35]2]. So far, more than
   140 phytoconstituents have been identified from honeysuckle extracts.
   In addition to essential oils, an abundance of flavones, organic acids,
   iridoids, and triterpenoid saponins have also been found [[36]3].
   However, the key component responsible for its antipyretic activity and
   its corresponding target remain unknown. Therefore, it is important to
   investigate the complex mechanism behind antipyretic activity exerted
   by these active compounds, as this will provide the knowledge required
   to precisely tailor therapies for various conditions, such as
   exopathogenic diseases, sores, carbuncles, and some infectious
   diseases.

   Natural products (NPs) remain the most prolific source of inspiration
   for the development of drugs [[37]4]. The novel modes of action
   exhibited by NPs have always made them a subject of great interest in
   medicine [[38]5]. With the exploration of NPs has become more common,
   increased access to biological and chemical data, and the development
   of data analysis algorithms, and integration of computational methods
   in NP drug discovery pipelines may be expected to increase
   significantly [[39]6]. Data pre-processing using convolutional neural
   networks reportedly performed better in peak alignment and
   identification from liquid chromatography–mass spectrometry (LC–MS),
   compound identification and quantification, and integration and
   interpretation of multi-omics data [[40]7]. Considering the complex and
   elusive nature of botanical products and their metabolism in the human
   body, metabolomic data generated for herbal drugs are large compared to
   those for single compounds. Combining information from different
   sources, that is, chemical, biochemical, biological, and in silico,
   with advanced computer algorithms and effectively handling big data can
   open new possibilities for NP research [[41]8]. Unfortunately, the
   integration of multi-omics data—those for the structures/components and
   for their targets—remains a challenge.

   Here, we used honeysuckle as a model herb and its extract to screen the
   key bioactive ingredients and potential protein targets in order to
   explore the antipyretic mechanism based on metabolites and metabolomic
   data. LC–MS coupled with molecular networking (MN) was adopted to
   analyze the prototype and metabolites in the blood and build a chemomic
   profiled network consisting of precursor ions. Virtual docking and
   annotation enrichment analyses were used to identify their structures
   and potential targets. Concurrently, a metabonomic assay was employed
   to detect the major reversal changes that were regulated by honeysuckle
   extractive preparation (HEP) administration in a lipopolysaccharide
   (LPS)-induced rat fever model. The interpretation of integrated data
   revealed chlorogenic acid (CA) and swertiamarin (SWE) as key compounds
   acting on both the phosphatidyl-inositol-3-kinase-protein kinase B
   (PI3K–AKT) and p38 mitogen-activated protein kinase (MAPK) pathways
   that are associated with fever.

2. Results

2.1. Global Metabolic Profiling Coupled with MN Identifies the Key
Metabolites

   Global metabolite profiling based on negative and positive ion modes
   was performed to identify key metabolites in plasma derived from HEP, a
   honeysuckle oral liquid freeze-dried powder. The MS data of different
   groups were integrated into the Global Natural Product Social Molecular
   Networking (GNPS) platform. As shown in [42]Figure 1A, the MN map
   contained 1371 precursor ions, including 297 clusters (node ≥ 2) and
   313 single nodes. Interestingly, compared with the overlapped
   metabolite distribution in HEP- and HEP-treated plasma ([43]Figure 1B),
   a total of eight main prototype components (P1-8) showed a significant
   difference in dysregulated metabolites. Based on the exact mass
   measurements and fragmentation patterns confirmed from references, as