Traditional Chinese medicine (TCM) is a class of medicine derived from natural medicine and its processed products, such as plants, animals and minerals. Since ancient times, TCM has played a significant role in preventing and treating diseases. TCM formulas have played an integral role in treating COVID-19 infections Since 2019 (Zhao et al., 2020; Huang et al., 2021). The theory of Chinese medicinal properties has received increased attention from researchers. The theory is mainly based on the system of “four Natures and five Flavors” and “Channel Tropisms” constructed by Shennong Herbal Classic of Materia Medica. “Four Natures” include “Warm, Hot, Cold and Cool”, Some research has shown that the important feature of the “Natures” is the regulation of the energy production process in the organism (Li, 1993). The “Warm” and “Hot” TCM can enhance substance metabolism and energy metabolism related to enzyme expression. In contrast, “Cold and Cool” TCM have the opposite effects. Some TCMs do not reflect the attributes of “four Natures” trends belong to the “Neutral”. The “five Flavors” refer to “Pungent, Sour, Sweet, Bitter and Salty” are directly related to the pharmacological effects. Some researchers have concluded that the “Flavors” are determined by their active ingredients (Liu and Yang, 2006). The “five Flavors” are the basis for the pharmacological efficacy of TCM and the “four Natures” are the reflection of the overall effect of TCM on the organism. The property of a TCM can have multi-“Flavors”, but only one “Nature”. The “Flavors” and “Natures” are different and some scholars have discussed that the “Natures and Flavors” can be split to studied separately (Kuang and Cheng, 2009). Due to the complex “four Natures five Flavors” theory, We need much research to establish a modern scientific understanding of the “Natures” and “Flavors” to serve the standardized medication and resource exploitation of TCM.

The characterizations of the “Flavors” and “Natures” directly by several components are difficult due to the complicated and different components of each TCM. The “Flavors” and “Natures” are the descriptions of the organism's pharmacological effects. Thus, It is a promising way to characterize the “Flavors and Natures” by metabolites using metabolomics. Metabolomics is a technique to study the metabolic network of a biological system by examining the metabolite profiles and their dynamics before and after stimulation or perturbation of the biological system (Su et al., 2016). The target metabolites of metabolomics are endogenous small molecules with relative molecular masses below 1000. The advantage of metabolomics is that metabolites are closely related to phenotypes and can provide information about biochemical activities by reflecting the substrates and products of cellular metabolism (Tang and Wang, 2006). Mass spectrometry, NMR spectroscopy, FTIR spectroscopy and Raman spectroscopy are the main analytical instruments in metabolomics. Q-Exactive mass spectrometry has a quadrupole and an Orbitrap mass analyzer. The tandem analyzer has the advantages of high sensitivity and rapid polarity switching features. Hundreds of trace components in complex matrices can be identified, quantified and confirmed by a single analysis using Q-Exactive. The metabolomics in TCM research showed that metabolomics can be used to analyze the physiological and pathological states of the response organism system, exploring the mechanism of TCM in the organism and gaining a deeper understanding(Zhang et al., 2010, Zhang et al., 2010; Wang et al., 2015, 2017).

In this study, we prepared the extracts of “Pungent-Neutral”, “Sweet-neural”, “Bitter-Neutral”, “Pungent-Warm”, and “Bitter-Cold” TCM given to rats by intragastrical injection. The serum metabolomics-integrated strategies combined with PCA and OPLS-DA statistical analysis were developed to screen potential biomarkers of the “Natures” and “Flavors” to characterize “Flavors” (Pungent, Sweet, Bitter) and “Natures” (Warm, Cold).