Due to the long time and wide range of use of TCM, especially the dual use of TCM, the use of a single TCM may reflect the toxicity of TCM (Shuai-nan et al. 2015; Yiliang 2016) (Port 2019). Modern pharmacological studies have found that some herbal ingredients of aristolochliaceae are toxic (Wei 2017). For example, aristolochliic acid in them shows relatively strong toxicity and even irreversible permanent carcinogenicity. In recent years, more and more attention has been paid to the evaluation of the safety of Traditional Chinese medicine (Zhang Guangping 2016; Jiang-cun et al. 2016) (Xiaohe 2019; Jiabo, Haibo, and Keyong 2018) and there are many new methods being explored, such as network pharmacology(Gui and Jie 2012), genomics (Xue-ping et al. 2015), comparative omics and metabonomics (Zhuo et al. 2014) (Li-hua et al. 2015) (Dan-dan et al. 2016) (Nai-xi et al. 2012). TCM safety evaluation research based on TCM syndrome classification theory(Hui 2008), evidence-based pharmacy(Li Ling 2016), data mining (Yuan-yuan et al. 2011), etc. Mammalian 90-day toxicity test is an important experiment for safety evaluation, and the traditional 90-day feeding test has relatively few endpoints, narrow coverage and. Nowadays, some toxicologists began to explore the use of invertebrates as an alternative of mammals for the long-term studies (Hong et al. 2020). We added intestinal microbial diversity detection and urine mass spectrometry to the traditional 90-day feeding test analysis in order to obtain more comprehensive data for safety evaluation of subjects.
The weight of experimental animals during their growth and development is one of the most basic and sensitive indicators to comprehensively reflect the health status of animals(Zhong-ren et al. 2011), so it is an important observation indicator in the toxicity test. As well as the balance of lipid metabolism. In the 90-day subchronic toxicity study of Elsholtzia Maxim, it was shown that it had no significant influence on the weight and growth of rats. However, the weight of male rats from the second week showed that the weight of the administration group was slightly lower than that of the normal control group and the TC, LDL levels of the male administration group were significantly lower than those of the normal control group, the HDL level increased. The results are in line with previous studies that MCM has the effect of lowering blood lipids (lijuan 2011).
The blood routine index is an important index to reflect whether the test substance has affected the hematopoietic function. The results of this experiment showed that the number of red blood cells in female rats with low and high dose and male rats with medium dose increased after 90 days, but no obvious dose-effect relationship was observed, so comprehensive analysis showed that feeding experiment of MCM for 90 days had no obvious influence on blood routine of rats.
In order to improve the overall understanding of the safety of traditional Chinese medicine, we increased the detection of serum energy metabolism, oxidation index and gastrointestinal motility index. Motilin (Mtl) is one of the hormones of the digestive system, its function is to increase the migrating motor complex (MMC), promote gastrointestinal movement and stimulate the secretion of pepsin, while promoting gastric emptying(Haba 1993; Melmed et al. 2015). The results of this experiment showed that Mtl levels in all the treatment groups were decreased, and the Mtl levels in the medium and high dose groups were significantly lower than those in the control group. Calcitonin gene-related peptide (CGRP) is a neuropeptide that is distributed in the brain and gastrointestinal system and has a variety of biological effects, it can protect gastrointestinal function by inhibiting gastric acid secretion, increasing gastric mucosal blood flow, slowing gastrointestinal movement, inhibiting inflammatory reaction, antagonizing free radical damage and regulating gastrointestinal hormone secretion. In this experiment, it was found that the CGRP level of male rats in the low-dose, middle-dose and high-dose groups and female rats in the high-dose group increased, and there were significant differences with the control group. It was reported that the increase of CGRP level could reduce the release of inflammatory factors TNF and IL-1 (Xiao et al. 2019), therefore, the results show that MCM can inhibit the release of inflammatory cytokines, to play a important role in regulating local inflammation. The results are in line with the clinical treatment of acute gastroenteritis and abdominal pain vomiting and diarrhea by MCM, which may have good resistance to intestinal accumulation heat and enhance the characteristics of intestinal peristalsis.
The levels of ALT, AST, ADA and CHE can reflect the damage and severity of liver cells. The biochemical indicators of liver function such as TP, ALB and CHE can reflect the anabolic function of cell protein, this indicates a decrease in Alb level in serum liver injur (Ying et al. 2018; Xian-ping, Wei-yan, and Cai-yan 2017); The levels of TB, DB, associates can reflect the function of liver excretion and secretion and detoxification, ALP and GGT levels can reflect cholestasis. AST and ALT are very important enzymes in the metabolic process of the body. When liver cells are damaged, AST and ALT are released, increasing the levels of AST and ALT in serum. However, when the increase of these two indicators does not exceed twice, there is no toxicological significance. This study found that AST and ALT increased in both the female and male administration groups, and the liver organ coefficient was larger than that of the normal control group. It shows that MCM has effect on the liver, but it is not toxic, and the pathological section shows normal.
BUN, CRE and UA were the indicators of renal function. BUN is the end product of amino acid and protein metabolism of the body, and is the main component of non-protein in blood. When the kidney is injured, the excretion of BUN decreases, leading to an increase in the level of BUN in the serum. CRE is a kind of small molecule product produced by muscle metabolism in the body, which can be filtered and excreted by the kidney. CRE level is an important indicator to detect the detoxification ability of the kidney (Yuliang et al. 2019). In this experiment, it was found that the effects of 90 days feeding of water extract from MCM on renal function had no significant toxicological significance.
Rich in the amount and type of gut microbes not only helps digestion and absorption of nutrients, it also has a huge impact on the ADME process of exogenous chemicals, at the same time these metabolites can adversely affect the abundance and diversity of the gut microbes, and analyzing the distribution of intestinal flora is the innovation of this experiment. From the results (Fig. 10), the distribution of intestinal flora in the administration group and the control group has changed. Compared with the normal control group, the flora with a significantly higher relative abundance of the MCM administration groups are Prevotella, Ruminococeaceae and Romboutsia (Fig. 11–12). In terms of urine metabolomics, it was found that the level of uric acid in the urinary metabolites of the administration group was significantly different from that of the normal control group, the results are similar to previous studies. Previous studies have found that the intestinal flora is involved in the regulation of uric acid excretion. Short-chain fatty acids (such as butyrate) regulate the proliferation and repair of intestinal epithelial cells, change the number and distribution of uric acid transporters in the body, and affect the transport and excretion of uric acid. Lim et al. (Lim et al. 2014) conducted a horizontal study and analysis of fecal microbes in the general population of Korea, and a two-year longitudinal study and analysis of fecal microbes in identical twin pairs, and confirmed that the level of human blood uric acid and the dominant intestinal type of intestinal flora It is closely related. Among them, the dominant intestinal type of Prevotella (the main decomposition products are butyric acid, succinic acid, lactic acid, etc.) has lower blood uric acid than the dominant intestinal type of Bacteroides. In 2016, Stern et al. (Stern et al. 2016) used broad-spectrum gene sequencing technology to compare and analyze the stools of 23 patients with kidney stones and 6 general population, and also found the correlation between hemorrhagic uric acid and the abundance of Prevotella and Bacteroides. In the same year, Guo et al. (Guo et al. 2016) studied the intestinal flora and metabolites and found that the type and quantity of intestinal flora in patients with gout and the decrease in the content of broken chain fatty acid butyric acid are associated with xanthine dehydrogenase and uric acid in the intestine. The decrease in the level of uric acid in the urine may be caused by changes in the distribution of intestinal flora.
N-acetyl-L-phenylalanine is the product of phenylalanine N-acetyltransferase in the phenylalanine metabolic pathway. Changes in the level of N-acetyl-L-phenylalanine indicate that exposure to MCM will affect the metabolism of phenylalanine.Indole acetic acid is the starting point and central compound of the tryptophan metabolism pathway. Tryptophan metabolism is closely related to the central nervous system and immune regulation, and plays an important role in maintaining the normal physiological functions of the central nervous system. Therefore, the change of indole acetic acid content suggests that MCM can interfere with the tryptophan metabolism of normal rats. Previous studies have shown that tryptophan and its metabolites are involved in the development of liver steatosis and steatohepatitis (Ritze et al. 2014). Changes in ALT and AST levels may be related to the metabolic pathways of tryptophan.