Evaluation of Anti-Inflammatory and Antioxidant Effectsof Chrysanthemum Stem and Leaf Extract on Zebrafish Inflammatory Bowel Disease Model

Present studies have shown that Flos Chrysanthemi has anti-inflammatory and other effects and regulates intestinal function, while the chrysanthemum stem and leaf as non-medicinal parts of chrysanthemum have similar chemical components with chrysanthemum, but the activity and mechanisms are rarely elucidated. Therefore, this study used a DSS-induced zebrafish inflammatory bowel disease model to study the anti-inflammatory and antioxidant effects of chrysanthemum stem and leaf extracts. The results indicate that DSS induction leads to increased secretion of acidic mucin in the intestines of juvenile fish, enlargement of the intestinal lumen and the emergence of intestinal inflammation. Compared with the model group, each administration group differentially inhibited the expression of IL-1β, IL-8 and MMP9 in DSS-induced zebrafish, while upregulating the activity of superoxide dismutase. The quantitative analysis results showed that the flavonoids (including Linarin, Diosmetin-7-glucoside, Tilianin, etc.) and phenolic acids (including Isochlorogenic acid C, Isochlorogenic acid A, 1,3-Dicaffeoylquinic acid, etc.) in the alcohol extract were closely related with both anti-inflammatory and antioxidant activity, while the polysaccharides were also shown a certain anti-inflammatory and antioxidant activity. In conclusion, this study suggests that the flavonoids, phenolic acids and polysaccharides from chrysanthemum stem and leaf extracts can improve inflammatory bowel disease of zebrafish by regulating the expressions of IL-1β, IL-8 and MMP9.


Introduction
Inflammatory bowel disease (IBD) includes Crohn's disease (CD) and ulcerative colitis (UC). Symptoms can include abdominal pain, vomiting, diarrhea, and weight loss [1][2][3]. With the incidence and prevalence of IBD increasing over time in different parts of the world, the disease has now become a global disease [4][5][6]. Some studies show that about 6.8 million people worldwide suffer from IBD [7]. In recent years, the pathogenesis of IBD has made some progress, involving environmental factors, psychological factors, genetic factors and intestinal microbes [8,9], but the specific pathogenesis is not clear. The main manifestations of the patient are abdominal pain, diarrhea, fever, weight loss, and possible sub-occlusion (spasm pain, vomiting, difficulty with feces/discharge) or other complications (i.e., abscess, fistula) [10]. At present, western medicine treatment drugs and means have been more common, such as corticosteroids, aminosalicylic acid, sulfasalazine, antibiotics [11] and immunosuppression [12]. These drugs can significantly reduce inflammation and improve clinical symptoms, but long-term use still has certain

Alcian Blue Staining
Whole mount alcian blue staining is a useful technique for visualizing the acidic mucin produced by zebrafish goblet cells [33]. Studies have shown that DSS induction leads to the accumulation of acidic mucin in the gut bulb [34]. The staining results showed that the intestinal fraction of DSS-induced zebrafish larvae was significantly stained blue compared to the control group, indicating the presence of acidic mucin accumulation ( Figure 1).

Hematoxylin Eosin Staining
By staining the pathological sections and placing them under the microscope and counting the proportion of the intestinal lumen area, it was seen that the intestinal tissue in the blank group did not show any obvious lesions. More serious intestinal epithelium damage was seen in the intestinal lumen after 72 h of 0.5% DSS modeling, with an increase in cupped cells and an enlargement of the intestinal lumen area compared to the blank group. The degree of intestinal epithelium shedding was reduced in the water extract administration group and alcohol extract administration group of chrysanthemum stem leaves compared with the model group, and some of the administration groups could reduce the enlargement of intestinal lumen caused by DSS modeling. The histological scores of the model group were significantly higher than those of the control group, and a comparison of the administration groups revealed a more pronounced reduction in the degree of pathology in S0.2, S0.6, C0.02 and C0.04. The HE staining results, histological score and percentage of intestinal lumen area are shown in Figure

Changes in Superoxide Dismutase Levels during DSS Induced Inflammation
The model was made by 0.5% DSS, and the concentration of 0.02, 0.04, 0.06 mg/mL alcohol extract of chrysanthemum stem and leaf and 0.1, 0.2, 0.4 mg/mL water extract of chrysanthemum stem and leaf were given to determine the corresponding SOD values. SOD activity in the model group was significantly lower than that in the blank group (p < 0.01). Compared with the model group, SOD activity in the alcohol extract groups of 0.02, 0.04, and 0.06 mg/mL and the water extract groups of 0.1, 0.2, and 0.4 mg/mL of chrysanthemum stem and leaf increased with the increase in drug concentration. There was no significant difference between the 0.1 mg/mL water extract administration group and the model group, while there were significant or extremely significant differences between the other groups and the model group, and the increase in SOD in the 0.06 mg/mL alcohol extract group and 0.4 mg/mL water extract group was close to that in the blank group, as shown in Figure 3. alcohol extract from chrysanthemum stem and leaf (0.02, 0.04, 0.06 mg/mL). (* p < 0.05, ** p < 0.01, *** p < 0.001).
It was concluded that 0.02, 0.04, and 0.06 mg/mL alcohol extract groups and 0.2 and 0.4 mg/mL water extract groups of chrysanthemum stem and leaf effectively inhibited intestinal injury of zebrafish and played a certain therapeutic effect on intestinal dysfunction induced by DSS. In conclusion, both alcohol and water extracts from chrysanthemum stem and leaf can inhibit oxidative damage of intestinal dysfunction zebrafish by changing the activity of superoxide dismutase (SOD), and they play a certain role in regulating intestinal dysfunction.

Inflammatory Cytokine Production in Larvae Exposed to DSS
The proinflammatory cytokines IL-1β, IL-8, and MMP9 were selected in this study; we then measured the expression levels of IL-1β, IL-8, and MMP9 using q-PCR. As shown in Figure 4, compared with the blank group, mRNA expressions of IL-1β, IL8, and MMP9 genes in the model group were significantly increased (p < 0.001), indicating that there was inflammation in the model group. Compared with the model group, the up-regulation of IL-1β, IL-8, and MMP9 induced by DSS was significantly inhibited in the water extract group and alcohol extract group (p < 0.05, 0.01, 0.001), and the inhibition effect of S0.2 in the water extract group and C0.04 in alcohol extract group was the most significant (p < 0.001).
In conclusion, these results suggest that water extract from chrysanthemum stem and leaf and alcohol extract from chrysanthemum stem and leaf have a protective effect on DSS induced inflammatory bowel disease model of zebrafish and inhibit the DSS-induced inflammatory response.  The results showed that the 19 flavonoids and phenolic acids showed a good linear relationship in the determination range. The regression equation, R 2 and linear range of 19 reference substances were given in Table 1. The TQ chromatograms of the components to be measured in the mixed standards and the components to be measured in the samples are shown in Figure 5.

Precision, Repeatability, and Stability
The precision test results showed that the RSD values of 19 phenolic acids and flavonoids were all less than 4.27%, indicating good precision of the instrument. Repeatability test results showed that the repeatability of 19 components in water extract (ST) and alcohol extract (CT) of chrysanthemum stem and leaf was less than 4.75% and 4.13%, respectively, indicating that the method had good repeatability. Stability test results showed that the stability of 19 components in water extract from chrysanthemum stem and leaf (ST) and alcohol extract from chrysanthemum stem and leaf (CT) of chrysanthemum stem and leaf was less than 4.50% and 4.95%, respectively, indicating that the tested solution had good stability within 24 h.
The results showed that both water and alcohol extracts of chrysanthemum stem and leaf were rich in flavonoids and phenolic acids. The contents of total flavonoids and phenolic acid in water extract of chrysanthemum stem and leaf were 11.96 ± 0.96 mg/g. Total flavonoids and total phenolic acid were 5.95 ± 0.66 mg/g and 6.00 ± 0.59 mg/g, respectively. The total flavonoid and phenolic acid contents of the ethanol extracts from the stems and leaves of chrysanthemum were 26.91 ± 1.17 mg/g. The total flavonoid and phenolic acid contents were 18.25 ± 1.11 mg/g and 8.66 ± 0.68 mg/g, respectively. The 11 flavonoids in the alcohol extract of chrysanthemum stem and leaf were higher than those in the water extract of chrysanthemum stem and leaf, the content of Isochlorogenic acid A, B, and C were higher than that of water extract from chrysanthemum stem and leaf, but the content of total phenolic acid was higher. Figure 6 shows the content contrast of components in alcohol extract from chrysanthemum stem and leaf (CT) and water extract from chrysanthemum stem and leaf (ST) of chrysanthemum stem and leaf.   The neutral polysaccharide content in chrysanthemum stem and leaf was determined by the phenol-sulfuric acid method, and the acid polysaccharide content in chrysanthemum stem and leaf was determined by the carbazole-sulfuric acid method. The content of neutral polysaccharide was 74.36 ± 4.51 mg/g, acid polysaccharide was 26.72 ± 1.84 mg/g, and total polysaccharide was 101.08 ± 5.86 mg/g. The above results showed that neutral and acidic polysaccharides were important components of total polysaccharides in chrysanthemum stems and leaves, and the content of neutral polysaccharides was higher.

Discussion
As one of the important vertebrate animal models, zebrafish has the advantages of small size, transparent embryos, and a gastrointestinal system highly similar to humans, many digestive organs, such as the intestine, liver, gallbladder, and pancreas, are functionally similar to their mammalian counterparts, and have been increasingly used in the study of IBD and gastrointestinal diseases [35][36][37]. In this study, we first evaluated the antiinflammatory and antioxidant effects of water and alcohol extracts from chrysanthemum stem and leaf using the zebrafish IBD model. The results showed that the DSS-induced zebrafish model had acidic mucin accumulation, enlarged intestinal lumen and broken epithelial cells in the intestinal fraction, while chrysanthemum stem and leaf extract inhibited the upregulation of various inflammatory factors (IL-1β, IL-8, MMP9) to varying degrees, showing its effective role in inflammatory regulation. Previously, TAO et al. [21,23] showed that chrysanthemum polysaccharide could improve ulcerative colitis by promoting the growth of beneficial intestinal flora, regulating intestinal microecological balance, and restoring the immune system, while significantly reducing the levels of inflammatory cytokines TNF-α, IL-6, IFN-γ, and IL-1β. In addition, the antioxidant function can be achieved by upregulating superoxide dismutase activity. The results suggest that chrysanthemum stems and leaves have a similar effect on intestinal inflammation as chrysanthemum. The anti-inflammatory effect of water extract of the chrysanthemum stem and leaf group increased with the decrease in concentration, while the anti-inflammatory effect of the alcohol extract group was the best at medium concentration, and the antioxidant effect of both extracts was good at high concentration.
As a non-medicinal part of chrysanthemum, the yield of chrysanthemum stem and leaf was significantly higher than that of chrysanthemum, but it could not be effectively used, resulting in resource waste. Zhou  In addition, neutral and acidic polysaccharides were determined by the phenol-sulfuric acid method and carbazol-sulfuric acid method, and the total polysaccharide content in chrysanthemum stems and leaves was obtained. The results showed that chrysanthemum stem and leaf contained similar components to chrysanthemum, among which the contents of total flavonoids and total phenolic acids in the alcohol extract of chrysanthemum stem and leaf were significantly higher than those in the water extract of chrysanthemum stem and leaf, but the water-soluble polysaccharides were significantly higher in the water extract of chrysanthemum stem and leaf.
In this present study, the experimental results showed that different extracts from chrysanthemum stem and leaf have certain anti-inflammatory and antioxidant effects, and the main bioactive components were analyzed, including flavonoids, phenolic acid, and polysaccharides. However, the anti-inflammatory and anti-oxidation mechanisms and targets of these bioactive components need to be further studied and explored.

Animal Care Ethics
All zebrafish experiments were conducted according to the guidelines of the Animal Ethics Committee of the Laboratory Animal Center, Nanjing University of Traditional Chinese Medicine.

Zebrafish Husbandry
The adult AB strain zebrafish (YSY Biotechnology Inc., Nanjing, China) were raised in a dark/light (10 h:14 h) [40], 26 • C environment, and live brine shrimp feed was cast twice a day. One day before the experiment, healthy zebrafish with reproductive ability were placed in spawning boxes with a 1:1 or 1:2 male to female ratio, separated by a partition. The partitions were pulled out at 9:00 the next morning, and the fertilized eggs were collected in the box at 11:30. The collected embryos were placed in a constant temperature incubator at 28 • C and kept under 14 h light. E3 culture water [41] (5 mM NaCl, 0.17 mm KCl, 0.33 mm CaCl 2 , 0.33 mm MgSO 4 , pH = 7.2) was prepared, and E3 was used as a specific medium for embryo culture, rather than zebrafish fish culture system water, to ensure stable buffer salt concentration and pH. Dead eggs were removed every 24 h, and fresh embryo culture medium was replaced.

Chemicals and Regents
The stems and leaves of chrysanthemum were collected from Yangma Town, Sheyang County, Yancheng City, Jiangsu Province in November 2019, and extracted and concentrated by Kanion Pharmaceutical using industrial percolation extraction, vacuum concentration, and other processes to obtain water extraction (ST) and 80% alcohol extraction (CT) extract respectively.
Deionized water was distilled and purified by an EPED super-purification system (Eped, Nanjing, China).
Water extract and alcohol extract from industry were taken, and the yield was measured after freeze-drying. The freeze-dried yield of water extract and alcohol extract was 16.13% and 16.94% respectively. The water-extracted freeze-dried powder and alcoholextracted freeze-dried powder were accurately weighed at about 1 mg each, and then placed in a 1 mL volumetric bottle, methanol was added to a constant volume to scale, and it was well mixed. It was then centrifuged at 13,000 r·min −1 for 15 min before injection, the supernatant was filtered through 0.22 µm microporous membrane, and the filtrate was used as the test solution and stored at 4 • C for later use.

Zebrafish IBD Modeling and Drug Treatment
Dextran Sulfate Sodium (DSS) is a sulfated polysaccharide that can cause chemical damage to the intestinal mucosa [35][36][37]. A zebrafish model with intestinal dysfunction was established using 0.5% DSS and the concentration was screened. The water extract and alcohol extract of chrysanthemum stem and leaf were prepared with different concentrations. According to the screening results, the concentration of alcohol extract from chrysanthemum stem and leaf was determined to be 0.02 mg/mL (CT0.02), 0.04 mg/mL (CT0.04), 0.06 mg/mL (CT0.06). The concentration of water was 0.2 mg/mL (ST0.2), 0.4 mg/mL (ST0.4) and 0.6 mg/mL (ST0.6). Then, 40-50 juvenile fishes on the third day post fertilization(dpf) were grouped into blank group, model group (immersed in 0.5% DSS), alcohol-extract group (immersion in different concentrations of alcohol extracts of chrysanthemum stems and leaves prepared with 0.5% DSS solution), and water-extract group (immersion in different concentrations of water extract solutions of chrysanthemum stems and leaves prepared with 0.5% DSS solution). Each group was repeated with 3 wells, the drug was administered for 72 h continuously, and the solution was changed every 24 h to observe the survival rate.

Alcian Blue Staining
DSS-induced intestinal injury in zebrafish leads to accumulation of acidic mucin in the intestinal bulb. Moreover, a significant feature of DSS-modeled zebrafish is alcian blue staining in intestinal globules, and the accumulation of acidic mucin in intestinal globules is called "mucinous phenotype". To observe the changes of intestinal goblet cells in the DSS model, alcian blue staining of acidic mucin was performed for comparison between the model group and the blank group [34]. Specific dyeing methods are as follows: (1) Fix the young fish in 4% paraformaldehyde at room temperature for 2 h.

Hematoxylin Eosin Staining
The 6 dpf zebrafish juvenile fish were immobilized overnight in 4% PFA at room temperature and then progressively dehydrated in ethanol. The juvenile fish were embedded in paraffin sections, cut into 3 µm sections, and stained with hematoxylin-eosin. Cross-sectional areas of the zebrafish intestine were scored using H&E staining and graded histologically. The analysis was modified as described in the literature and included grade of inflammation (0-3), degree of mucosal edema (0-3), degree of vacuolation (0-3), epithelial cell damage (0-3) and percentage of cupped cells (0-3) [42]. At least three juvenile fish were examined under each treatment condition.

Superoxide Dismutase (SOD)
SOD is a key enzyme in the antioxidant system, which can inhibit oxidative damage caused by external stimuli to the body, and the level of SOD can reflect the degree of the body's anti-inflammatory response. Seventy-two hours later, 25 juvenile zebrafish in each group were collected and washed in a 1.5 mL centrifuge tube with normal saline 3 times and treated with 1:19 (zebrafish quality:normal saline volume). Then, 5% tissue homogenate was prepared by automatic sample rapid grinding machine, centrifuged at 4 • C for 5000 RPM × 15 min, and the supernatant was taken.
Before the test, 5% tissue homogenate was diluted into different concentrations for pre-experiment, and the concentration with an inhibition rate of 40-60% was selected. The pre-experiment results showed that the inhibition rate of 1% tissue homogenate met the requirements. The protein concentration of 1% tissue homogenate was determined according to the BCA method protein concentration determination kit. The activity of superoxide dismutase (SOD) in 1% tissue homogenate of each group was detected according to the instruction of the SOD assay kit.

RNA Extraction and q-PCR
The 6 dpf zebrafish tissues were collected and placed into a homogenizer, and the RNA of each sample was extracted. The RNA of each group was reverse transcribed to obtain cDNA. The expression levels of genes related to inflammation were determined by the Bio-rad CFX96 real-time system. The results of β-actin were used as the internal reference. PCR primers of interleukin (IL) -1β, IL-8, Matrix metallopeptidase 9(MMP9), and reference gene β-actin were synthesized and purified by Shanghai Sangon Bio-Technology Co., Ltd (Shanghai, China), and their quality was detected. Primer information of RT-QPCR is shown in Table 2. After the RT-QPCR reaction, the original data were derived. The original data were imported into Excel, and the 2 −( CT) calculation method was used for data processing. SPSS software was used to conduct one-way ANOVA, and p < 0.05 indicated a significant difference. GraphPad 8 software was used to draw experimental data pictures.

Linearity
The preparation solution of mixed reference substance was diluted to prepare mixed reference substance solution with a series of mass concentrations. The retention time of each component was obtained from the mixed standards as a way to identify the components in the extracts. UPLC-TQ/MS analysis was performed according to the chromatographic conditions and the mass spectrometric conditions. With the peak area as Y and the concentration of reference solution as X, linear regression analysis was performed to calculate the correlation coefficient.

Precision and Accuracy
Precision test: the mixed reference solution was injected 6 times in one day to determine the peak area of each component to be measured, and the intraday precision was evaluated by the relative standard deviation (RSD%) of each peak area.
Repeatability test: the last 6 samples of freeze-dried powder, each about 1.0 mg, were weighed accurately, prepared for the test solution, and the content and RSD value of each component to be measured in the test solution after UPLC-TQ/MS analysis were calculated.
Stability test: a sample solution from the repeatability test was injected into the liquid chromatography at 0, 2, 4, 8, 12, and 24 h, separately. After the UPLC-TQ/MS analysis, the peak area and RSD values of each component to be measured were calculated.

Determination of Total Polysaccharide Content
Using glucose and glucuronic acid as control, the neutral polysaccharide content in chrysanthemum stem and leaf was determined by the phenol-sulfuric acid method, and the acid polysaccharide content in chrysanthemum stem and leaf were determined by the carbazol-sulfuric acid method. Total polysaccharide content = neutral polysaccharide content + acid polysaccharide content.

Statistical Evaluation
MassLynx4.2 (Waters Corporation, Milford, Massachusetts state, USA) software was used to analyze the contents of total flavonoids and phenolic acids in water extract and alcohol extract of chrysanthemum stem and leaf, and SPSS22.0 was used for statistical analysis. Independent sample T test was used for comparison. A value of p < 0.05 was considered significant.

Conclusions
In summary, our study confirmed the anti-inflammatory and antioxidant effects of Chrysanthemum morifolium leaf extract in a zebrafish inflammatory bowel disease model and verified that its flavonoid, phenolic acid, and polysaccharide components were closely related to both anti-inflammatory and antioxidant activities. This study may also be helpful to develop the therapeutic agents for IBD chrysanthemum stem and leaf resources.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.