Amomum villosum Lour. (Figures 1A,B) was collected in Jingping County, Honghe Prefecture of Yunnan Province, China. The plants were identified as A. villosum Lour. by Jie Yu, an Associate Professor at Yunnan University of Traditional Chinese Medicine. Voucher specimens were deposited in the Herbarium of Pharmacognosy, Yunnan University of Traditional Chinese Medicine. BA (Figure 1C), was purchased from Jingzhu Biotechnology Co., Ltd. (Nanjing, China, purity > 98%, GC). 5-FU was obtained from XuDong HaiPu Pharmaceutical Co., Ltd. (Shanghai, China). Live combined Bifidobacterium and Lactobacillus tablets (CBL) were purchased from Inner Mongolia Shuangqi Pharmaceutical Co., Ltd. (China).

Powder (100 g) of A. villosum fruit was soaked with 800 mL distilled water for 12 h (4°C). VOA, gathered by steam distillation, was stored at 4°C after drying with Na₂SO₄.

Gas chromatography-mass spectrometry (GC-MS) was used to characterize VOA (HP6890GC/5973MS, Agilent). Chromatographic separation was achieved on an HP-5MS (30 mm × 0.25 mm, 0.25 μm) fused-silica capillary column. The injector temperature was 250°C and the column pressure was 100 kPa. Helium was used as the carrier gas (1.0 mL/min) and the injection volume was 0.1 μL (split ratio of 50:1). The temperature was 80°C for 2 min and increased to 280°C for 20 min (3°C/min).

Mass spectrometry conditions were as follows: ion source temperature, 230°C; electron energy, 70 eV; interface temperature, 250°C; quadrupole temperature, 150°C; and mass scan range, 30–500 amu. Wiley7n.l mass spectral database was used to compare data with a standard spectrum to identify the components of every peak.

Fifty-four, 8-week-old male Spraque–Dawley rats (Da Shuo Biotech Co., Ltd., Chengdu, China, Certificate of Quality No: 0016254) were kept in a temperature-controlled room with free access to water and food, and they fasted for 2 h before all of the treatments. The study was approved by the Institutional Ethical Committee on Animal Care and Experimentation of Yunnan University of Traditional Chinese Medicine (R-0620150028). All reasonable efforts were made to minimize animal suffering.

After adaptive feeding for 3 days, the rats were randomized to one of nine groups (N = 6/group): normal controls (CON); 5-FU-induced intestinal mucositis model (MOD); live combined Bifidobacterium and Lactobacillus tablets (CBL, 450 mg/kg) positive controls; VOA low, medium, and high groups (VOA.L, 8 mg/kg; VOA.M, 16 mg/kg; and VOA.H, 32 mg/kg); BA low, medium, and high groups (BA.L, 2 mg/kg; BA.M, 4 mg/kg; and BA.H, 8 mg/kg). All of the rats, except for the normal controls, received 5-FU (35 mg/kg, daily, i.p.) for 5 days. Researchers used personal protective gear to prevent exposure to 5-FU and CBL. At the end of the study, the drugs were destroyed by the Experimental Center of Yunnan University of Traditional Chinese Medicine.

The dose of A. villosum was 3–6 g/kg in clinical medium and the BA concentration was required to be not less than 1% in A. villosum according to the Commission of Chinese Pharmacopoeia (2015). The yield of VOA was 3.9% from A. villosum in our study and BA was calculated as 1%, which was the minimum content of BA regulated by the Commission of Chinese Pharmacopoeia (2015). The clinical dose for humans was calculated as 4.5 g/kg in this study. These factors were considered in our dosage conversation, and we then converted the human dosage into the rat dose according to the body surface area. We concluded that the dose of VOA was 16 mg/kg and BA was 4 mg/kg in rats, which was considered to be the medium dose. Finally, the low, medium, and high dose of each administration group was calculated by 1:2:4. VOA and BA were dissolved in distilled water with 0.1% Tween-80 by an ultrasonic mixer at 16°C and then stored at 4°C. Before chemotherapy (4 h), the rats received CBL, VOA, and BA (via gavage) once daily for 12 days. Figure 2 depicts the treatment schedule.

Whether or not the rats had diarrhea, and their weight and food intake were recorded daily. Rats were killed 12 days after treatment using 10% chloral hydrate anesthetization. Blood samples and small intestinal tissue were collected.

Segments of the jejunum were collected, fixed, and stained with hematoxylin and eosin to measure the villus height and crypt depth. Three tissues from different rats in each group and three images (10×) per section were analyzed for morphological studies.

Blood samples were collected from the retro-orbital venous plexus and hepatic portal vein at 2 h after treatment in the morning at the end of the study. Serum was centrifuged at 12,000 × g for 15 min and analyzed immediately. Serum interleukin-6 (IL-6) and reactive oxygen species (ROS) were measured with ELISA kits purchased from Cusabio Biotech Co., Ltd. (China).

At the end of the treatment, the rats were killed via 10% chloral hydrate (0.3 mL/100 g, ip). Small intestine tissue samples were excised, weighed, and washed in 0.9% saline. Tissues (100 mg) were rinsed with PBS and homogenized in 1 mL of PBS and then stored overnight at -20°C. Two freeze-thaw cycles were performed to break the cell membranes, and homogenates were centrifuged for 10 min at 6,500 × g, 4°C. The supernatant was collected for biochemical analysis. ROS, tumor necrosis factor-α (TNF-α), nuclear factor of kappa b (NF-κB), and myeloperoxidase MPO in the intestinal homogenate were measured with ELISA kits purchased from Cusabio Biotech Co., Ltd. (China).

Hepatic portal vein blood samples were centrifuged at 6,500 × g for 10 min at 4°C after being collected under anesthesia. Lipopolysaccharide (LPS) was measured using a tachypleus amebocyte lysate test purchased from Chinese Horseshoe Crab Reagent Manufacturers Co., Ltd. (Xiamen, China).

A single-cell suspension was prepared by cutting and disrupting one representative intestinal sample from each group through a 70-μm filter membrane. Cells were diluted with staining buffer (1 × 10⁶ cells/mL) after blockage with 3% FBS. For analysis, the cells were incubated with an anti-occludin antibody (EPR8208, Abcam, United States), anti-ZO-1 antibody (21773-1-AP, Proteintech, United States) and an anti-caspase antibody (25546-1-AP, Proteintech, United States) for 2 h, and then they were incubated with fluorescein isothiocyanate (FITC, SA00003-2) in the dark for 1 h. Occludin, ZO-1, and caspase-3 expression was measured using flow cytometry (FACSCalibur, Becton, Dickinson, and Company, United States).

Intestinal tissues (100 mg) were homogenized using 1 mL PIRA lysis buffer, and centrifuged (12,000 × g, 10 min, 4°C). Then, samples were kept on ice for 20 min. The supernatant was collected for Western blotting, and the extracted proteins were quantified using the bicinchoninic acid (BCA) method. Next, 50 μg of total protein was added to each sample. Proteins were resolved with 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Transferred membranes were blocked with 5% albumin bovine V (BSA) to inhibit non-specific proteins. Then, the primary antibodies (9212S, 1:1.000 dilution; Cell Signaling, Danver, MA, United States) were added, with β-actin (20536-1-AP, 1:1.000 dilution; Proteintech Group, United States) used as an internal reference. After incubating with primary antibodies, the membranes were washed with TBS/Tween-20 (TBST) three times and then incubated with the secondary antibodies (SA00001-2, 1:10.000 dilution; Proteintech Group, United States). Each protein band was visualized with an enhanced chemiluminescence (ECL) detection system (Proteintech Group, United States) and quantified with Quantity One Analysis Software (Bio-Rad Laboratories, Inc.).

Rat feces samples were collected on the last day and placed in a sterile centrifuge tube. They were then evenly ground in liquid nitrogen and preserved at -80°C for future inspection. All of the fecal samples from the treatment groups were blended, and transferred to a mortar and ground to a fine powder. DNA was extracted according to the instructions of the stool DNA kit (Omegea Bio-tek, United States).

To determine bacterial diversity and composition in the feces samples, we used the protocol described by Caporaso’s group (Caporaso et al., 2010). PCR amplifications were conducted with a 515f/806r primer set that amplifies the V4 region of the 16S rDNA gene. This primer set has few biases and yields accurate phylogenetic and taxonomic information. The reverse primer contains a 6-bp error-correcting barcode unique to each sample. DNA was amplified as described in the literature (Magoč and Salzberg, 2011). Sequencing was conducted on an Illumina MiSeq platform (Novogene, Beijing, China).

Data are means ± SD. One-way analysis of variance (ANOVA) was used to compare multiple groups (p < 0.05, < 0.01, and < 0.001). Graphics were created with Origin 6.1 (MicroCal Software, United States). A 95% confidence interval (CI) was used as a threshold to identify potential outliers in all of the samples, and clustering was analyzed with TMEV Clustering (Mev Development Team).

A total of 65 compounds were determined, and 58 compounds were successfully identified, which represented over 99% of the total oil composition. As shown in Table 1, BA (54.54%), camphor (17.92%), camphene (6.76%), limonene (5.25%), borneol (4.07%), myrcene (1.97%), α-pinene (1.50%), β-caryophyllene (0.85%), β-pinene (0.80%), and α-pepperene (0.54%) were detected in VOA. BA was considered as the representative compound of VOA.

Initial animal weight and food intake were similar among the groups, and the controls gained weight over time (Figure 3A). Rats treated with 5-FU had decreased weight from the fourth to the eighth day and the weight of these animals was the lowest until the end of the experiment. Treatment with CBL, VOA, and BA prevented (p < 0.01) significant weight loss in the MOD group. During the study, the average MOD food intake was 10.81 g/day, which was roughly half of the normal group (Figure 3B). CBL, VOA, and BA treatments reversed the appetite reduction in the MOD group. Compared with the MOD group, the food intake of the CBL, VOA, and BA groups was increased by about 50%.

Fecal appearance and shape indicated diarrhea in the MOD group, and all of the treatments relieved 5-FU-induced watery diarrhea (Figure 4A). Compared with the controls, the MOD group had histopathological changes in the jejunum, such as mucosa with shortened villi with vacuolated cells, crypt necrosis, and intense inflammatory cell infiltration. Treatment with VOA, BA, and CBL for 12 days reversed these changes in limited fashion (Figure 4B).

The administration of 5-FU induced significant decreases in villus height, increased crypt depth, and decreased villus: crypt ratio (Figure 4C). Figure 4C shows that CBL, VOA, and BA treatment reversed 5-FU-induced reductions in villus height. Similarly, a smaller villus: crypt ratio was observed in the MOD group, and this was reversed with the drug treatments, especially with the VOA.H treatment (p < 0.01).

After an injection of 5-FU, we observed a significant increase (p < 0.01) in the serum IL-6 concentration (3.07 ± 0.79 pg/mL) compared with the controls (1.54 ± 0.39 pg/mL), suggesting that the inflammation was elevated in the MOD rats. Treatment with VOA and BA significantly reduced the IL-6 levels by 26.71%, 40.06%, 38.76%, 30.94%, 45.60%, and 42.34% (Figure 5A). In the meantime, we found a significantly elevated ROS concentration in the sera of the MOD group (2.48 ± 0.41 U/mL) compared with the control group (1.93 ± 0.18 U/mL); this indicated that the model rats induced by 5-FU were in an inflammatory status. The increase was significantly inhibited after treatment with CBL (1.98 ± 0.31 U/mL) (Figure 5B). In brief, VOA.M, VOA.H, and BA suppressed the 5-FU-mediated increase in IL-6, and VOA.H suppressed the ROS increase compared with the MOD group.

As shown in Figures 5C,D, the concentrations of ROS and TNF-α in the small intestinal tissue of the intestinal mucositis rats was increased significantly (to 20.42 ± 1.10 U/mL and 184.7442 ± 25.71 pg/mL, respectively). Similarly, MPO and NF-κB concentrations were also increased by 67.44% and 51.21%, respectively, in the intestinal mucositis rats. All of the treatments could effectively reduce (p < 0.001) these inflammatory factor levels. Figure 5C shows that treatment with BA significantly reduced ROS concentrations by 24.06%, 44.62%, and 53.18%, while Figure 5D shows that treatment with BA significantly reduced TNF-α concentrations by 69.43%, 72.02%, and 73.77%, respectively. VOA treatment reduced MPO (55.63%, 60.03%, and 49.46%, respectively) and NF-κB concentrations (44.68%, 46.74%, and 43.84%, respectively) (Figures 5E,F). Thus, VOA and BA prevented inflammation in the small intestinal tissue after 5-FU chemotherapy.

The concentrations of p38 MAPK in the small intestine were detected by Western blotting. Our results revealed that p38 MAPK expression in the MOD group was significantly higher than the CON (control) group (p < 0.01), while the therapeutic drugs could successfully inhibit the expression of p38 MAPK. Treatment with VOA and BA reduced (p < 0.01) the p38 MAPK levels by 37.29%, 39.83%, 60.17%, 12.71%, 23.73%, and 41.53%, respectively (Figure 6A).

Caspase-3 protein in the small intestinal tissue was measured with flow cytometry. Figure 6B shows that 5-FU significantly increased the expression of caspase-3 compared with the control. In contrast, both VOA and BA could effectively reduce (p < 0.01 and p < 0.001) the expression of caspase-3 in a dose-dependent manner. These data suggested that they could significantly inhibit cell apoptosis and alleviate the development of endoenteritis.

The 5-FU treatment increased LPS in the MOD group more than CON group (p < 0.001; Figure 7A). VOA (p < 0.01) and BA (p < 0.05) showed a satisfactory effect with respect to the inhibition of LPS rise. This inhibition effect may be related to its beneficial effects on gut microbiota equilibrium.

Figures 7B,C show that the expression of ZO-1 and occludin was significantly lower (p < 0.001) in MOD rats compared to the CON rats, and this agreed with a previous study (Song et al., 2013). VOA and BA increased ZO-1 and occludin protein expression to normal levels. VOA and BA improved the intestinal mucosal barrier by increasing the expression of ZO-1 and occludin protein and blocking endotoxin translocation-induced endotoxemia.

The structure and composition of gut microbiota were dramatically altered by 5-FU injection. Results of the microbial classification at the level of family showed that the relative abundance of Bacteroidaceae, Helicobacteraceae, Enterobacteriaceae, Porphyromonadaceae, and Streptococcaceae in the CON group was 1.19%, 0.32%, 0.02%, 0.25%, and 0.02%, respectively. However, their relative abundance in the MOD group increased to 10.40%, 1.54%, 0.28%, 1.09%, and 0.07%, respectively. The relative abundance of Helicobacteraceae, Enterobacteriaceae, Porphyromonadaceae, and Streptococcaceae was significantly reduced after treatment with BA, and Bacteroidaceae was reduced by VOA.M, VOA.H, and BA. Meanwhile, the relative abundance of Lactobacillaceae, which are usually considered probiotics, decreased from 4.69% in the CON group to 3.15% in the MOD group. VOA and BA treatment reincreased the abundance of Lactobacillaceae to 5.35–13.45% and 14.03–18.63%, respectively (Figure 8A).

Results of the microbial classification at the level of genus showed that the relative abundance of the Bacteroides (Bacteroidaceae), Ruminococcus (Ruminococcaceae), Helicobacter (Helicobacteraceae), Escherichia (Enterobacteriaceae), and Parabacteroides (Porphyromonadaceae) in the CON group was 1.19%, 1.48%, 0.03%, 0.01%, and 0.25%, respectively. However, their relative abundance in the MOD group increased to 10.40%, 2.14%, 1.24%, 0.28%, and 1.09%, respectively. The relative abundance of Ruminococcus, Helicobacter, Escherichia, and Parabacteroides was significantly reduced after treatment with BA, and Bacteroides was reduced by VOA.M, VOA.H, and BA. The relative abundance of Lactobacillus (Lactobacillaceae) in the CON group was 4.69%, and it decreased to 3.15% in the MOD group. After treatments with VOA and BA, the abundance of Lactobacillus increased to 5.35–13.45% and 14.03–18.63%, respectively (Figure 8B).

In addition, according to the species annotations and the abundance information at the level of genus, the heat map of the most abundant 35 genus was plotted and cluster analysis was conducted (Figure 9). They could be divided into four categories: BA.L/BA.M/BA.H; CON/CBL; MOD/VOA.H/VOA.L; and VOA.M/BA, and we found that BA.M and CBL were the closest. We concluded that BA increased the abundance of probiotics, such as Lactobacillus and Bifidobacterium, while VOA increased the abundance of Roseburia and Coprococcus. We suggested that BA and VOA might regulate intestinal microecology in two ways not just one way.

Pearson correlation and cluster analyses were performed with clean operational taxonomic unit (OTU) data and we identified 52 key variables, which were significantly altered after the VOA and BA treatments. These were correlated to alternations in ROS, MPO, LPS, and caspase-3 (Figure 10). Among them, Bacteroidaceae (four OTUs, from Bacteroidetes) were positively correlated with ROS, MPO, and caspase-3. S24-7 (eight OTUs, from Bacteroidetes), Ruminococcaceae (five OTUs, from Firmicutes), and Desulfovibrionaceae (one OTU, from Proteobacteria) were positively correlated with LPS. In addition, a few bacteria from Bacteroidetes (three OTUs) and Firmicutes (one OTU) were negatively correlated with LPS (Figures 10A,C).

Clustering analysis of the OTUs (Figure 10B) showed that among the 52 key OTUs, most of them in the MOD rats were increased when compared to the CON group. Using the Euclidean distance metric, the MOD group was at a long distance from the CON group. However, all of the treatment groups could shorten the distance to the CON group and the VOA and BA groups were clustered into two trees, suggesting that VOA and BA had a significant effect on the regulation of the 52 key OTUs. Thus, gut microbiota in the MOD rats were significantly altered compared to CON rats, and CLB, VOA, and BA treatment could partially recover the gut microbiota equilibrium.