Effects of ginseng soluble dietary fiber on serum antioxidant status, immune factor levels and cecal health in healthy rats
ABSTRACT
As an important component of ginseng, the in vivo benefits of ginseng water-soluble dietary fiber (ginseng-SDF) have not been fully revealed. To explore these benefits, healthy rats were given ginseng-SDF (200, 400, and 800 mg/kg body weight/day) by gavage for 15 days. The results showed that ginseng-SDF significantly improved the rats’ growth performance and serum antioxidant status. Insulin-like growth factor (IGF-1 and IGF-2) and immunoglobulin (IgA, IgM, and IgG) levels in the ginseng-SDF groups were increased. High-dose ginseng-SDF significantly increased the cecal butyric acid proportion compared with the K group. Ginseng-SDF increased the abundance of Firmicutes and promoted the proliferation of probiotics such as Lactobacillus, and cellulose de- composers such as Ruminococcus and Clostridium in cecal microflora. These altered microflora were correlated with growth performance, antioxidant status and immunoglobulin indexes. The above results suggested that ginseng-SDF might have positive effects on growth, oxidative-immune levels and cecal health in rats.
1. Introduction
Dietary fiber (DF) is usually not digested and absorbed by the small intestine, but serves as an important fermentation substrate and energy source for intestinal microorganisms. DF can be fermented by intestinal flora to produce small-molecule metabolites, such as short-chain fatty acids (SCFAs), which are absorbed by intestinal epithelial cells and play roles in different tissues and organs (Yang et al., 2020; Bultman, 2017; Correa-Oliveira, Fachi, Vieira, Sato, & Vinolo, 2016). Recent studies have confirmed that DF plays prominent roles in regulating metabolism and oxidative stress, reducing the risk of cardiovascular diseases and regulating the immunity and structure of the intestinal flora (Makki, Deehan, Walter, & Ba¨ckhed, 2018). DF can also affect substance meta- bolism, energy homeostasis and host health through the intestinal microbiome-gut-brain axis (Makki, Deehan, Walter, & Backhed, 2018; Desai et al., 2016). An important feature of DF is its utility for certain intestinal floras, which has many effects on the intestinal environment (Koh, De Vadder, Kovatcheva-Datchary, & Backhed, 2016; Wu et al., 2011). Hooda et al. (2012) confirmed that even a short-term (21 days) DF intervention had a significant impact on human intestinal flora. However, different types of DFs regulate various types of microorgan- isms. So et al. (2018) reported that a DF-supplemented diet in healthy adults could significantly improve the abundance of Bifidobacteria and Lactobacillus and affect the bacterial structure and the levels of metabolites such as SCFAs. However, no significant difference in α-diversity was found.
Although the health effect of DF has been proven in many aspects, the monosaccharide compositions and polysaccharide structures of different DFs vary considerably, and the specific functions and clinical manifestations of each DF must be verified in a large number of experiments. The development of natural DF with different health effects is a hot topic (Desai et al., 2016). Ginseng (Panax ginseng C. A. Mey.) is popular in Asia and around the world because of its health benefits and is one of the most widely used medicinal plants. In recent years, people have continued to pursue new and healthy methods of ginseng consumption. Sliced or whole fresh food has become a recent trend, and ginseng DF has become an important ingredient in ginseng food products. At present, ginseng DF is mainly derived from ginseng roots and their cooking residue. The water-soluble dietary fiber (ginseng-SDF) content in the residue is approximately 10%, the molecular weight is approximately 30,000–200 Da, and the main monosaccharide component is glucose (Hua et al., 2020). Our previous study confirmed that ginseng-SDF had various antioxidant properties in vitro (DPPH, ABTS and FRAP), suggesting its potential health effect in vivo (Hua et al., 2020). In addition, we also found that certain probiotics, such as Lactobacillus plantarum and Streptococcus acidophilus, can be cultured in MRS medium with ginseng-SDF as the only carbon source (unpublished data), suggesting that ginseng-SDF might have a prebiotic effect. How- ever, the in vivo efficacy of ginseng-SDF remains unclear to date.
The purpose of this study was to explore the effects of ginseng-SDF on the growth performance, oxidative-immune levels and cecal health of healthy rats, reveal its potential health effects, and lay a foundation for its future application in food.
2. Materials and methods
2.1. Materials
Ginseng residue was obtained from boiled ginseng (1:10 w/v water, 2 h/time × twice), dried at 60 ◦C and ground with a medicinal material crusher (FW135, Taisite Instrument Co., Ltd, Tianjin, China). Ginseng- SDF was extracted according to the method of Hua et al. (2020), and its nutritional composition and ginsenoside content were determined.
2.2. Animal experimental design
Animal experiments were approved by the Laboratory Animal Management and Ethics Committee of the Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences (NO. ISAPSAEC-2020–021-01). The feeding conditions were set strictly in accordance with the national standards for the environment and facilities of laboratory animals (China, GB14925-2010). The experimental animals were SPF male SD rats (6 weeks old, 150.0 ± 5.0 g, qualification number of SYXK (Ji) 2018–0001), which were purchased from Chang-sheng Biotechnology Co., Ltd. (Liaoning, China). Bedding materials were also purchased from the same company. Drinking water was treated by high-temperature and high-pressure sterilization.
Thirty rats were raised in an SPF animal house (20–22 ◦C temperature and 50–55% humidity) with light conditions programmed for 12-h day/12-h night. The rats were provided a sufficient standard chow (Beijing Keao Xieli Feed Co., Ltd., Beijing, China) diet containing 24% calories from protein, 13% from fat, and 63% from carbohydrates (Supplementary Table S2) and drinking water, and their bedding was changed regularly. After 7 days of adaptation, the rats were randomly divided into 5 groups (n = 6/group), including the control group (physiological saline, K group), the low-dose group (200 mg/kg bw, SDFL group), the medium-dose group (400 mg/kg bw, SDFM group), the high-dose group (800 mg/kg bw, SDFH group), and the fructo- oligosaccharide (F8370, Solarbio Science & Technology Co., Ltd., Beijing, China) positive-control group (400 mg/kg bw, FOS group).
Every morning, 2 mL of physiological saline was given to the K group, and 2 mL of various doses of ginseng-SDF or FOS (dissolved in physiological saline solution) was given to the intervention groups for 15 days. During gavage, the general health status of the rats was observed, and weight and food intake were recorded.
2.3. Sample collection
Before the end of the experiment, rats were fasted for>12 h with unlimited drinking water. The next day, all rats were anesthetized with pentobarbital sodium (0.5–1.0 mg/kg bw). Weight and the body length were recorded. Blood samples were collected by heart puncture and then centrifuged at 4 ◦C and 3000 rpm for 15 min to collect the serum. Levels of serum antioxidant status indicators (Nanjing Jian-cheng Bioengineering Research Institute, Jiangsu, China), insulin-like growth factor (IGF)-1 and IGF-2, and immunoglobulins IgA, IgM, and IgG (Shanghai Enzyme-linked Biotechnology Co., Ltd., China) were tested following the manufacturer’s instructions. The liver, spleen and thymus of rats were weighed and stored at —80 ◦C. Cecal contents were collected in a 2.0-mL sterile tube and stored at —80 ◦C.
2.4. Content analysis of SCFAs
Next, 2.0 mL of precooled PBS solution (4 ◦C) was added to 0.4 g of cecal contents, and the mixtue was fully shaken and ground with 5–6 steel balls in a high-throughput Tissue Lyser (TissuePrep TP24, Tianjin, China) at low temperature (20 Hz/s 60 s × 3 times). Then, the samples were centrifuged at 13,000 rpm (4 ◦C) for 15 min, and the supernatant was stored at —20 ◦C.
SCFAs were detected according to the method of Inoue et al. (2019) by gas chromatography with slight modification. In short, a series of standard substances with a concentration gradient were prepared, including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid (Sinopharm Chemical Reagent Co, Ltd., Shanghai, China). Then, 1.0 mL of cecal content extract and standards were added to 0.2 mL of the internal standard 2-ethylbutyric acid (TE007025ML, Sinopharm Chemical Reagent Co, Ltd., in 25% meta- popic acid, final concentration 2.88 mmoL/L). Then, the samples were centrifuged at 13,000 rpm (4 ◦C) for 5 min. The supernatant was filtered through a 0.22-μm Millipore membrane and tested on an Agilent 7890A gas chromatograph system. The chromatographic separation was per- formed on a 30 m × 0.32 mm × 0.32 μm DB-FFAP capillary column with high-purity nitrogen carrier gas (1.5 mL/min), and the injection inlet temperature was 220 ◦C. A flame ionization detector was set to a temperature of 250 ◦C. For heating conditions, the initial temperature was 60 ◦C, which was increased to 180 ◦C at 10 ◦C/min.
2.5. Microbiome analysis based on 16S rRNA sequencing
Approximately 0.25 g of cecal contents was stored in dry ice and sent to Shanghai Personal Biotechnology Co., Ltd. for 16S rRNA detection of intestinal microbes. Microbial genomic DNA was extracted from the samples using the QIAamp DNA Fecal Mini Kit (No. 51504, QIAGEN China (Shanghai) Co., Ltd) according to the manufacturer’s instructions, and the quality inspection of DNA samples was performed by agarose gel electrophoresis. The 16S rRNA V3-V4 region was selected to design polymerase chain reaction amplification primers (forward primer: ACTCCTACGGGAGGCAGCA; reverse primer: GGACTACHVGGGTWTC- TAAT).
The Illumina NovaSeq platform was used for paired-end sequencing. The obtained sequences were denoised, merged and non- chimeric by the DADA 2 method of QIIME 2 software, and then, high- quality reads were divided into operational taxonomic units (OTUs) with 97% similarity. The OTUs were compared and annotated in the Greengenes database (Release 13.8, http://greengenes.secondgenome. com/). For taxonomic composition analysis, which was performed on a feature table after removing singletons, the composition distribution of each sample was visualized at the six classification levels of phylum, class, order, family, genus, and species. R language and the Venn Diagram package were used to draw Venn diagrams of common/unique OTUs between different groups. R language and the PheatMap package were used to calculate the clustering results of each sample and each taxon, and the results of the species composition heat map were presented in the form of an interactive graph. Correlations between the microbial community and oxidative-immune factors were analyzed by redundancy analysis (RDA), which was performed by Genescloud tools, a free online platform for data analysis (https://www.genescloud.cn) from Shanghai Personal Biotechnology Co., Ltd.
2.6. Statistical analysis
The values are presented as the mean ± SD. Statistical software SPSS 19.0 was used for one-way analysis of variance (ANOVA), and the LSD post-mortem test was used to compare the differences between groups. *P＜0.05 or Δ P < 0.05 were considered significant, **P < 0.01 or ΔΔ P < 0.01 were considered extremely significant. 3. Results 3.1. Effects of ginseng-SDF on the growth performance of rats During the 15-day experiment, all rats were in good condition and showed no abnormal behaviors. Compared with the K group, ginseng- SDF groups showed in a slight increase in the weight growth rate (P > 0.05) and a significant increase in feed intake (P < 0.05 and P < 0.01). Moreover, Lee’s index in the SDFH group was significantly increased (P < 0.01). Notably, FOS also promoted the growth performance of rats and significantly increased their weight growth rate and feed intake (P < 0.01). However, ginseng-SDF had no significant effects on rat liver, spleen or thymus indexes (P > 0.05).
3.2. Effects of ginseng-SDF on the antioxidant status of rats
Free radicals can be generated in the course of normal life activities and due to diet, and their excessive accumulation can cause body damage through various mechanisms. Therefore, maintaining a normal oxidative stress (OS) level is particularly important for health (Davalli, Marverti, Lauriola, & D’Arca, 2018). Compared with the K group, ginseng-SDF groups showed significantly reduced serum malondialdehyde (MDA) levels (P < 0.01, Fig. 1b) and significantly increased serum glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) levels (P < 0.01, Fig. 1c and d). In addition, the effect of SDFM was significantly stronger than that of the same dose of FOS (P < 0.01. In this study, the effect of ginseng-SDF appeared to have a negative correlation with the dose level, and ginseng-SDF at a concentration of 200 mg/kg had the best effect on OS. 3.3. Effects of ginseng-SDF on growth- and immune-related factors in rats Ginseng is currently one of the most widely studied and promising plant immune supplements. Ginseng can enhance active and passive immunity levels in the host and can be used as a vaccine adjuvant to fight a variety of infections and autoimmune diseases (Ratan et al., 2021). IGF in the human body plays an important role in normal cell growth, proliferation, differentiation and apoptosis. In recent years, IGF has also been found to be involved in the occurrence and development of a variety of tumors (Xu et al., 2017). Immunoglobulin is closely related to innate immunity and the proliferation and migration of tumor cells (Sun et al., 2018). In this study, the SDFH group had significantly increased IGF-1 (P < 0.05), IGF-2, IgM, and IgG (P < 0.01) contents compared with the K group. The effects of SDFM group on IGF-2 (P < 0.01), IgA (P < 0.05) and IgG (P < 0.05) were significantly stronger than those of the same dose of FOS group. 3.4. Effects of ginseng-SDF on cecal health in rats 3.4.1. Effects of ginseng-SDF on the quality of cecal contents The large intestine is the main fermentation site of DF, and the cecal environment is especially affected by this fermentation process. Compared with those in the K group, the wet weight and moisture content of cecal contents in the SDFL, SDFM and FOS groups were significantly increased (Table 1). The effect of SDFM group on wet weight was stronger than that of the same dose of FOS group (P < 0.05). In addition, the pH values of the cecal contents in the SDFM (P < 0.05) and SDFH (P < 0.01) groups decreased significantly, suggesting that drastic metabolism might have occurred. 3.4.2. Effects of ginseng-SDF on cecal SCFAs SCFAs are the main metabolites of DF in the intestine. They are not only important factors for maintaining the energy balance but also signal molecules and metabolic regulators that affect host health (Bander, Nitert, Mousa, & Naderpoor, 2020). In this study, the total SCFA contents in the SDFH group were significantly increased compared with those in the K group (P < 0.01, Fig. 3a). Ginseng-SDF changed not only the contents of SCFAs but also their proportions (Fig. 3b–e). The SDFH group showed significantly altered proportions of acetic acid and butyric acid. The former’s proportion decreased from 64.04% in the K group to 55.37% (P < 0.01), and the latter’s proportion increased from 12.36% in the K group to 40.04% (P < 0.01). Moreover, the proportions of propionic acid, isobutyric acid, valeric acid and isovaleric acid decreased significantly (P < 0.01) in the SDFH group. According to the results above, ginseng-SDF mainly promoted the production of butyric acid among cecal SCFAs, suggesting an important prebiotics properties. 3.4.3. Effects of ginseng-SDF on the cecal microflora We analyzed structural changes in the intestinal flora to further characterize the effect of ginseng-SDF on cecal health. By sequencing the V3-V4 regions from 16S rRNA of intestinal flora, 430,693 (K group), 437,776 (SDFL group), 401,207 (SDFM group), 426,086 (SDFH group) and 295,316 (FOS group) high-quality sequences were obtained, and the diversity of each group was analyzed. The results showed that ginseng- SDF had no significant effect on α-diversity compared with the K group (P > 0.05), but FOS group significantly decreased the Chao1 and observed species indexes (P < 0.05) and showed a significant difference compared with the same dose of SDFM group (P < 0.01). Principal coordinate analysis (PCoA) of each sample can clearly show the differences between groups. As shown in Fig. 4b, community dif- ferences began to appear between the SDFL and SDFM groups compared with the K group. The SDFH and FOS groups were completely separated from the K group and no longer overlapped with each other. In addition, the SDFM group was completely separated from the FOS group and presented significant differences with the latter. Bacteroidetes or the ratio of Firmicutes/Bacteroidetes (F/B). The FOS group had a significantly increased F/B ratio, and its effects on Firmi- cutes and Bacteroidetes were significantly stronger than those of the same dose of SDFM group (P < 0.01). Ginseng-SDF increased the abundance of Lactobacillus (P < 0.01), Ruminococci (P < 0.01), Clostridiaceae_Clostridium and Coprococcus and reduced the abundance of Oscillospira and Desulfovibrio (P < 0.01), suggesting that ginseng-SDF showed prebiotic properties and promoted the proliferation of cellulose-decomposing bacteria. Effects of ginseng-SDF on different characteristics of cecal microflora The Venn diagram (Fig. 5a) shows the characteristic OTUs in each group: 335 OTUs were shared in all groups, accounting for 3.71–5.55% in the ginseng-SDF groups, 9.48% in the FOS group and 3.47% in the K group. The unique OTUs of the FOS group were fewer than those of the K group, which is consistent with the decline in its Chao1 index in Fig. 4a. The composition heat map showed that compared with those in the K group, ginseng-SDF reduced the abundance of Bacteroidetes, Elusimicrobia, Verrucomicrobia, Proteobacteria, and Fusobacteria while increasing the abundance of Chlorobi, Spirochaetes, and Deferri- bacteres to different degrees. The SDFL group had an increased abun- dance of Coprococcus, Paraprevotella, Prevotella, and Ruminococcus. The SDFM group had an increased abundance of Turicibacter, Lactobacillus, Treponema, and Bifidobacterium. The SDFH group had an increased abundance of Lactobacillus, Treponema, Bifidobacterium, Clostridium and Acinetobacter. Fig. 5b shows the evolutionary relationships of the predicted biomarkers in each group. The biomarker in the ginseng-SDF groups changed from Bacteroidetes (such as Paraprevotella in the SDFL group and Turicibacteraceae in the SDFM group) to Firmicutes (such as Clostridium and Ruminococcus in the SDFH group). The random forest model is especially applicable for calculating the community relationships that are relatively discrete and discontinuously distributed to obtain a more robust and accurate classification (Breiman, 2001). As shown in Fig. 5e, high-content Clostridium was the most important representative strain of the SDFH group, which was different from the K group. While high-content Bifidobacterium was the most important representative strain of the SDFM group. Ginseng-SDF reduced the abundance of Bilophila, Prevotella and Shigella, which was also the major factor resulting in significant differences from the K group. These results were consistent with the composition heat map and LEfSe analyses, suggesting that ginseng-SDF resulted in enrichment of probiotics such as Lactobacillus, Bifidobacterium, and Coprococcus and cellulose-decomposing bacteria such as Clostridium and Ruminococcus in the cecal microflora. Moreover, RDA of cecal microflora and physio- logical indicators in the K and SDFH groups was conducted (Fig. 5f and g). The results showed that Lactobacillus, Clostridium and Ruminococcus in the SDFH group were positively correlated with high levels of acetic acid, butyric acid, weight gain, feed intake and antioxidation indexes such as superoxide dismutase (SOD), GSH-Px, and T-AOC. The above bacteria were negatively correlated with propionic acid, valeric acid and MDA levels. The reducing Oscillospira in the SDFH group was positively correlated with the low propanoic acid, valeric acid and MDA levels (Fig. 5f). The significantly increasing Lactobacillus, Clostridium and Coprococcus in the SDFH group were positively correlated with the high IGF-1, IGF-2, IgA, IgM and IgG levels. The above results suggested the possible role of ginseng-SDF in regulating the physiological indexes of rats through the intestinal flora. 4. Discussion 4.1. Effects of ginseng-SDF on growth promotion of rats In this study, ginseng-SDF promoted weight gain and feed intake in rats. At the same time, the Firmicutes content in the cecal microflora increased significantly. Firmicutes is one of the most important members of the human intestinal flora and is mainly responsible for absorbing energy from the diet (Almeida et al., 2019). Studies have shown that intestinal Firmicutes levels increased in overweight men following the consumption of a high-resistance starch diet (Walker et al., 2011). The intestinal flora of obese animals and human individuals generally showed an increased F/B ratio. The results of this study suggested that the increase in Firmicutes may be one of the mechanisms through which ginseng-SDF helps the host absorb more energy from food, produce more metabolites and gain weight. Notably, FOS had similar effects on body weight and the Firmicutes content in cecal microflora to those of ginseng-SDF in this study. Lin et al. (2015) found that DF may also be a factor influencing obesity. In their study of approximately 1,804 adolescents, total DF intake was directly proportional to the body fat rate; for every 1-kcal/day increase in total DF, the body fat rate increases by 0.355, which may explain the effects of ginseng-SDF and FOS on 6-week-old rats in the exuberant growth stage in this study. Notably, although dietary adjustments can cause rapid changes in the structure of the human intestinal flora within 24 h, the formation of a stable flora structure requires long-term (>12 weeks) dietary interventions (John et al., 2018). The changes in the intestinal flora in this study could be regarded as short-term intervention results of low-dose ginseng-SDF administration in healthy rats (SDF = 2.0 ~ 8.0 g/ 60 kg/day for humans). In summary, the results of this study showed that a ginseng-SDF diet intervention for 15 days resulted in structural changes in the intestinal flora in rats, with enhanced growth performance and increased energy absorption.
4.2. Effects of ginseng-SDF on antioxidant status and immune factor levels of rats
The number of hydroxyl and carboxyl groups in a polysaccharide structure has an important influence on the polysaccharide’s antioxidant activity (Zheng, Tian, Li, Wang, & Shi, 2021). Our previous study confirmed the presence of carboxyl groups and plenty of hydroxyl groups in ginseng-SDF, conferring a good antioxidant capacity in vitro (Hua et al., 2020), which should also be the structural basis for the in vivo antioxidant effect of ginseng-SDF in this study. Studies have shown that long-term intake of ginseng extract can change the secretion levels of cytokines, such as IL-4 and IL-10, and immunoglobulins in rats, affecting B cell proliferation. This change results in an increase in pro- biotics, such as Lactobacillus, which promotes the secretion of IgA (Sun et al., 2018).
This study confirmed that ginseng-SDF also produced effects similar to those of ginseng extract on immune factors in rats, suggesting that ginseng might have various components involved in immune regulation. This finding is also consistent with the idea in traditional Chinese medicine that ginseng “strengthens the body”, suggesting the health value of ginseng-SDF. In addition, the regulation of intestinal flora by ginseng-SDF might also affect the bidirectional communication between the gut and the immune system (Fung, 2020). Immune cells can regulate the abundance of intestinal flora, while the metabolites of the intestinal flora can in turn affect the secretion of immune cells (Yu, Jia, Li, Bi, & Liu, 2018). Therefore, the immune effects of ginseng-SDF could be impacted by its regulation of intestinal flora, which is mainly achieved by intestinal metabolites such as SCFAs.
4.3. Effects of ginseng-SDF on cecal health in rats
At present, the effect of DF on the intestinal flora of healthy and diseased individuals is a research hotspot (Makki, Deehan, Walter, & Backhed, 2018; Koh, De Vadder, Kovatcheva-Datchary, & Backhed, 2016). On the one hand, the intestinal flora can produce harmful me- tabolites related to intestinal barrier dysfunction and disease occurrence; on the other hand, it can produce beneficial metabolites with anti- inflammatory and intestinal barrier-maintaining functions to affect host health and disease. Therefore, the balance of the intestinal flora struc- ture is considered an important factor affecting host health, and diet is the main factor determining the structure of the intestinal flora (Koh, De Vadder, Kovatcheva-Datchary, & Backhed, 2016). This study found that ginseng-SDF increased the abundance of Lactobacillus, Ruminococcaceae_Ruminococcus and Clostridiaceae_Clostridium in the cecal microflora. Clostridium is considered one of the most important families of butyrate- producing bacteria (Leylabadlo et al., 2020). The RDA results of the SDFH group showed that Clostridium was positively correlated with acetic acid and butyric acid contents (Fig. 5f). This flora regulation effect suggested that ginseng-SDF has prebiotic properties. In addition, the more important prebiotic effect of ginseng-SDF was reflected in the promotion of Lactobacillus proliferation. Lactobacillus in the SDFH group was positively correlated with the acetic acid, butyric acid, SOD, GSH-Px and other physiological indexes (Fig. 5f). Similar effects were obtained in a study exploring the effects of Panax ginseng polysaccharide (WGP) on intestinal flora regulation in mice (Qi et al., 2019). WGP (similar to ginseng-SDF and mainly composed of neutral glucan) can significantly change the intestinal flora structure of mice with antibiotic-associated diarrhea, increase the abundance of Lactobacillus, and correct carbohydrate, amino acid and energy metabolism to normal levels and has a repair effect on intestinal mucosal injury caused by diarrhea (Qi et al., 2019). Sun et al. (2018) also proved that long-term intake of ginseng extract can significantly increase the abundance of Lactobacillus and Bifidobacterium in the intestinal flora of rats, improve the level of immunoglobulin IgA, and enhance intestinal immune function. In conclusion, ginseng-SDF is an important prebiotic component in ginseng that promotes probiotic proliferation.
In addition to affecting the flora structure, the effect of ginseng-SDF on cecal health promotion is further manifested by improvements in the quality of cecal contents. The increase in the wet weight of cecal con- tents was consistent with the increase in feed intake. The increase in the moisture level of the cecal contents was related to the water-holding property of ginseng-SDF (Hua et al., 2020). More importantly, ginseng-SDF effectively increased the cecal butyric acid content. Butyric acid is the most efficient SCFA, and 75% of the oxygen consumed by colonic epithelial cells originates from butyrate oxidation. Butyric acid is mainly involved in gluconeogenesis, ketone body generation and tri- glyceride synthesis and has the functions of promoting colon cell pro- liferation, maintaining intestinal mucosal integrity, and fighting colitis and colorectal cancer (Bultman, 2017; Liu et al., 2020). In recent years, numerous studies have been conducted on the health effects and mechanisms of SCFAs (Michaudel and Sokol, 2020; Correa-Oliveira, Fachi, Vieira, Sato, & Vinolo, 2016). Butyric acid affects host immunity and inflammation through two main mechanisms. One mechanism is inhibition of histone acetylase (HDAC) activity, where butyric acid acts as a direct inhibitor, which increases histone acetylation and then reg- ulates gene transcription and the release of proinflammatory cytokines (Yang et al., 2020). The other mechanism is regulation of both the development and function of intestinal epithelial cells and white blood cells through activation of G protein-coupled receptors (GPCRs, Ffar2, Ffar3, Gpr109a and Olfr78) and the activity of enzymes and transcrip- tion factors to establish a connection between the intestinal flora and the host immune system (Corrˆea-Oliveira, Fachi, Vieira, Sato, & Vinolo, 2016; Michaudel and Sokol, 2020).
In animal and human studies, butyric acid has been confirmed to inhibite activation of nuclear transcription factor kappa B (NF-κB) and the degradation of NF-κB inhibitory protein α (IκBα), thereby reducing the expression of proinflammatory factors and exerting therapeutic effects on ulcerative colitis (Cryan et al., 2019). Thus, the effect of ginseng-SDF on butyric acid suggests that it might play a role in the prevention and treatment of intestinal diseases. Last but not the least, the effect of ginseng-SDF on promoting cecal health is also manifested in regulation of the antioxidant status in rats through intestinal probiotics. In this study, ginseng-SDF increased the proportions of Lactobacillus and Bifidobacterium in the cecal microflora, and the antioxidant status and immunoglobulin secretion levels in rats were positively related to the promotion of probiotics such as Lactobacillus. Lactobacillus and Bifidobacterium are the most widely studied probiotics and have the most explicit functions. They can improve host glucose and lipid metabolism, inhibit gastrointestinal infection and tumor cell proliferation, and reduce the occurrence and development of enteritis and colorectal cancer (Nowak, Paliwoda, & Błasiak, 2019; Kim et al., 2020). Reactive oxygen species (ROS) have been recognized as important carcinogens.
The body’s antioxidation mechanism includes enzymatic reaction systems such as SOD, glutathione peroxidase (GPx), glutathione reductase (GR) and peroxidase, as well as nonenzymatic antioxidant systems in cells such as vitamin E, vitamin C and glutathione (GSH) (Zerrouki and Benkaci-Ali, 2018). The most attractive properties of Lactobacillus and Bifidobacterium involve their anticancer potential, which is closely related to their antioxidant activities (Nowak, Paliwoda, & Błasiak, 2019). Lactobacillus and Bifido- bacterium have been reported to enhance antioxidant enzyme activity or regulate the oxidative stress cycle to protect cells from carcinogenic attack by ROS (Kong, Olejar, On, & Chelikani, 2020). Lactobacillus and Bifidobacterium can secrete SOD, catalyze the disproportionation of the promoter of the free radical reaction (O—•), and reduce the generation of free radical metabolites (such as MDA) (Mishra et al., 2015). They also have a complete GSH system for glutathione synthesis and transport.
As a major cellular nonenzyme antioxidant, GSH can protect cell mem- branes and other tissues from peroxidation damage by cooperating with selenium-dependent GSH-Px (Zerrouki and Benkaci-Ali, 2018). In addition, Lactobacillus and Bifidobacterium can hydrolyze proteins in food to produce active antioxidant peptides and can prevent lipid per- oxidation of cell membranes. They bond with ROS, which are formed during food digestion, and produce antioxidant molecules through their own anabolism (such as exopolysaccharides) (Kim et al., 2020). Finally, the antioxidant effects of Lactobacillus and Bifidobacterium are also re- flected in their participation in restoring the intestinal microecology after ROS-induced disturbances (Feng and Wang, 2020). Our study suggested that ginseng-SDF might be a health-promoting prebiotic whose health effects include antioxidation, immunity-enhancing and intestinal health-promoting properties.
5. Conclusion
In this study, the in vivo effects of ginseng-SDF were explored in terms growth performance, serum antioxidant status, immune factor levels and cecal health. The results showed that a ginseng-SDF diet intervention in healthy rats for 15 days can enhance weight gain, feed intake and Lee’s index, significantly improve serum antioxidant status and immunoglobulin levels, and increase cecal SCFA concentrations, especially butyric acid. Ginseng-SDF also altered the cecal microflora structure by increasing the abundance of Firmicutes, Lactobacillus, Ruminococcus and Clostridium and reducing the abundance of Oscillospira and Desulfovibrio, thereby improving cecal health.
In the SDFH group, these proliferating microflora were positively correlated with growth, immune factors and antioxidant status indexes, such as SOD, GSH-Px, T-AOC, and cecal acetic acid and butyric acid concentrations. In addition, Sodium butyrate the reduction in Oscillospira was positively correlated with serum MDA and cecal propionic acid and valeric acid concentrations. The above results suggested that ginseng-SDF has prebiotic properties and plays beneficial roles in antioxidant status, immunity improvement and cecal health promotion. In conclusion, our study supports the trend of chewing fresh and whole- stick ginseng, which can increase the intake of ginseng-SDF.