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Effects of Aqueous Extracts and Essential Oil from Cinnamomum cassia on Rat Colonic Mucosal Morphology and Bacteroidales

Shaoting Li, Cong Wang, Xichun Peng*, Xiyang Wu

Department of Food Science and Engineering, Jinan University, Guangzhou, China

Abstract: Recently, the preventive effects of Cinnamomum cassia on several metabolic diseases, e.g. obesity, diabetes, hypertension, have been reported. However, no literature reported on the interaction of phytochemicals from C. cassia and colonic microecology.This study aimed to explore the impact of essential oil and aqueous extracts of C. cassia on rat colonic mucosal morphology and microbiota. After 4-week feed, the rats administrated with aqueous extracts of C. cassia (AEC) resulted in less body-weight gain (45.33g, P<0.01) and those administrated with essential oil of C. cassia (EOC) had no difference in weight gain compared with controls (85.33g vs. 87.67g, P>0.05). AEC rather than EOC decreased villous area and crypt count of rat colonic mucosa significantly (P<0.01). According to the TRFLP analysis, both of the AEC and EOC increased total peak areas of Bacteroidales (P<0.05), as well as changed terminal restriction fragments in different degrees (mainly 76 bp and 614 bp, P<0.01). AEC altered the tested rats’ colonic mucosa and community structure of probably obesity-associated gut microbiota (Bacteroidales). This change potentially resulted in a decreased energy harvest from diet as the rats gained less body weight. Despite EOC up-regulated the total amount and diversity of colon Bacteroidales, its influence on weight gain had scarcely been detected.

Keywords: cinnamomum cassia, essential oil, aqueous extracts, colon, mucosal morphology, bacteroidales

Received May 08, 2013; Revised May 17, 2013; Accepted May 18, 2013

Citation: Shaoting Li, Cong Wang, Xichun Peng, Xiyang Wu, Effects of Aqueous Extracts and Essential Oil from Cinnamomum cassia on Rat Colonic Mucosal Morphology and Bacteroidales. Journal of Food and Nutrition Research, 1 (2). 7-12. January 2013. doi:10.12691/jfnr-1-2-1

1. Introduction

Cinnamomum cassia, also known as Chinese Cinnamon, is commonly used as spice in Chinese cooking due to its specific sweetness and health function [1]. It has also been shown that C. cassia possesses a wide variety of biological functions, including anti-oxidant [2], anti-inflammation [3], anti-tumor activity [4], and especially antimicrobial [5] and anti-diabetic effects [6]. Thus it has been used in Chinese medicine for treating dyspepsia, gastritis, blood circulation disturbances, and inflammatory diseases [7]. Aqueous extracts of C. cassia (AEC) contains various aromatics, diterpenes, polyphenols (e.g. anthocyan, flavonoid and tannin) [7,8], mucus and carbohydrates [9]; while essential oil of cinnamon (EOC) mainly contains cinnamaldehyde (60%-80%) [10,11], cinnamic acid, cinnamyl alcohol, and coumarin [12].

Studies have suggested that 80%-90% of dietary polyphenols in AEC are not absorbed in small intestine. Instead, they directly reach the large intestine and are metabolized by gut microbiota [13]. The main bioactive components in EOC will be rapidly absorbed from the gut [14]. Numbers of evidences have demonstrated the interaction of phytochemicals and gut microbiota. The gut microbiota has been shown to be essential for the transformation of some phytochemicals e.g. polyphenols [13]. On the other hand, phytochemicals and their derived products can also affect the composition of the gut microbiota [15]. Furthermore, the intestinal bacteria were associated with the colonic mucosal architecture according to the finding that the fructans-containing diet increased colonic crypt depth in bacteria-associated rats [16]. However, No literature reported on the impacts of phytochemicals from C. cassia on colonic morphology and microbiota changes.

The human gut is populated by a vast number of bacterial species (more than 800) that reach the highest concentrations in the colon (up to 10¹² cells per gram faeces) [15]. 98% of these bacteria belonged to phylum Bacteroidetes and Firmicutes [17]. Moreover, evidence suggested that the relative abundance of the two predominant bacterial divisions differs between lean and obese animals: mice that are genetically obese have fewer amount of Bacteroidetes and more Firmicutes than their lean siblings [18]. Furthermore, Bacteroidales is one of the most dominant groups in Bacteroidetes. It has been shown that Bacteroidales have significant impacts on human health, involving in carbohydrate fermentation and polysaccharides catabolism [19].

The aim of the study is to investigate the impacts of AEC and EOC of C. cassia on rat colonic mucosal morphology and Bacteroidales of colon microbiota by morphological observation and terminal restriction fragment length polymorphism (TRFLP) analysis.

2. Experimental

2.1. Preparation of AEC and EOC

C. cassia that obtained from Er Tiantang Co. (Guangzhou, China) were shattered and meshed with 10 meshes. AEC was prepared as follows: C. cassia powder was added with 10-fold volumes of distilled water and boiled for 20min at 100°C, followed by filtering. The residue was subsequently added with 4-fold volumes of distilled water and continued to be boiled for 20 min at 100°C. All of the filtrates was collected and mixed together, concentrated to 2g/mL and then kept at 4°C for further use. EOC was prepared by using hydrodistillation in a Clevenger-type apparatus, according to the method described previously [20] with modification: 50g of cinnamon bark powder was mixed with 200mL of distilled water and then heated at 100°C for 3h. The volatile essential oil was collected and stored at 4°C for further use.

2.2. Animals and Diets

Eight week-old healthy male Sprague-Dawley (SD) rats were obtained from the Animal Center of Southern Medical University (Guangzhou, China). The average weight of rats was 200g ± 20g. After one adaptive week, all animals were randomly classified into 3 groups (10 rats per group): Control group, which was fed with normal diet; AEC group, which was treated with 2mL of aqueous extracts of C. cassia, equivalent to about 4.0g raw C. cassia bark per kg body weight; EOC group, which was treated with 0.1mL essential oil of C. cassia, equivalent to about 0.38g essential oil per kg body weight; The two tested groups were fed with normal diet, too. During the 30 days of feeding, the environment was maintained at a temperature of 23 ± 2°C and relative humidity was 50% to 60%, with a light/dark cycle of 12h. Padding was renewed three times a week, and the rats had free access to food and water during the experiment. The animal experiments were approved by the Department of Care and Use of Laboratory Animal in Jinan University (Guangzhou, China).

2.3. Sampling Collection

After 30 days of feeding, 3 rats were randomly selected from each group and etherized after weighed. The colons of all rats were removed aseptically from the abdominal cavity and dissected free from fat and mesentery. The colon was sampled at 1cm proximal to the ileal valve. Their colonic contents were individually sampled and stored at -20°C for microbiota analysis. Then the colon tissue was fixed immediately with 10% formalin, then embedded with paraffin, sectioned and stained with haematoxylin and eosin for further morphology analysis.

2.4. Colonic Mucosal Analysis

Microstructure analysis was conducted with a light microscope (OLYMPUS X51, Olympus Co., Tokyo, Japan) at 40× and 100× magnification. Computerized morphometric measurements were made of the following: villous height, villous area, mucosal thickness, crypt count, crypt depth, fold thickness were carried out on random and in double blind method by using the image analyzer (DP2-BSW, Olympus Co.).

2.5. Total DNA Extraction and Purification of Colonic Microbe

The bacterial DNA of each sample was extracted by Stool DNA-out kit (AndyBio, Itasca, IL, USA) according to manufacturer’s instructions. The total DNA samples were characterized with 1% agarose gel electrophoresis for integrity and size. The DNA was adjusted to 40ng/μL and stored at -20°C before used as templates for PCR.

2.6. TRFLP Analysis

Bacteroidales specific 16S rRNA genes was amplified by the primers (AACGCTAGCTACAGGCTT) which is labeled with FAM, and 708r (CAATCGGAGTTCTTCGTG) [21]. Amplification was performed using an Eppendorf MastercyclerTM thermal cycler (Hamburg, Germany) with the following program: an initial 3 min denaturation at 94°C followed by 30 cycles of 45s denaturation at 94°C, 30s annealing at 56°C, and 2 min extension at 72°C. After the final cycle, 3 min elongating at 72°C completed the PCR. The following reaction cocktail was used: 20ng template DNA; 0.025U/μL Taq DNA polymerase (TaKaRa, Otsu, Shiga, Japan); 10× reaction buffer (Mg²⁺ plus) as supplied by manufacturer; 0.25mM of each primer (synthesized by Shanghai Sangong, China); and 0.2mM of each of the dNTPs (TaKaRa). The reactions were carried out in a final volume of 50μL.

PCR products were further purified by the NewProbe PCR purification kit (New Probe, Beijing, China) using the manufacturer’s protocol. The PCR cleanup products were quantified by determining absorption of samples at 260nm. An enzyme digestion was performed on each PCR product using Hha I (TaKaRa). Each 40μL digestion used 75ng DNA, 1 U enzyme and 4μL 10× buffer (0.4μL 20μg/mL BSA were added to Hha I digestions). Samples were digested for 4h at 37°C and inactivated for 20 min at 65°C. TRFLP was performed by Invitrogen (Carlsbad, NM, USA).

2.7. Data Analysis of TRFLP Profiles

Terminal restriction fragments (TRFs) smaller than 50 bp were excluded from the further analysis. Fragments differing by less than 1 bp length were clustered. Relative peak areas of each TRF were determined by dividing the area of the peak of interest by the total area of peaks within the following threshold: a lower threshold at 50 bp and an upper threshold at 600 bp. Only TRFs with relative abundances above 0.5% were included in the remaining analyses. Putative identities for the most dominant peaks were predicted by in silico digestion with Hha I in the Ribosomal Database Project II (http://rdp.cme.msu.edu) using the TRFLP analysis program TAP.

Replicate profiles from separate DNA extractions and PCR reactions for each sample were compared to identify the subset of reproducible fragment sizes. The average area of each reproducible peak was calculated. The standardized binning criteria used to identify the subset of reproducible peaks were as previously described [22,23]. Ribotype richness (S) was calculated according to the total number of distinct fragments in each sample. The Shannon diversity index (H) and evenness (E) were calculated according to the methods described previously [23,24]. Jaccard similarity index was calculated using EXCEL to evaluate the similarity for microcosm samples between two groups [25]. Similarity percentage (SIMPER) analysis was further used to identify the fragments that were mainly responsible for the dissimilarity between samples [26,27].

2.8. Statistics

Results are expressed as mean values with their standard deviation (SD). Statistical analyses were conducted with the Statistical Package for Social Science (SPSS for Windows, version 8.0; SPSS Inc., Chicago, IL, USA) to determine if variables differed among treatment groups.

3. Results

There were no mortalities during the experiment and no periods of weight loss or diarrhea observed throughout the study. Moreover, no organ of the rats was found morbid after dissection. The AEC group resulted in less body-weight gain (45.33g, P<0.01) after 4 weeks feed intake, the EOC group had no difference in weight gain compared with controls (85.33g vs. 87.67g, P>0.05) (Figure 1). Additionally, there was no difference in food intake among groups during the experiments.

Figure 1. SD rats’ body-weight gain of the control, AEC and EOC groups. Values are expressed as means ± SD. ** P<0.01, compared to the control group

Table 1. The colon mucosal morphometry of rat under light microscope at 40× magnification

.

Figure 2. The pictures of rat colonic mucosa. These morphological pictures were captured with the aid of a light microscope. “A” and “B” are used respectively to refer to the pictures amplified 40× and 100×

The morphological pictures of rat colonic mucosa were presented as followed (Figure 2). The crypt count and villous area of the AEC group were significantly lowered (P<0.01) than EOC and control groups. The other parameters (e.g. villous height and mucosal thickness) were also reduced with no significance (P>0.05). The crypt count, crypt depth and fold thickness in EOC group were increased comparing to control group (P<0.05) (Table 1).

Figure 3. Total peak areas of colonic Bacteroides from the control, AEC and EOC groups. Values are expressed as means ± SD. * P<0.05, compared to the control group

After treated with EOC and AEC, both of their total peak areas of Bacteroidales significantly increased (P<0.05) (Figure 3). The total average numbers of terminal restriction fragments (Hha I digest) in the three groups were all 6. Shannon diversity index and evenness of Bacteroidales were individually calculated and presented in Table 2. The value of H and E of the AEC group was significantly higher than those of the control and EOC group, which showed that the diversity of Bacteroidales significantly was improved in the AEC group.

Table 2. Comparison of diversity and evenness indices for the TRFLP profiles (Hha I digest) of colonic Bacteroides from different groups

The Jaccard index of similarity of Bacteroidales was quite low in colon. The similarity of the AEC group was about 32.56%, slightly lower than that of the EOC group (34.68%). The low Jaccard similarity reflected a big individual variation of community structure of Bacteroidales.

Figure 4. Terminal restriction fragments (Hha I digest) and mean relative abundance (%) of colonic Bacteroides from the control, AEC and EOC groups. Values are expressed as means ± SD. ** P<0.01, compared to the control group

Table 3. Results of SIMPER analysis of TRFLP profiles from different groups

Rat colons of both the two tested groups had different Bacteroidales community structure from those of the control group, according to the TRFLP profiles (Hha I digest) of detectable Bacteroidales (Figure 4). The SIMPER analysis revealed that the differences of TRFLP profiles (Hha I digest) between the control group and the two tested groups were almost driven by the variation in fragments of 76 bp and 614 bp (Table 3). Both EOC and AEC group had a tendency to lower down fragments of 614 bp (P<0.01). On the contrary, the EOC and AEC both increased the relative abundance of 76 bp (P<0.01). In addition, the fragments of 70 bp, 72 bp, 74 bp and 89 bp were changed by aqueous extracts and essential oil without significant difference (P>0.05) (Figure 4).

4. Discussion

The histologic appearance of the colonic mucosa revealed no inflammatory changes such as ulcerations, crypt abscesses, changes in the lamina propia, or alterations in the surface epithelium and crypt epithelium. Morphometric measurements of colonic mucosa (Table 1) showed that AEC diet significantly decreased the measurements of the villous area and the crypt count compared with the controlled.

The rats fed with AEC were prominently leaner than the controlled. This may be explained by the inhibitory effects of phenolic compounds against obesity [28].The bioactive cassia-derived phytochemicals and metabolites could partially accumulate in the contents of rat colon. It has been verified that phytochemicals and their derived products can affect the intestinal ecology as a majority of them can’t be fully absorbed and metabolized by the small intestine and the liver, but accumulated in the ileal and colorectal lumen [15]. Therefore, we examined the effects of EOC and AEC on the colonic Bacteroidales community.

Lee and Ahn [29] had reported that C. cassia bark-derived cinnamaldehyde revealed significant inhibition in vitro against specific human intestinal bacteria. However, our study reports the impacts of C. cassia extracts on gut microbiota in vivo. After 30-day feeding, the amount of colon Bacteriodales in both of the test groups increased (Figure 3) and the composition of colon Bacteriodales changed as well (Figure 4). This change may be due to the effects of the phytochemicals and metabolites. Unabsorbed dietary phenolics and their metabolites have been shown to exert varying degrees of bacteriostatic activities on different strains of intestine. Growth of certain pathogenic bacteria such as Clostridium perfringens, Clostridium difficile and Bacteroides spp. was significantly repressed by tea phenolics, while commensal anaerobes like Clostridium spp., Bifidobacterium spp. and probiotics such as Lactobacillus spp. were less severely affected [30].

According to the TRFLP profiles (Hha I digest) of detectable Bacteroidales (Figure 4), there showed a considerable variation of the terminal restriction fragments among samples. The influence of the two kinds of extracts differed from each other. Two possible explanations can be used to account for this response. Firstly, there do exist a substantial uniqueness in the gut bacterial community among individuals, which has been verified in several others studies [17,18]. We had tried to reduce the impacts came from these individual differences by increasing the repetitions in this study despite it was inevitable. Secondly, the phytochemicals from plant extracts may have inhibitory or promoting effects on different species of bacteria [15]. Given that the components of EOC and AEC differ from each other, it is not hard to understand that one of them would inhibit growth of some species of Bacteroidales while stimulate the others. Additionally, the following fragments contributed the main drive to the variation of colon Bacteroidales: 76 and 614 bp (Table 3). These fragments deserve to be cloned and identified for further study. It was somehow regretful that only Hha I was available to hydrolyze the PCR products well when we tried three restriction enzymes (Hha I, Alu I, and Hind III) to obtain TRFs, which might cause underestimation of microbial diversity.

Finally, the reason we focused on the effects of extracts from C. cassia on Bacteroidales was because C. cassia has been tested for its anti-obesity properties in humans and rats [31,32]. Bacteroidetes was also widely reported its association with obesity [17,18]. In our study, the AEC was observed to be functional in upgrading the amount of rat colonic Bacteroidales, down-regulating the several parameters of colon mucosa, and decreasing the rats’ body-weight gain. This experimental evidence implied that AEC could change both rat colonic mucosa and obesity-associated gut microbiota (Bacteroidales). This alteration potentially caused a decreased capacity for energy harvest from the diet as the rats gained less body weight. Despite essential oil also up-regulated the total amount and diversity of colon Bacteroidales, its influence on weight gain had scarcely been detected.

In conclusion, the paper reported the effect of AEC and EOC on rat colonic mucosal morphology and Bacteroidales. It was found that AEC rather than EOC can change both rat colonic mucosa and probably obesity-associated gut microbiome, which might cause a decreased capacity for energy harvest from the diet, as less body-weight gain was observed in AEC group. Despite essential oil also up-regulated the total amount and diversity of colon Bacteroidales, its influence on weight gain had scarcely been detected. However, the abuse of C. cassia might have negative effect on human health based on the changes of rat colonic mucosa by the treatment of AEC.

Acknowledgments

The program was supported by the funds of the National Natural Science Funds (No. 31271849), National Innovation Experiment Program for University Students (No. 101055902) and the Fundamental Research Funds for the Central Universities (No. 21611414). We thank Honglin Zhan, Kaijie Song, Lizhu Huang and Zhichao Zhang from the Department of Food Science and Engineering, Jinan University, for their contribution to the experiments of this study.

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