Edible Lily Bulb Modulates Colonic Barrier Functions , Microflora and Fermentation in Rats Fed a High-Fat Diet

Consumption of certain dietary fibers has been reported to increase intestinal mucins and IgA concentrations, both of which play a role in the maintenance of gut barrier function and health [7,8]. The increased production of mucins and IgA has been considered to be associated with a lower risk of colon cancer [9,10]. Consumption of dietary fiber has also been reported to reduce fecal activity of mucinase, which degrades the protective mucin coat in the gut wall, thereby exposing colonic mucosal cells to toxic and carcinogenic substances [11,12]. Several types of dietary fibers and low digestible starch are known to modulate colonic microflora profile, and promote the organic acid fermentation [13-16]. Increases in the beneficial bacteria, such as lactobacilli and bifidobacteria, and in the organic acid such as butyrate and propionate were also associated with a reduced risk of colon cancer and ulcerative colitis [17-19].


Introduction
The Maximowicz's lily (Lilium leichtlinii var.maximowiczii Baker) belongs to the genus Lilium of the family Liliaceae.Its bulbs have been traditionally consumed in Asia as a root vegetable for hundreds of years.Edible lily bulb contains physiologically active components, such as dietary fibers, flavonoids, and saponins and is also rich in nutrients including starch and protein [1][2][3][4][5].Lily bulb has been reported to contain around 20% highmolecular weight (MW=230 kDa) glucomannan per dry weight, which consists of D-glucose and D-mannose units joined by β-glycosidic bound linkages [4].Lily bulb has been also used in herbal medicine for the treatment of chronic gastritis, chronic bronchitis, pertussis and sedative, and it is assumed that lily bulb has physiological and pharmacological actions [6].Recent in vitro lyophilized sample was powdered to produce raw lily bulb powder.Steamed lily bulb powder was prepared as follows: The lily bulb cloves were peeled and steamed for 25 min using an electric steamer (Groupe SEB Japan Co., Ltd., Tokyo, Japan).The steamed sample was powdered after being freeze-dried.The composition of the raw and steamed lily bulb powders is shown in Table 1.In experiment 1, the amount of dietary fibers in the raw lily bulb was measured using an enzyme-gravimetric method [25].In experiment 2, the dietary fibers, in the raw and steamed lily bulb, including low molecular weight soluble dietary fibers were measured using the enzyme-gravimetric method in combination with HPLC (AOAC method 2001.03)[26].

Animals and diets
Four-wk old male Sprague Dawley (SD) rats were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and maintained according to the "Guide for the Care and Use of Laboratory Animals" established by Fuji Women's University and approved by the ethics committee of the same university.The rats were individually housed in an air-conditioned room at 23-24°C and 50%-60% relative humidity, with 12-h light cycle (light, 08:00-20:00 h).The rats were acclimated by feeding a nonpurified commercial rodent powder diet (CE-2; CLEA Japan, Inc., Tokyo, Japan) for 6 d, before commencing the experimental diet.In experiment 1, the rats after acclimatization were divided into three groups of 8 rats each and assigned to a HF (30%, w/w) diet or the same diet containing 5% or 10% raw lily-bulb powder.In experiment 2, the rats after acclimatization were divided into three groups of 8 rats each and assigned to a HF (30%, w/w) diet, with or without 7% raw or steamed lily bulb powder.The composition of the experimental diets is shown in Table 2.The levels of dietary carbohydrate, protein, fat, minerals, and fibers in the raw and steamed lily bulb diets were adjusted by reducing the amount of dietary corn starch, casein, beef tallow, and cellulose.To prevent difference in food intake, equal amounts of the experimental diets were provided daily in food cups at 19:00 (9 g for day 1, 10 g for days 2-4, 12 g for days 5-7, 14 g for days 8-13, and 15 g for day [14][15][16][17][18][19][20][21].The entire amount of food provided was consumed each day before the next day's food was provided.The weight of spilled diet was recorded daily and was included in the calculation of food intake.Feces were collected during the last 3 d.They were stored at -20°C and then freeze-dried and milled.At the end of the feeding period, the rats were anesthetized with sodium pentobarbital and then killed by collection of whole blood from the abdominal aorta.The serum was separated at 2,000 × g for 20 min and stored at -80°C.The cecum, liver, and adipose tissues (epididymal, perirenal and mesenteric) were removed, weighed, frozen immediately with liquid nitrogen and stored at -80°C for subsequent analysis.

Measurements
The mucins in cecal digesta and feces were extracted using the method of Bovee-Oudenhoven IM, et al. [27].After extraction of the mucin fraction, the O-linked oligosaccharide chains were quantitated using a fluorometric assay that discriminated between O-linked glycoproteins (mucin) from N-linked glycoproteins [28].Fecal mucinase activity was determined using previously described method [29].
Cecal and fecal IgA concentration was measured by ELISA using a Rat IgA ELISA quantitation kit (Bethyl Laboratories, Montgomery, Texas, USA) as according to the instructions of the manufacturer.Serum TNF-α, IL-6, and lipopolysaccharidebinding protein (LBP) were determined using ELISA quantitation kits (a Kit for TNF-α manufactured by Sibayagi, Gunma, Japan, a kit for IL-6 manufactured by Immuno-Biological Laboratories Co. Ltd., Gunma, Japan and a kit for LBP manufactured by Biometec GmbH, Greifswald, Germany).
Serum triglyceride, total cholesterol, and HDL-cholesterol were quantified using enzymatic kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan).Hepatic lipids were extracted by the method of Folch J, et al. [30] and quantified using enzymatic kits.The hepatic activities of glucose 6-phosphate dehydrogenase and malic enzyme were determined as previously described [31].Fecal triglyceride was measured using an enzymatic kit after lipid extraction according to the method of Folch J, et al. [30].

Statistical analyses
Data are expressed as the means ± SEM.Data that followed a normal distribution were analyzed using one-way ANOVA and data that did not follow a normal distribution were analyzed by the nonparametric Kruskal-Wallis one-way ANOVA.The Tukey-Kramer post hoc-test was performed when a significant effect was determined by one-way ANOVA.Steel-Dwass post-hoc test was performed when a significant effect was found in the Kruskal-Wallis one-way ANOVA.The data were also analyzed using Spearman rank correlation analysis (Rs; Spearman rank correlation coefficient).All the analyses were performed using Excel Statistics 2010 for Windows (Social Survey Research Information Co., Ltd, Tokyo, Japan).Statistical significance for differences among means was estimated at P < 0.05.

Results and Discussions Experiment 1
Food intake and final body weight did not differ between the HF and the HF + 5% raw lily bulb groups, but were significantly lower in the group receiving HF + 10% raw lily bulb powder than in the HF group (P < 0.05, Table 3).The HF + 10% raw lily bulb diet caused a significant increase in weight of pancreas, whereas the HF + 5% raw lily bulb diet did not.Raw lily bulb is known to contain a trypsin inhibitor, which induces growth and food intake depressions and pancreatic enlargement in animals [32].Therefore, the presence of excess trypsin inhibitor in the HF + 10% raw lily bulb diet, compared with that in the HF + 5% raw lily bulb diet, may be associated with the depressions of growth and food intake, and increase in weight of the pancreas.
The cecal digesta weight was significantly higher in the group receiving HF + 10% lily bulb powder than in that of the HF group (P < 0.05, Table 3).In addition, the group receiving HF + 5% raw lily bulb powder had an intermediate in the cecal digesta weight.The pH of cecal digesta was significantly lower in the HF + 5% and HF + 10% raw lily bulb groups compared to the HF group (P < 0.05).These results suggest that dietary raw lily bulb may increase intestinal fermentation.The fecal dry weight was unaffected by dietary treatment.Dietary raw lily bulb significantly increased cecal and fecal mucin content in a dose dependent manner (Table 4).Cecal IgA markedly increased in the HF + 5% and HF + 10% raw lily bulb groups in a dose dependent manner.Fecal IgA content was also significantly higher in the HF + 5% and HF + 10% raw lily bulb groups than in the HF group (P < 0.05).These results suggest that raw lily bulbs have a favorable effect on intestinal barrier and immune functions.The fecal activity of mucinase was unaffected by dietary raw lily bulbs.The HF + 5% and HF + 10% raw lily bulb diets contained 0.99% and 1.98% dietary fibers, respectively.These levels are below those required to increase the level of IgA and mucins [7,8].The lily bulb samples using in this study contained raw starch and saponins as well as dietary fibers.Raw starch has relatively low digestibility and it has been suggested that undigested starch may increase intestinal production of mucins and IgA [15].Furthermore, our recent experiment [33] showed that dietary saponins increased fecal mucins and IgA levels in rats fed a HF diet.Therefore, the promoting effect of dietary lily bulb on IgA and mucins might be complexly related to its dietary fibers, undigested starch, and saponins.Further study is necessary to confirm this possibility.The HF + 5% and HF + 10% raw lily bulb diets markedly increased the cecal ratio of Lactobacillales in a dose dependent manner (P < 0.05, Table 4).The ratio of Bifidobacterium in the cecum was also elevated by dietary supplementation of 5% and 10% raw lily bulb.The cecal ratio of Clostridium was not significantly different among the three groups.These results provide evidence that consumption of raw lily bulb has a prebiotic effect.Cecal Lactobacillales significantly correlated with cecal mucins (Rs = 0.74, P < 0.001), fecal mucins (Rs = 0.71, P < 0.001), cecal IgA (Rs = 0.56, P < 0.005), and fecal IgA (Rs = 0.46, P < 0.05).It has been previously reported that lactobacilli increases the expression of intestinal mucin and prevents attachment of pathogenic bacteria [34].The administration of lactic acid bacteria as Lactobacillus has also been shown to stimulate the IgA immune response in the gut [35].Taken together, these results suggest the possibility that higher ratio of Lactobacillales in the groups receiving lily bulb may at least in part contribute to increases in the levels of intestinal mucins and IgA.

Experiment 2
Because lily bulb is usually consumed after heating, we further examined whether heating treatment of lily bulb, as well as raw lily bulb, could affect colonic luminal environment in rats fed a high-fat diet.In experiment 1, the effect of HF + 10% raw lily bulb group on colonic luminal environment was prominent compared with that of the HF + 5% raw lily bulb group, whereas dietary 10 % lily bulb caused slight inhibitions of growth and food intake.Therefore, in the second experiment, the lily bulbs were added to HF diets at the level of 7% to avoid growth and food intake depressions.As expected, food intake and final body weight did not differ among the three groups (Table 5).Liver, pancreas, and fecal dry weights were also unaffected by dietary treatment.The wet weight of cecal digesta was markedly higher, and the pH of the digesta significantly lower in both the HF + raw and HF + steamed lily bulb groups than in the HF group (P < 0.05).Both the steamed and lily bulb diets caused markedly increased in cecal and fecal mucin content compared with that in the HF diet (P < 0.05, Table 6).In accordance with the experiment 1, fecal IgA was significantly increased in the HF + raw lily bulb group (P < 0.05), with a similar trend being observed in the HF + steamed lily bulb group.These results suggest that heating treatment of lily bulb have also a favorable effect on the intestinal barrier and its immune functions.Interestingly enough, the fecal activity of mucinase was significantly reduced in the HF + steamed lily bulb group compared with the HF group (P < 0.05, Table 6).The bacterial enzyme mucinase degrades the protective mucin coat in the gut wall, reduces barrier function, and therefore is considered a risk factor for colon cancer [12].The results of our study suggest that the increase in the mucosal mucin barrier caused by the steamed lily bulb may be related, in part, to the inhibition of mucin degradation.Heat treatments of several vegetables have been reported to increase flavonoids contents and physiological activities [36][37][38].Therefore, the enhanced flavonoids in steamed lily bulb might be associated with the suppression of mucinase activity.Further study is necessary to examine the molecular mechanism by which steamed lily bulb and its bioactive compounds suppress the enzyme activity, and to compare bacterial load in the raw and steamed lily bulbs.
The inflammatory cytokines, serum TNFα and IL-6 were both unaffected by the HF + raw and HF + steamed lily bulb diets (Table 5).The serum level of LBP, a pro-inflammatory LPS transporter used as a marker of endotoxemia [39], was also not different in the three groups.Therefore, the raw and steamed lily bulbs appear to modulate mucin and IgA levels in the gut without affecting inflammatory factors.
As in experiment 1, the HF + raw lily bulb diet significantly increased cecal Lactobacillales (P < 0.05), with similar trend being observed in the HF + steamed lily bulb group (Table 6).Bifidobacterium was detected in the cecum of the groups consuming HF + raw and HF + steamed lily bulbs, whereas it was hardly detected in the HF group.The cecal content of Clostridium was not different among the three groups.The present study demonstrated that the heat treatment of lily bulb also can modulate the cecal microflora profile.
We have previously demonstrated that a HF diet decreases fermentation of organic acids in the rat cecum [20].This decrease in intestinal fermentation leads to a higher risk for colon diseases [17,18].Because the HF + raw and HF + steamed lily bulb diets increased the weight of cecal digesta and decreased the pH, we further investigated whether the bulbs affected cecal organic acids.The results demonstrated that both HF + raw and HF + steamed bulbs caused significant increases in cecal n-butyrate (+253% and +189%, respectively), succinate (+257% and +378%, respectively), lactate (+286% and +496%, respectively), and total organic acids (+169% and +174% respectively) per g wet cecal digesta (P < 0.05, Table 7).Dietary raw and steamed lily bulbs caused significant increase in cecal acetate (+201% and +163%, respectively), propionate (+161% and +171%, respectively), n-butyrate (+363% and +253%, respectively), lactate (+388% and +700%, respectively) and total organic acids (+242% and +245%, respectively) per total cecal digesta (P < 0.05).When the data were expressed relative to total cecal digesta, the increase in cecal succinate level was prominent in the HF + steamed lily bulb group (+556%).Because lactate and succinate are absorbed more slowly in the gut than other organic acids [40], the pH of cecal digesta is considerably lower, with this acidic environment favoring acid-resistant bacteria such as lactobacilli and bifidobacteria.Furthermore, organic acids such as butyrate, acetate, and propionate are also considered to play a key role in colonic health [13].Dietary fibers and undigested starch have been reported to increase the levels of organic acids [11,13].Kishida T, et al. [41] reported that a type of heatmoisture treated starch decreased enzymatic digestibility and increased the levels of cecal organic acids to the same degree as raw starch.Therefore, it is necessary to test whether dietary fiber and undigested starch in raw and steamed lily bulbs contribute to observed effect on cecal organic acids.
Unexpectedly, the HF + steamed lily bulb diet caused a significant reduction in mesenteric adipose tissue weight (P < 0.05), whereas the HF + raw lily bulb diet did not cause any reduction (Table 8).The same tendency was observed for epididymal adipose tissue weight.Serum triglyceride, cholesterol and free fatty acids were unaffected by dietary treatment.Liver triglyceride, cholesterol, and the activities of glucose 6-phosphate dehydrogenase and malic enzyme were also unaffected.Therefore, these findings indicate that the suppressive effect of the steamed lily bulb on the adipose tissue weight cannot be explained by changes in hepatic lipogenesis.Consumption of steamed lily bulb significantly increased fecal excretion of triglyceride and neutral sterol (P < 0.05), but the HF + raw lily bulb diet did not.It is therefore possible that the reduction in the adipose tissue weight  associated with the dietary steamed lily bulb may at least be mediated by a reduction in lipid absorption.Several studies have reported that heat treatments enhance flavonoids contents in vegetables [36,37].Dietary flavonoids are known to suppress fat accumulation in obese animal [42].Therefore, it is of interest to test the possibility that steam treatment may increase flavonoids contents in lily bulb, and the increase may be associated with the reduction of adipose tissue weight.
It has been recently suggested that obesity is related to dysbiosis of the gut microbiota [43].Several studies [44,45] have shown a higher ratio of Firmicutes and reduction in the ratio of Bacteroidetes in overweight and obese animals than among lean animals.Further study is in progress in rats fed a HF diet to test whether dietary supplementation with steamed lily bulbs modulates the intestinal ratios of Firmicutes and Bacteroidetes, and whether this modulation is related to a decrease in the adipose tissue weight.

Conclusion
This study provides first evidence that dietary lily bulb increases the levels of intestinal mucin, IgA, cecal Lactobacillales and organic acid fermentation.An interesting finding in this study of rats fed a HF diet is that steam treatment of the lily bulbs significantly reduced mesenteric adipose tissue weight and fecal mucinase activity, a risk factor for colon cancer.These findings suggest that lily bulb can be considered as a novel functional food for colon health.Further studies are in progress to isolate the dietary fiber, low digestible starch, flavonoids, and sapponins from lily bulb that may be responsible for the functions described in this study.

f
Direct incineration method.

Table 1 :
Chemical composition (%, w/w) of raw and steamed lily bulb powders a .
c Kjeldahl method.d Soxhlet method.

Table 2 :
Composition of the experimental diet.

Table 3 :
Effect of dietary addition of 5% or 10% raw lily bulb on body weight, food intake, cecal and fecal weight in rats fed a HF diet 1 (Experiment 1).Values are means ± SEM, n=8.Means in a row with superscripts without a common letter (a, b) differ, P <0.05 (Tukey-Kramer post-hoc test or Steel-Dwass test). 1

Table 4 :
Effect of dietary addition of 5% or 10% raw lily bulb on cecal and fecal mucins, IgA, and cecal microflora in rats fed a HF diet 1 (Experiment 1).

Table 5 :
Effect of dietary addition of 7% raw and steamed lily bulb on body weight, food intake, cecal and fecal weight and serum inflammatory parameters in rats fed a HF diet 1 (Experiment 2).Values are means ± SEM, n=8.Means in a row with superscripts without a common letter differ (a, b), P <0.05 (Tukey-Kramer post-hoc test or Steel-Dwass test). 1

Table 6 :
Effect of dietary addition of 7% raw and steamed lily bulb on cecal and fecal mucins, IgA and cecal microflora in rats fed a HF diet 1 (Experiment 2).

Table 7 :
Effect of dietary addition of 7% raw and steamed lily bulb on cecal organic acids in rats fed a HF diet 1 (Experiment 2).Values are means ± SEM, n=8.Means in a row with superscripts without a common letter (a, b) differ, P <0.05 (Tukey-Kramer post-hoc test or Steel-Dwass test). 1