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Hypolipidaemic Effect of Processed Sulfur, Allium tuberosum Rottl. and Fermented Allium tuberosum Rottl. in Rat



Eun-Jeong Son, Uttam Kumar Pal, Prabhat Kumar Mandal, Go-Eun Hong, Soo-Ki Kim and Chi-Ho Lee
 
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ABSTRACT

This study was performed to determine the hypolipidaemic effects of Allium tuberosum Rottl. (ATR), fermented Allium tuberosum Rottl. (FATR) and processed sulfur. Thirty male Wistar rats were divided into five groups: (i) CON (control, normal diet); (ii) HFD (high fat diet); (iii) HS (HFD+processed sulfur); (iv) AS (HFD+ATR+processed sulfur) and (v) FAS (HFD+FATR+ processed sulfur). Animals were fed the diets for six weeks. Body weights of the animals were recorded in the beginning and end of experiment. The animals were sacrificed, organ and tissue weights recorded and blood and liver samples were analyzed for various biochemical parameters. Body weight gain in the AS group was significantly (p<0.05) lower than the other groups. Spleen and kidney weights in FAS group were significantly (p<0.05) higher than the other groups. The weight of epididymal adipose tissue in AS group was significantly (p<0.05) lower than that of HFD group. The weight of retroperitoneal fat in HFD groups was significantly (p<0.05) higher than other groups. The concentration of aspartate amino transferase (AST) was significantly (p<0.05) higher in HFD group compared to HS, AS and FAS groups. Total liver lipid contents in the HFD group was significantly (p<0.05) higher than the other groups. AS and FAS group rats had significantly (p<0.05) lower triglyceride and total cholesterol compared to HFD group. All together, these results suggest that FATR and processed sulfur may play an important role in reducing body fat deposition and altering the serum and liver lipid profile in rats.

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  How to cite this article:

Eun-Jeong Son, Uttam Kumar Pal, Prabhat Kumar Mandal, Go-Eun Hong, Soo-Ki Kim and Chi-Ho Lee, 2012. Hypolipidaemic Effect of Processed Sulfur, Allium tuberosum Rottl. and Fermented Allium tuberosum Rottl. in Rat. Asian Journal of Animal and Veterinary Advances, 7: 812-821.

DOI: 10.3923/ajava.2012.812.821

URL: https://scialert.net/abstract/?doi=ajava.2012.812.821
 
Received: December 09, 2011; Accepted: March 22, 2012; Published: May 10, 2012



INTRODUCTION

General fatness of animals increases with intake of excess calorie as it is converted into fat and accumulate in various parts of the interior of the body in the form of hypodermic or intra abdominal fats. Excessive accumulation of fat has adverse effects on physiological and biochemical functions of the body (Ryu et al., 2006). The WHO (World Health Organization) and other international authorities have recognized that diseases such as arthritis and cardio vascular disorder are increasing globally due to excessive fatness (Lee et al., 2008; Jang and Jeong, 2010). Recent developments in functional food ingredients isolated from natural substances such as flavonoid from plants, chitosan from shell fishes and catechin of green tea have shown great promise in reducing fatness (Zacour et al., 1992). Hung et al. (2008) reported hypolipidaemic effect of fermented soy meal with low serum cholesterol and triglyceride in pigs.

Allium tuberosum Rottl. (ATR) is a plant in the Allium group, and from old time it is being used habitually as herbs for its unique scent and taste. It is also reported to be effective in dismantling of fat cell (Hwang et al., 2001). Sulfur-containing compounds present in garlic, onions and ATR of the Allium group are reported to have various physiologic effects including antioxidation, detoxification and cardiovascular protection (Kim et al., 2005; Park et al., 1998; Pinto et al., 1997; Senapati et al., 2001; Lee et al., 2002). Onion (Allium cepa) is reported to have hypoglycemic, hypolipidaemic and cardioprotective effect and its potential use in diabetes and cardiovascular diseases is suggested (Sohail et al., 2011). Allicin, an organosulfur compound derived from Allium sativum has been reported to have hepatoprotective effects in rats (Attia and Ali, 2006). Abdel Gadir et al. (2006) has reported the dietary effect of Allium cepa and Allium sativum in rats. Abdel Gadir et al. (2007) has reported that Allium cepa, Allium sativum or sodium selenite in combination with potassium bromate has no adverse effect in Wistar rats. ATR have high concentration of chlorophyll, dietary fiber and adenosine, an antithrombotic component which have effects to decrease serum cholesterol and triglyceride (Hong and Wang, 2000). It is also reported to have anticarcinogenic effects on human cancer cells (Park et al., 2002).

After removal of toxicity through processing, sulfur has been prescribed for homeostasis, paralysis and known to prevent osteoporosis and experimental bone disease (Chae et al., 2007; Kim and Seo, 1996). Sulfur-containing ducks are currently raised in domestic production facilities to get the benefit of sulfur containing diet.

This study was carried out to determine the effects of Allium tuberosum Rottl. (ATR), fermented Allium tuberosum Rottl. (FATR) and sulfur added to a high fat diet on growth, body fat deposition, organ weights, serum and liver biochemistry and lipid profile in rats.

MATERIALS AND METHODS

This study was carried out during the period from February 2010-February 2011.

Preparation of ATR and FATR: Allium tuberosum Rottl. (ATR) was purchased from the local market (Seoul, Korea) and processed sulfur was received from Ebada Eco Technology (Seoul, Korea). The tubers of ATR were cleaned, de-skinned and then ground in a blender (Angel-juicer, Busan, Korea) and filtered through gauze (Daegu, Korea). This filtrate was used as ATR extract. For preparation of fermented Allium tuberosum Rottl. (FATR), the filtrate was added to de Man, Rogosa and Sharpe (MRS) medium inoculated with Leuconostoc mesenteroides at 1:1(v/v) ratio and incubated at 30°C for 16 h. ATR and FATR extracts were freeze-dried (Ilshin Lab. MCFD 8510, Suwon, Korea) and the powder was used in the experimental diets.

Analysis of processed sulfur, ATR and FATR: The processed sulfur content of freeze-dried ATR and FATR was measured without preprocessing using an EA 1110 (ThermoQuest CE Instruments, Italia) analysis instrument.

The moisture, ash, crude protein, crude fat, crude fiber and carbohydrates percentage in ATR and FATR powder were estimated according to Association of Official Analytical Chemists (AOAC, 2005) methods.

Table 1: Composition of experimental diets (g kg-1 diet)
1ATR: Allium tuberosum Rottl., 2FATR: Fermented Allium tuberosum Rottl. CON: Control diet, HFD: High fat diet, HS: High fat diet+processed sulfur, AS: High fat diet+Allium tuberosum Rottl.+ processed sulfur, FAS: High fat diet+fermented Allium tuberosum Rottl.+processed sulfur

Moisture (%) was determined using the drying method at 105°C; ash content was estimated by dry ashing at 550°C; crude protein was determined by the micro-Kjeldahl method; crude fat by the Soxhlet method and crude fiber was determined using a fiber extractor (Fibretherm, Gerhardt, USA). Carbohydrate content was calculated by subtracting the sum total of moisture, ash, crude protein, crude fat, crude fiber contents (%) from 100 (carbohydrate = 100-(moisture+protein+fat+ash).

Experimental animals and diets: A total of 30 male, five-weeks-old Wistar rats, obtained from Central Laboratory Animal Inc., Seoul, Korea, were acclimatized for one week. Then the animals were randomly assigned to one of five diet groups (n = 6):( i) CON (control, normal diet);( ii) HFD (high fat diet, AIN-93G diet); (iii) HS (HFD+processed sulfur); (iv) AS (HFD+ATR+processed sulfur) and (v) FAS (HFD+FATR+processed sulfur). The rats were fed a restricted diet (20 g) and allowed free access to tap water. The animal room was maintained at controlled temperature (22±2°C) and humidity (50±5%) with a 12 h light-dark cycle. The experiment continued for 6 weeks. The sulfur content of the feed was 30 mg/day/rat (Lee et al., 2008). The other ingredients of the experimental diets are detailed in Table 1.

Parameter analysis: Body weight was recorded weekly using an electronic scale (Adventurer AR2140; OHAUS Corp., USA). Food intake (g) was calculated by subtracting the amount (g) of food left in the next day from the amount (g) of food supplied in the previous day. After the experimental feeding period was over the animals were sacrificed and the spleen, liver, kidney, epididymal adipose tissue and retroperitoneal fat were removed and weighed. Liver samples were stored at -80°C until analyzed.

Blood was collected from a postcaval vein and centrifuged at 3,000 rpm for 15 min. The serum was frozen at -20°C until analyzed for Total Cholesterol (TC), Triglyceride (TG), High-Density Lipoprotein Cholesterol (HDLC), Low-Density Lipoprotein Cholesterol (LDLC), Atherogenic Index (AI), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and glucose. AI was calculated as follows: [TC-HDLC]/HDLC (Haglund et al., 1991). All assays were performed by Biotoxtech Co.

Total liver lipids were extracted as per (Folch et al., 1957). A 50 mg sample of liver and 3 mL of distilled water were homogenized (HS-30E; Daihan Sci., Korea) and 7 mL of chloroform added. After centrifugation, the supernatant was removed and incrassated using Nitrogen (N2) gas to measure total lipid weight.

To measure total cholesterol in the liver, samples were incrassated using N2 gas, dissolved with 2 mL of ethanol and processed using a reagent kit (Asan Pharmaceutical Co., Korea). Enzyme reagent (3 mL) was added to liver samples (20 μL) and allowed to stand for 5 min at 37°C after which absorbance was measured at 500 nm.

For liver triglyceride analysis, dissolved samples (500 μL) and ethanol (2 mL) were mixed in Eppendorf tubes and the ethanol was then removed using N2 gas and a solution (50 μL) containing triton X-100 and chloroform (1:1) were added and mixed with chloroform (450 μL). Enzyme reagent (3 mL) (Asan Pharmaceutical) was added and absorbance was measured at 550 nm after keeping at 37°C for 15 min.

Statistical analyses: The data were analyzed using the SAS (Statistic Analytical Institute, Cary, NC, USA) program. Significance was set at p<0.05 and Tukey’s multiple range test (Snedecor and Cochran, 1989) was used for 95% significant difference comparisons.

RESULTS AND DISCUSSION

This experiment was conducted to find the effects of Allium tuberosum Rottl. (ATR), fermented Allium tuberosum Rottl. (FATR) and sulfur added to a high fat diet on growth, body fat deposition, organ weights, serum and liver biochemistry and lipid profile in rats.

Sulfur content (0.6±0.02%) of ATR was significantly (p<0.05) higher than that of FATR (0.4±0.01%). The proximate composition of ATR and FATR powder including crude fiber are shown in Table 2. Crude protein and crude fat percentage in ATR were significantly (p<0.05) higher than those of FATR, whereas, moisture and carbohydrate contents showed reverse trend.

The body weight and feed intake results for six weeks are depicted in Table 3. Initial body weight of all rats was approximately 181 g. Body weight gain in AS and CON group of rats was significantly (p<0.05) lower than that of FAS, HFD and HS group rats after 6 weeks, although feed intake (g) was similar for all the groups. Similar findings were reported by Jang and Jeong (2010) recording higher body weight gain in mice fed with high fat diet compared to mice fed with normal diet and diet containing phyto-extract mixture.

The results related to epididymal adipose tissue, retroperitoneal fat and some internal organ weights (g) are presented in Table 4 and Fig. 1a and b. Liver weights did not vary among the groups. FAS group rats had significantly (p<0.05) higher kidney weight compared to rats of other groups. Similarly the rats belonging to FAS group had significantly (p<0.05) higher spleen weight than other groups. Rats of HFD group deposited significantly (p<0.05) higher amount of retroperitoneal and epididymal fats compared to all other groups except HS and FAS group in respect of epididymal fat only.

Table 2: The proximate composition (%) of ATR and FATR powder
Means with different superscripts differ significantly (p<0.05), All values are mean ± SD of triplicate determinations

Table 3: Body weight gain and food intake (g) of rats fed CON, HFD, HS, AS and FAS diets for six weeks
Value which have different letters are significantly different (p<0.05), All values are Mean±SD (n = 5)

Table 4: Organ weights (g) of rats fed CON, HFD, HS, AS and FAS diets for six weeks
Value which have different letters are significantly different (p<0.05), All values are mean±SD (n = 5)

Ryu et al. (2006) reported that no difference in abdominal fat, paratesticular fat and perirenal fat weights of mice fed with HFD+Misaengtang (a Korean herbal formula) compared to HFD only. However, in the present study results revealed that ATR, FATR and processed sulfur had better anti-obesity effect than the traditional Korean herbal formula resulting in lower fat deposition in the experimental mice. Results on liver weights were similar to those reported by Ryu et al. (2006) but the present findings indicated positive effect of FAS on spleen and kidney weights.

Table 5 and 6 depict the results of enzyme and lipid profiles of rats belonging to different experimental groups. There was no effect on ALT in any group; however, AST in all groups (HS, AS, FAS) was significantly (p<0.05) lower than HFD group and lowest in the FAS group. HS group rats had significantly (p<0.05) lower blood glucose level compared to rats of AS group, however, rats of other groups did not show any significant difference in blood glucose level. Most importantly atherogenic index (AI) was lowest (2.3) in FAS group rats followed by AS (2.5), HS (2.7), CON (3.1) and HFD (3.3) group rats, although differences were statistically non-significant.

Fi g. 1(a-b): (a) Epidermal adipose tissue and (b)Retroperitoneal fat weights (g) of rats fed CON, HFD, HS, AS and FAS diets for six weeks, Values which have different letters are significantly different (p<0.05), All values are Mean±SD (n = 5)

Table 5: Serum ALT, AST and glucose levels of rats fed with CON, HFD, HS, AS and FAS diets for six weeks
Values which have different letters are significantly different (p<0.05), All values are Mean±SD (n = 5)

Similar to the present findings, Ryu et al. (2006) reported that there was no effect of diets (HFD and HFD+Korean herbal formula) at different levels on ALT and glucose concentration but significant effect of diet containing herbal formula on AST was reported by the same authors. Choudhary (2008) recorded significant positive effects of A. sativum and A. tuberosum on atherogenic index in guinea pigs.

Serum LDL-C level in AS and FAS group rats were significantly (p<0.05) lower than in the control group. But levels did not vary significantly among various treatment groups, although FAS group rats had lowest LDL-C level followed by AS, HFD and HS groups, respectively.

Serum HDL-C levels were significantly (p<0.05) lower in FAS and AS group rats compared to CON and HFD group rats. On the contrary, serum Total Cholesterol (TC) levels were significantly (p<0.05) higher in FAS group rats than in rats belonging to other groups. In case of serum TG rats fed with CON diet exhibited significantly (p<0.05) lower level compared to rats fed with HS, AS and FAS diets.

Choudhary (2008) reported significant reduction in cholesterol, triglycerides, LDL-C levels and AI in serum samples of guinea pigs fed with diet containing A. sativum and A. tuberosum but no effect was seen on HDL-C level. On the other hand Ryu et al. (2006) did not find any significant change in the serum levels of TC, HDL, LDL and TG in mice fed with diet of HFD+Misaengtang, a Korean herbal preparation.

Concentration of total lipid in liver of rats belonging to different groups is shown in Fig. 2. Total lipid content was significantly (p<0.05) higher in HFD group rats than that in other groups including control. AS group rats showed significantly (p<0.05) lower total lipid concentration in liver than in CON, HFD and HS group rats.

The results of adding sulfur, ATR and FATR to a high fat diet on total cholesterol are presented in Fig. 3. Total cholesterol in the AS and FAS groups were significantly (p<0.05) lower than in the HFD group. Liver triglyceride values are depicted in Fig. 4.

Table 6: Serum levels of TC, TG, HDL-C, LDL-C, and AI of rats fed with CON, HFD, HS, AS, and FAS diets for six weeks
Values which have different letters are significantly different (p<0.05), All values are Mean±SD (n = 5)

Fig. 2: Total lipid levels in the livers of rats fed with CON, HFD, HS, AS and FAS diets for six weeks. Values which have different letters are significantly different (p<0.05), All values are Mean±SD (n = 5)

Fig. 3: Total cholesterol levels in the livers of rats fed with CON, HFD, HS, AS and FAS diets for six weeks, Values which have different letters are significantly different (p<0.05), All values are Mean±SD (n = 5)

Fig. 4: Triglyceride levels in the livers of rats fed CON, HFD, HS, AS and FAS diets for six weeks. Values which have different letters are significantly different (p<0.05), All values are Mean±SD (n = 5)

Triglyceride concentration in liver of rats belonging to AS and FAS groups was significantly (p<0.05) lower than that in rats of CON, HFD and HS groups.

CONCLUSION

This study was conducted to determine the effects of feeding Allium tuberosum Rottl. (ATR), fermented Allium tuberosum Rottl. (FATR) and processed sulfur added to a high fat diet for six weeks on body fat accumulation, blood and liver lipid profiles, body weight, organ weights, blood and liver biochemical parameters. Based upon the results, it can be concluded that rats experienced improvement in liver function and a reduction in body weight, intra-abdominal fat and serum LDL-C by consuming a high fat diet with the addition of FATR and processed sulfur. This study provides preliminary data regarding the effects of utilizing processed sulfur and ATR as food additives.

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