COMPOSITION OF DECREASING THE ACCUMULATION OF FAT AND USE THEREOF

Abstract

The present invention provides a natural composition of reducing weight gains and decreasing the accumulation of fat, comprising Bacillus subtilis BS139 (Storage ID: NRRL NO.B-50347) and Coleus forskohlii extract. Moreover, this composition can be used in the treatment of obesity, helping them healthy reduce fat accumulation, to achieve the effect of weight loss.

Claims

1. A natural composition of reducing weight gains and decreasing the accumulation of fat, comprising 0.01 wt % to 0.05 wt % Coleus Forskohlii extract and 0.02 wt % to 0.08 wt % Bacillus subtilis BS139 (Storage ID: NRRL NO.B-50347), wherein the natural composition could reduce 20% to 40% rate of body weight gain in an animal or a human body.

2. The natural composition of claim 1, the natural composition could reduce 10% to 80% weight of abdominal fat in an animal or a human body effectively.

3. The natural composition of claim 1, the natural composition could reduce 5% to 12% Triglyceride in an animal or a human body effectively.

4. The natural composition of claim 1, the natural composition could reduce 15% to 25% caloric intake rate in an animal or a human body effectively.

5. The natural composition of claim 1, the natural composition is applied to a pharmaceutical product, health food, health-care food, food or animal feed.

6. A preparation method of a pharmaceutical composition for treating obesity by a plant extract composition of, wherein the plant extract comprising: 0.01 wt % to 0.05 wt % Coleus forskohlii extract and 0.02 wt % to 0.08 wt % Bacillus subtilis BS139 (Storage ID: NRRL NO.B-50347), wherein the natural composition could reduce 20% to 40% rate of body weight gain in an animal or a human body.

7. The preparation method of claim 6, the natural composition could reduce 10% to 80% weight of abdominal fat in an animal or a human body effectively.

8. The preparation method of claim 6, the natural composition could reduce 5% to 12% Triglyceride in an animal or a human body effectively.

9. The preparation method of claim 6, the natural composition could reduce 15% to 25% caloric intake rate in an animal or a human body effectively.

10. The preparation method of claim 6, the natural composition is applied to a pharmaceutical product, health food, health-care food, food or animal feed.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 Illustration of the weight change of female rats after feeding the different feeds in different days.

[0017] FIG. 2 Illustration of the weight change of female rats after feeding the different feeds in different weeks.

[0018] FIG. 3 Illustration of the feed intake of female rats after feeding the different feeds in different weeks.

[0019] FIG. 4 Illustration of the feed efficiencies of female rats after feeding the different feeds.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Experimental Animal and Feeding Environment

[0021] Eighteen 5-weeks old Wistar female rats were used as experimental animals, each rat was 160 g and was kept in the rodent animal feeding room of Agricultural Technology Research Institute (Hsinchu City, Taiwan R.O.C). The temperature of the rodent animal feeding room was controlled in 23° C. (20-26° C.) and the light cycle was 12-hours lighted. The experimental rats were kept in cages, which were sterilized by high temperature and high pressure; besides, the feeding water was provided with water bottles, which were also sterilized by high pressure.

[0022] The Construction of Different Groups of Wistar Female Rats Feeding with Different Feeds

[0023] Two rats were kept in one cage, and the animal numbers, cage numbers were labeled. The six-week experiment was followed by 7-days habitual period. The rats were randomly into 3 groups: control group, oil-added feed group, oil-added and drug treated feed group. There were 3 cages in total, and there were 6 rats in each group. The rats in the control group were feed with imported granular rodent feed (Lab Diet® 5001 Rodent Diet). The rats in oil-added feed group was feed with 10% soybean oil containing high-fat feed, wherein the high-fat feed was prepared by adding 10% soybean oil after smashing the imported granular rodent feed and shaping into a lump. The high-fat feed was prepared two days before feeding, so as to avoid the problem of oil oxidation. The rats in the oil-added and drug treated feed group were feed with high-fat feed, and the Coleus forskohlii extract and Bacillus subtilis BS139 were then added. The main nutritious ingredients in the feeds were listed as Table 1, wherein all the data in the table were average values.

TABLE-US-00001 TABLE 1 Main nutritious ingredient in the feeds Granular rodent feed group (Lab Diet ® 5001 Oil-added Group Rodent Diet) feed group Total Calories (kcal/kg) 4,070 4,560 Total Proteins (%) 23.7 21.8 Total Fat (%) 5.2 14.4 Total Calcium (%) 0.94 0.86 Total Phosphorous (%) 0.68 0.60

[0024] Experiment Analysis

[0025] The rats were weighted in the beginning of the experiment (Day 1) and every week respectively, and the weekly feed consumption of each rats were recorded. The daily clinical symptoms were observed during experiment, and whether the clinical symptoms or death were existed in each animal was recorded. The tail vein blood was collected in the end of experiment (Day 42), and the glucose level, triglyceride level, aspartate aminotransferase (AST) level, and alanine aminotransferase (ALT) level of each rats were tested by Automated Clinical Chemistry Analyzer (Kodak Ektachem DT-II System, Rochester, N.Y., USA). Then the rats were sacrificed for pathological observation, and the tissue weight was recorded.

Example 1. The Intake Amounts of Feeds and Calories

[0026] Please refer to the Table 2, the feed intake amounts were slightly different between different groups during experiment period, but there was no statistical difference (P>0.05), wherein the values in Table 2 were average values.

TABLE-US-00002 TABLE 2 The intake amounts of feeds (g/rat/week) and calories (kcal/rat) Total Total calories feed intake amount Week Week Week Week Week Week intake (calculated groups 1 2 3 4 5 6 amount value) Control group 151 185 185 209 204 198 1,132 4,607 Oil-added feed 138 134 210 224 244 218 1,168 5,326 group oil-added and drug 177 134 192 194 262 183 1,142 5,207 treated feed group

[0027] To compare the rodent feed group with control group, the oil-added feed group was feed with 10% soybean oil containing high-fat feed. In the oil-added feed group, the feed intake amount was 8.6% lower than control group during week 1, the amount was even 27.6% lower than control group during week 2; however the feed intake amount was higher than control group afterward, the amount was increased 7-19% during week 3 and week 6. The feed intake amount of the oil-added feed group was decreased during week 1 and week 2, which seemed to be affected by the added oil; however, after two weeks habituation, the feed intake amounts during following 4 weeks were higher than control group. During experiment period, the total feed intake amount of oil-added group was similar to the total feed intake amount of control group; however, the total calories intake amount was 15.6% higher than control group, which was matched with the purpose of high-fat feed to increase the rat's calories intake amount.

[0028] The rats in the oil-added and drug treated feed group were then feed with Coleus forskohlii extract and Bacillus subtilis BS139, and the intake amount was measured, the test result of intake amount change in this group is not consistent with the test result of oil-added group. During week 1, the rat's feed intake amount of oil-added and drug treated feed group was 17.2% higher than the control group, and was 28.3% higher than the oil-added feed group. During week 2, the rat's feed intake amount of oil-added and drug treated feed group was similar to the rats in oil-added feed group, but 27.6% lower than the rats in control group. Then, during the week 5, the rat's feed intake amount of oil-added and drug treated feed group was 28.4 higher than the rats in control group. During week 4 and week 6, the rat's feed intake amount of oil-added and drug treated feed group was both 7% lower than the rats in control group. The added Coleus forskohlii extract and Bacillus subtilis BS139 seem to inhibit the rat's feed intake amount after one week (that is, week 2, 4, 6). During the whole experiment period, the rat feed intake amount of oil-added and drug treated feed group was similar to rats in control group, however, the total calories intake amount was 13% higher than the rats in control group but 2.6% lower than the rats in the oil-added feed group.

Example 2: Growth Curve and Weight Changes

[0029] Please refer to the FIG. 1, all the rats in different groups were similar to each other in weight, which is about 117 g in the beginning; however, all the rats in different groups gain weight as more feed days. The rats in the control gain weight gradually slow down as more feed days, and the growth curve of the oil-added feed group was different with the control group. It's noteworthy that the rat's weight of the oil-added and drug treated feed group was lower than control group and oil-added feed group from day 21, the difference between the oil-added and drug treated feed group and control group, oil-added feed group was increase as more feed days but the increase of the difference was not equidistant.

[0030] Please refer to the Table 3, all the data in the Table 3 were average values, the superscripts showed the significant differences in the same row. Wherein the P value of a and b was smaller than 0.05, the P value of a and c was smaller than 0.01, and the P value of a and d was smaller than 0.001.

TABLE-US-00003 TABLE 3 Weekly Weight gain (g/rat) Total Week Week Week Week Week Week weight Group 1 2 3 4 5 6 gain Control 24.2.sup.a 22.2.sup.a 16.5.sup.a 16.5 7.7 3.2 90.2.sup.a group Oil-added  8.0.sup.b 42.3.sup.d 20.2.sup.a 12.5 7.8 7.7 98.5.sup.a feed group oil-added 14.3.sup.ab 35.2.sup.cd  5.2.sup.c 10.3 4.7 5.2 74.8.sup.b and drug treated feed group

[0031] The rats in the control group gained weight over 22 g during week 1 and 2, gained weight about 16.5 g during week 3 and 4, gained 7.7 g during week 5, and gained only 3.2 g during week 6. The rats in oil-added feed group gained only 8 g during week 1, which is significantly lower than control group (P<0.05). On the other hand, the gained weight in oil-added feed group was significantly higher than control group during week 2, which is 42.3 g (P<0.001). Then, the weekly weight gain in control group and oil-added feed group was no significant difference between two groups during week 3-6. The weight gain in oil-added and drug treated feed group was no difference to the other two groups during week 1, but the weight gain in oil-added and drug treated feed group was significantly higher than control group during week 2, which is 35.2 g (P<0.01). During week 3, the rats in oil-added and drug treated feed group only gain 5.2 g, which was significantly lower than control group and oil-added feed group (P<0.01). Then, the weekly rate of weight gain in oil-added and drug treated feed group was no difference to the other groups in statics, but the values of weight gain in oil-added and drug treated feed group was lower than in control group and oil-added feed group during week 4-6. During the experiment period, the total weight gain in control group and oil-added feed group was 90.2 g and 98.5 g respectively. In contrast, the oil-added and drug treated feed group only gain 74.8 g, which was significantly lower than the other groups (P<0.05). The result showed that the weight curve of oil-added and drug treated feed group was lower than the control group and oil-added feed group since week 3, which prove that the feed of the composition of 0.01 wt % to 0.05 wt % Coleus forskohlii extract and 0.02 wt % to 0.08 wt % Bacillus subtilis BS139 could help reduce weight gain.

[0032] Please refer to FIG. 2 and FIG. 3, the rat in the group of high-fat feed, the amount of feed intake could be reduced due to accommodation in early stage, so as to reduce the weight gain of the rat (week 1). However, the amount of feed intake in oil-added and drug treated feed group was 17% higher than the control group, but the weight gain was 40% lower than the control group, which represented that the added Coleus forskohlii extract and Bacillus subtilis BS139 composition seemed to have the function of inhibit the weight gain. Except for week 2, similar result was observed during week 3, 4, 5 and 6. In comparison with control group, the amount of feed intake in oil-added and drug treated feed group was 3.8% higher, but the weight gain was 68.5% lower during week 3; the amount of feed intake in oil-added and drug treated feed group was 28.4% higher, but the weight gain was 38.7% lower during week 5. In comparison with oil-added group, the amount of feed intake in oil-added and drug treated feed group was 8.6% higher, but the weight gain was 74.3% lower during week 3. Furtherly, the amount of feed intake in oil-added and drug treated feed group was 7.4% higher, but the weight gain was 39.7% lower during week 5. The result showed that the amount of feed intake was increased in the oil-added and drug treated feed group, but the increase rate of weight gain was lower than control group and oil-added feed group. That is, the composition of 0.01 wt % to 0.05 wt % Coleus forskohlii extract and 0.02 wt % to 0.08 wt % Bacillus subtilis BS139 in the present invention could reduce 20% to 40% rate of body weight gain in an animal or a human body.

Example 3. Tissue Weight

[0033] All the animals were healthy and not dead, nor observed any clinical symptoms during experiment period. Please refer to Table 4, all the data in the Table 4 were average values, the superscripts showed the significant differences of statics in the same row. Wherein the P value of a and b was smaller than 0.05, the P value of a and c was smaller than 0.01. The heart and kidney weight was no difference between all the groups after animal sacrificed. However, the abdominal fat weight of oil-added feed group was 58.5% higher (P<0.05) than control group; but the abdominal fat weight of oil-added and drug treated feed group was 44.5% lower (P<0.01) than oil-added feed group, and was 12.1% lower (P>0.05) than control group, while the rats in oil-added and drug treated feed group were also feed with high-fat feed. The Coleus forskohlii extract and Bacillus subtilis BS139 composition was added in the oil-added and drug treated feed group, which inhibit the abdominal fat accumulation under similar calories intake condition. The abdominal fat accumulation in the oil-added and drug treated feed group was significantly lower than control group and oil-added feed group, which proved that the Coleus forskohlii extract and Bacillus subtilis BS139 composition of the present invention could reduce the fat intake and reduce the fat accumulation effectively. That is, the composition of 0.01 wt % to 0.05 wt % Coleus forskohlii extract and 0.02 wt % to 0.08 wt % Bacillus subtilis BS139 in the present invention could reduce 10% to 80% weight of abdominal fat accumulation in an animal or a human body.

TABLE-US-00004 TABLE 4 Tissue weight (g) Oil-added Control Oil-added and drug treated Tissue group feed group feed group Heart 0.86 0.88 0.90 Kidney 2.42 2.44 2.26 Abdominal fat 3.23.sup.b 5.12.sup.a 2.84.sup.bc

Example 4. Biochemical Profile of Blood

[0034] Please refer to Table 5, all the data in the Table 5 were average values. The superscripts showed the significant differences of statics in the same row, for example, the P value of a and c was smaller than 0.01. In comparison with control group, the blood glucose level was lower (P<0.01) in the oil-add feed group, which means that the high-fat feed could reduce the blood glucose level, but the triglycerate level was slightly higher (16.5%). The blood glucose level in oil-added and drug treated feed group was lower (P<0.01) than control group; but the increase level of triglyceride (5.5%) in oil-added and drug treated feed group was 11% lower than oil-added feed group. Therefore, the added Coleus forskohlii extract and Bacillus subtilis BS139 composition could reduce the triglycerate when the rats eat more high-fat food, wherein the mechanism might not be related to glucose generation, but related to lipid metabolism and generation. That is, the composition of 0.01 wt % to 0.05 wt % Coleus forskohlii extract and 0.02 wt % to 0.08 wt % Bacillus subtilis BS139 in the present invention could reduce 5% to 12% triglyceride in blood either an animal or a human body.

TABLE-US-00005 TABLE 5 Biochemical Profile of Blood Oil-added and drug Control Oil-added treated Item group feed group feed group Glucose (mg/dL) 232.sup.a 146.sup.c 151.sup.c Triglycerate (mg./dL)  69.7  81.2  73.5 aspartate aminotransferase 160 135  93.2 (AST)   alanine  41.3  49.5  40.3 aminotransferase(ALT)

[0035] Besides, the aspartate aminotransferase (AST) level and alanine aminotransferase (ALT) level were important markers to evaluate the effectiveness of liver disease treatment, which were often used to evaluate the liver cell damage level and classify the liver disease as acute or chronic. Aspartate aminotransferase (AST) abundantly existed in liver, and alanine aminotransferase (ALT) exists in heart, liver and muscle cell. When these cells were damaged, especially when hepatic cells were damaged, the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of the serum would increase significantly. In this example, although the aspartate aminotransferase (AST) level in oil-added feed group was lower than control group, alanine aminotransferase (ALT) level remained high. Moreover, the alanine aminotransferase (ALT) level in oil-added feed group was slightly increased, on the other side, the alanine aminotransferase (ALT) level in oil-added and drug treated feed group showed slightly decreased. However, the differences were not much between different groups.

Example 5. Feed Efficiency

[0036] Please refer to FIG. 4, the total weight gain was divided by total intake amount, so as to calculate the efficiency of feed converted into weight. The calculated result of control group was similar to oil-added feed group. The feed efficiency of oil-added and drug treated feed group was 19% lower than the other groups, which means the rats feed with the feed containing Coleus forskohlii extract and Bacillus subtilis BS139 needed to eat 19% more feed to achieve similar weight gain. Therefore, the Coleus forskohlii extract and Bacillus subtilis BS139 composition of the present invention could reduce the feed nutrition intake and reduce the reaction of feed convert into weight effectively. That is, the composition of the present invention could reduce 15% to 25% caloric intake rate in an animal or a human body effectively.

[0037] According to the experiment of the present invention, the Wistar female rats feed with high-fat feed would increase weight and abdominal fat accumulation; meanwhile, the Coleus forskohlii extract and Bacillus Subtilis BS139 composition of the present invention could control the high-fat feed induced rate of weight gain (24.1%), and inhibit the lipid metabolism (triglyceride) and abdominal fat accumulation (44.5%), the inhibition level is greater than the increase level of weight gain and abdominal fat accumulation in normal feed group. Besides, after 6 weeks, there is no any clinical symptoms (severe side effects) in the rats feed with the Coleus forskohlii extract and Bacillus subtilis BS139 composition and high-fat feed. The composition of the present invention could inhibit user's weight gain, reduce the fat intake, reduce the fat accumulation, without reducing the appetite and without having any side effect, so that the user could control his/her body weight in a healthy style. As mention above, the present invention could reduce the weight gain in animal or human body, reduce triglyceride in animal or human body, reduce the food intake rate in animal or human body through specific percentage range of the composition of 0.01 wt % to 0.05 wt % Coleus forskohlii extract and 0.02 wt % to 0.08 wt % Bacillus subtilis BS139 (Storage ID: NRRL NO. B-50347).

[0038] Although the present invention has been described in terms of specific exemplary embodiments and examples, it will be appreciated that the embodiments disclosed herein are for illustrative purposes only and various modifications and alterations might be made by those skilled in the art without departing from the spirit and scope of the invention as set forth in the following claims.