Composition and use thereof in manufacture of product for improving intestinal function

Abstract

The present invention relates to the technical field of health care products, in particular to a composition and use thereof in the manufacture of a product for improving intestinal function. The present invention provides a composition and use thereof in the manufacture of a product for improving intestinal function. In the present invention, probiotics, prebiotics, Crataegi Fructus and Dioscoreae Rhizoma are reasonably compounded, wherein each component acts synergistically, thus allowing the obtained composition to have good effects of preventing and/or treating diarrhea as well as improving intestinal flora. The efficacy thereof is significantly superior to (P<0.05) that of the Comparative Examples.

Claims

1. A composition, comprising probiotics, prebiotics, Crataegi fructus and Dioscoreae rhizoma, wherein the mass ratio of the probiotics, the prebiotics, the Crataegi fructus and the Dioscoreae rhizoma is (2.0-40.0):(30.0-96.0):(1.0-30.0):(1.0-30.0), the probiotics are selected from Lactobacillus and Bifidobacterium, and the prebiotics are inulin.

2. The composition according to claim 1, wherein the probiotics are Lactobacillus and Bifidobacterium, the mass ratio of the Lactobacillus to the Bifidobacterium is (2.0-20.0):(2.0-20.0).

3. The composition according to claim 1, wherein the probiotics are added in an amount of 10.sup.6 CFU/g-10.sup.12 CFU/g based on the total weight of the composition.

4. A method for treating diarrhea, comprising administering the composition of claim 1.

5. The method according to claim 4, wherein the diarrhea is antibiotic-associated diarrhea and/or diarrhea induced by spleen deficiency.

6. A product for treating diarrhea, comprising the composition of claim 1.

7. A method for improving intestinal flora, comprising administering the composition of claim 1.

8. The method according to claim 7, wherein the improving intestinal flora is improving intestinal flora disorder induced by antibiotics administration and/or improving intestinal flora disorder induced by spleen deficiency.

9. A product for improving intestinal flora, comprising the composition of claim 1.

Description

DETAILED EMBODIMENTS OF THE INVENTION

(1) The present invention provides a composition and use thereof in the manufacture of a product for improving intestinal function. Those skilled in the art can use the content herein for reference and suitably modify the process parameters to achieve. It is to be specifically noted that all similar alternatives and modifications will be apparent to those skilled in the art and are considered to be included in the present invention. The method and use of the present invention have been described by way of preferred embodiments, and related personnel obviously can alter or appropriately change and combine the methods and uses described herein so as to realize and apply the technology of the present invention without departing from the content, spirit and scope of the present invention.

(2) All the materials and instruments used in the present invention are ordinary commercial products and are commercially available.

(3) The Crataegi Fructus powder is prepared by drying and crushing the fresh fruit of Crataegi Fructus, and the particle size is 40 mesh. The Dioscoreae Rhizoma powder is prepared by drying and crushing the the Dioscoreae Rhizoma, and the particle size is 40 mesh.

(4) The Lactobacillus used in the present invention has a bacterial density of no less than 10 billion CFU/g.

(5) The Bifidobacterium used in the present invention has a bacterial density of no less than 10 billion CFU/g.

(6) The present invention is further illustrated by the following Examples:

Example 1

(7) 20.0 g of Lactobacillus, 3.0 g of Bifidobacterium, 25.0 g of Crataegi Fructus powder, 5.0 g of Dioscoreae Rhizoma powder and 47.0 g of inulin.

(8) The above components were pulverized and mixed evenly to prepare a composition.

Example 2

(9) 10.0 g of Lactobacillus, 5.0 g of Bifidobacterium, 5.0 g of Crataegi Fructus powder, 15.0 g of Dioscoreae Rhizoma powder and 65.0 g of inulin.

(10) The above components were pulverized and mixed evenly to prepare a composition.

Example 3

(11) 7.0 g of Lactobacillus, 12.0 g of Bifidobacterium, 8.0 g of Crataegi Fructus powder, 25.0 g of Dioscoreae Rhizoma powder and 48.0 g of inulin.

(12) The above components were pulverized and mixed evenly to prepare a composition.

Example 4

(13) 2.0 g of Lactobacillus, 20.0 g of Bifidobacterium, 3.0 g of Crataegi Fructus powder, 25.0 g of Dioscoreae Rhizoma powder and 50.0 g of inulin.

(14) The above components were pulverized and mixed evenly to prepare a composition.

Example 5

(15) 2.0 g of Lactobacillus, 2.0 g of Bifidobacterium, 3.0 g of Crataegi Fructus powder, 3.0 g of Dioscoreae Rhizoma powder and 90.0 g of inulin.

(16) The above components were pulverized and mixed evenly to prepare a composition.

Comparative Example 1

(17) 50 g of Crataegi Fructus powder and 50 g of Dioscoreae Rhizoma powder were pulverized and mixed evenly to prepare a composition.

Comparative Example 2

(18) 2.0 g of Lactobacillus, 2.0 g of Bifidobacterium, and 96.0 g of inulin were pulverized and mixed evenly to prepare a composition.

Example 8

(19) The effect of the composition prepared by the composition of the present invention for improving and preventing diarrhea was studied.

(20) 1. Experimental Materials

(21) 1.1 Experimental Samples and Preparations

(22) Using the compositions prepared in Example 1, Example 4, Example 5 and Comparative Examples 1 to 2 as the experimental samples, the comparative experiments of the Comparative Examples and the Examples were carried out at the same dose. In the experiments, the untreated normal murines were used as the normal controls, and the murines which did not receive the experimental samples after the establishment of the model were used as the model controls, as specifically shown in Table 1:

(23) TABLE-US-00001 TABLE 1 Dosage for preventing and improving diarrhea induced by spleen deficiency Administration dosage of Groups the composition (g/kg) Normal control group Distilled water Model control group Distilled water Comparative Example 1 0.33 Comparative Example 1 0.33 Example 1 0.33 Example 4 0.33 Example 5 0.33

(24) 1.2 Experimental Reagents

(25) Rhubarb root and rhizome, 3% hydrogen peroxide, ampicillin for injection, EMB medium, BDS medium, TPY agar medium, TSC medium, LBS agar and Enterococcus agar.

(26) 1.3 Experimental Animals

(27) SPF grade SD rats, body weight 180-220 g, male and female in half;

(28) SPF grade BALB/c mice, three weeks of age, male, 18 g22 g.

(29) 1.4 Statistical Analysis of Experimental Results

(30) The experimental data were expressed as meanSD (xs). SPSS 16.0 software was used for statistical analysis. Differences between the experimental groups were analyzed by t test, with P<0.05 indicating that the difference is statistically significant, and P<0.01 indicating that the difference is extremely statistically significant.

(31) 2. Experimental Method

(32) 2.1 Healthy SD rats were selected and randomly divided into 7 groups, normal control, model control, Comparative Example 1, Comparative Example 2, Example 1, Example 4 and Example 5, with 10 rats in each group.

(33) Except the normal control group, other rats were prepared for establishment of model of spleen deficiency: rhubarb root and rhizome was washed with water to remove dust, and boiled twice in water (boiled for 15 min each time) and filtrated. The two filtrates were combined and concentrated in a water bath to a crude drug concentration of 1 g/ml, and stored in a refrigerator at 4 C. for later use. Animals were normally fed for 3 days, and then administered with rhubarb root and rhizome decoction (1 g/ml) by oral gavage. The administration volume was 1 ml/100 g body weight, twice daily for 10 consecutive days.

(34) After the model establishment, animals were administered according to Table 1, with an oral gavage volume of 1 ml/100 g body weight, once a day for 10 consecutive days; the normal control group and the model control group were administered the same amount of distilled water. Fatigue and huddling, accidie, dull hair color, loose stool and other behaves occurred during the establishment of rhubarb root and rhizome-induced spleen deficiency model. 10 days after establishment of the models, the above symptoms of the model control group were relieved and the fecal characters of rats tended to be normal, while for the animals of Examples 1, Examples 4 and Examples 5, the animal activities and the fecal characters were normal and the dietary and body weight were not significantly different from that of the normal animals.

(35) 2. BALB/c mice after one week of acclimating were randomly divided into 7 groups, normal control, model control, Comparative Example 1, Comparative Example 2, Example 1, Example 4 and Example 5, with 15 mice in each group.

(36) The mice were prepared for the antibiotic-associated diarrhea model: mice were administered daily with ampicillin at a dose of 22.4 g/kg (mouse weight) by oral gavage for 5 days, twice daily at a dose of 11.2 g/kg; 5 mice in the normal control group were administered the same volume of saline as the reference. The criteria for the successful establishment of the model are: less luster hair, reduced feed intake, curled up, less activity, unresponsive, slower movement, and clear stain on the buttocks.

(37) On day 6, the mice of each group were observed and weighed after establishment of the model. Mice which were not ideal for the model establishment were removed and ten mice were left in each group. On day 6, experiments for counting loose stools of the mice in each group were carried out before administration: after fasting for 12 h, 25 mL/kg body weight of ampicillin (0.3 g crude drug/mL) was administered to each mouse in each group by oral gavage, and the same amount of physiological saline was administered to the normal control group. The mice were then placed in observation cages for observation, with each animal in a single cage (the bottom of the cage is covered with special filter papers). The number of loose stools, the number of dry stools, and the total number of stools within 5 hours after oral gavage of ampicillin solution (timely replacing the filter papers on the bottom of the cage as appropriate) were recorded, and the loose stool grade of each loose stool and the diarrhea index of each mouse were measured. The judgement standard of dry (formed) stool and loose (wet) stool is based on the absence or presence of stains on the filter paper.

(38) TABLE-US-00002 TABLE 2 Judgement standard of loose stool grade Diameter of stain (cm) Loose stool grade <1 1 1-1.9 2 2-2.9 3 3-3.9 4 4-4.9 5

(39) Calculation formula of loose stool rate: loose stool rate=the number of loose stools/total number of defecation within a certain period of time.

(40) Calculation formula of diarrhea index: diarrhea index=loose stool rateloose stool grade.

(41) From day 7, the mice in the Comparative Examples and the Examples were treated. The mice were administered with a dose of 0.33 mg/kg by oral gavage once a day, for 14 consecutive days. The diarrhea model control group and the normal control group were administered with the same amount of physiological saline by oral gavage. After 14 days treatment, experiments for counting loose stools were carried out to calculate the loose stool grade, the loose stool rate and the diarrhea index. The results are shown in table 3.

(42) TABLE-US-00003 TABLE 3 Loose stool grade and Diarrhea index of diarrhea mice Loose stool grade Diarrhea index After model After treatment After model After treatment establishment (%) (%) establishment (%) (%) Normal control 1.00 0.00*** 1.00 0.00*** 0.00 0.00*** 0.00 0.00*** group Model control 1.67 0.11 1.83 0.10 1.42 0.18 1.59 0.10 group Comparative 1.70 0.09 1.58 0.08* 1.45 0.17 1.35 0.06* Example 1 Comparative 1.66 0.07 1.51 0.03** 1.43 0.15 1.30 0.04** Example 2 Example 1 1.71 0.10 1.27 0.06*** 1.45 0.13 0.87 0.06*** Example 4 1.69 0.09 1.29 0.07*** 1.46 0.16 0.90 0.08*** Example 5 1.67 0.08 1.30 0.05*** 1.44 0.15 0.95 0.05*** Note: Compared with the model control group, *P < 0.05, **P < 0.01, ***P < 0.001

(43) It can be known from Table 3 that, the difference between the loose stool grade and diarrhea index of the diarrhea model control group and that of the normal control group is significant (P<0.05). Compared with the model control group, the compositions of the Comparative Examples and the Examples were able to decrease the loose stool grade and diarrhea index to some extent, and the difference was significant. Furthermore, the effects of Example 1, Example 4 and Example 5 were significantly superior to that of Comparative Examples 1 and 2 (p<0.01). The results show that the compositions provided by the present invention have synergistic effects and may improve the symptoms of antibiotic-induced diarrhea.

Example 8

(44) The effect of the composition prepared by the composition of the present invention for improving and preventing diarrhea was studied.

(45) 1. Experimental Materials

(46) 1.1 Experimental Samples and Preparations

(47) Using the compositions prepared in Example 1, Example 4, Example 5 and Comparative Examples 1 to 2 as the experimental samples, the experiments of the Comparative Examples and the Examples were carried out at the same dose. In the experiment, the untreated normal animals were used as the normal controls, and the animals which did not receive the experimental samples after the establishment of model were used as the model controls, as specifically shown in Table 4:

(48) TABLE-US-00004 TABLE 4 Dose for preventing and improving diarrhea Administration dose of Groups the composition (g/kg) Normal control group Distilled water Model control group Distilled water Comparative Example 1 0.33 Comparative Example 2 0.33 Example 1 0.33 Example 4 0.33 Example 5 0.33

(49) 1.2 Experimental Reagents

(50) Rhubarb root and rhizome, 3% hydrogen peroxide, ampicillin for injection, EMB medium, BDS medium, TPY agar medium, TSC medium, LBS agar and Enterococcus agar.

(51) 1.3 Experimental Animals

(52) SPF grade SD rats, body weight 180-220 g, male and female in half;

(53) SPF grade BALB/c mice, three weeks of age, male, 18 g22 g.

(54) 1.4 Statistical Analysis of Experimental Results

(55) The experimental data were expressed as meanstandard deviation (xs). SPSS 16.0 software was used for statistical analysis. Differences between the experimental groups were analyzed by t test, with P<0.05 indicating that the difference is statistically significant, and P<0.01 meaning that the difference is extremely statistically significant.

(56) 2. Experimental Method

(57) 2.1 Healthy SD rats were selected and randomly divided into 7 groups, normal control, model control, Comparative Example 1, Comparative Example 2, Example 1, Example 4 and Example 5, with 10 rats in each group.

(58) Except the normal control group, other rats were prepared for establishment of model of spleen deficiency: rhubarb root and rhizome was washed with water to remove dust, and boiled twice in water (boiled for 15 min each time) and filtrated. The two filtrates were combined and concentrated in a water bath to the crude drug concentration of 1 g/ml, and stored in refrigerator at 4 C. for later use. Animals were normally fed for 3 days, and then administered with rhubarb root and rhizome decoction (1 g/ml) by oral gavage. The administration volume was 1 ml/100 g body weight, twice daily for 10 consecutive days.

(59) After the establishment of the model of spleen deficiency, animals were administered according to Table 1, with an oral gavage volume of 1 ml/100 g body weight, once a day for 10 consecutive days; the normal control group and the model control group were administered the same amount of distilled water. After the administration, fresh feces were collected from all groups, and all samples were diluted 10 times in succession. 0.2 mL of each dilution was plated on the LBS, TPY, EMB, Enterococcus agar plate and TSC, and incubated at 37 C. under anaerobic conditions. After 48 h incubation, Lactobacillus, Bifidobacterium, Enterobacter, Enterococcus and Clostridium perfringens were plate-counted.

(60) The results are shown in Table 5:

(61) TABLE-US-00005 TABLE 5 Effects of the compositions on the culture of bacteria from feces of spleen deficiency rats (unit: 1 gCFU/g) Clostridium Groups Bifidobacterium Lactobacillus Enterobacter Enterococcus perfringens Normal control 8.49 0.20 9.26 0.15 6.56 0.25 3.92 0.46 1.09 0.30 group Model control 7.24 0.18** 7.60 0.06** 7.10 0.66 3.42 0.39 3.48 0.64* group Comparative 8.10 0.49 8.20 0.08# 7.77 0.56 4.68 0.68 2.86 0.61 Example 1 Comparative 8.32 0.16# 8.16 0.14# 7.57 0.51 4.77 0.41 2.82 0.54 Example 2 Example 1 8.67 0.08## 9.03 0.23## 7.61 0.51 4.64 0.66 2.97 0.61 Example 4 8.70 0.09## 8.89 0.22## 7.69 0.99 4.82 0.39 2.91 0.62 Example 5 8.57 0.12## 8.93 0.23## 7.59 0.98 5.03 0.38 3.11 0.66* Compared with the normal control group, *P < 0.05, **P < 0.01; Compared with the model control group, #P < 0.05, ##P < 0.01

(62) The results of culture of the bacterial from the feces of rats (Table 5) show that, compared with the normal control group, the number of Lactobacillus and Bifidobacterium in stool of spleen deficiency model rats were significantly decreased, and the number of Clostridium perfringens increased significantly; Comparative Example 1 can significantly increase the number of Lactobacillus in the feces of model rats, but had no significant effect on other bacteria; Comparative Example 2 can significantly increase the number of Lactobacillus and Bifidobacterium in the feces of the model rats but had no significant effect on other bacteria; Example 1, Example 4 and Example 5 can significantly increase the number of Lactobacillus and Bifidobacterium in the feces of model rats but had no significant effect on other bacteria, and the effect was significantly superior to that of Comparative Example 1 and 2(P<0.01). The result shows that the probiotics composition provided by the invention has a synergistic effect, can improve the symptoms of diarrhea induced by spleen deficiency, and effectively improve the balance of intestinal flora.

(63) 2.2 BALB/c mice after one week of acclimating were randomly divided into 7 groups, normal control, model control, Comparative Example 1, Comparative Example 2, Example 1, Example 4 and Example 5, with 15 mice in each group.

(64) The mice were prepared for the model of antibiotic-associated diarrhea: mice were administered daily with ampicillin at a dose of 22.4 g/kg (mouse weight) by oral gavage for 5 days, twice daily at a dose of 11.2 g/kg; 5 mice in the normal control group were administered the same volume of saline as the reference. The criteria for the successful establishment of the model are: less luster hair, reduced feed intake, curled up, less activity, unresponsive, slower movement of the mice, and clear stain on the buttocks.

(65) On day 6, the mice of each group were observed and weighed after establishment of the model. Mice which were not ideal for the model establishment were removed and ten mice were left in each group. On day 6, experiments for counting loose stools of the mice in each group were carried out before administration: after fasting for 12 h, 25 mL/kg body weight of senna leaf liquid (0.3 g crude drug/mL) was administered to each mouse in each group by oral gavage, and the same amount of physiological saline was administered to the normal control group.

(66) From day 7, the mice in the Comparative Examples and the Examples were treated and studied. The mice were administered with a dose of 0.33 mg/kg by oral gavage once a day, for 14 consecutive days. The diarrhea model control group and the normal control group were administered with the same amount of physiological saline by oral gavage. After 14 days of oral gavage, the stool samples were taken aseptically before and after oral gavage on day 15. All samples were diluted 10 times in succession. 0.2 mL of each dilution was plated on the LBS, TPY, EMB, Enterococcus agar plate and TSC, and incubated at 37 C. under anaerobic conditions. After 48 h incubation, Lactobacillus, Bifidobacterium, Enterobacter, Enterococcus and Clostridium perfringens were plate-counted.

(67) The results are shown in Table 6:

(68) TABLE-US-00006 TABLE 6 Effects of the compositions on the culture of bacteria from feces of antibiotic induced diarrheal rats (unit: 1 gCFU/g) Clostridium Group Bifidobacterium Lactobacillus Enterobacter Enterococcus perfringens Normal Pre-administration 7.85 0.23 7.22 0.13 6.83 0.23 4.88 0.17 5.68 0.37 control Post-administration 7.81 0.43 7.18 0.41 6.87 0.19 4.79 0.32 5.71 0.46 group Model Pre-administration 5.72 0.18** 4.88 0.31** 7.86 0.28** 6.52 0.23** 6.99 0.15** control Post-administration 7.02 0.25*# 6.18 0.32*# 7.41 0.52* 6.26 0.51* 6.41 0.19* group Comparative Pre-administration 5.71 0.22** 4.92 0.35** 7.94 0.46** 6.54 0.19** 7.01 0.35** Example 1 Post-administration 7.01 0.37# 6.45 0.53# 7.11 0.25 5.24 0.46*# 5.89 0.27# Comparative Pre-administration 5.75 0.32** 4.89 0.29** 7.75 0.31** 6.53 0.19** 6.97 0.31** Example 2 Post-administration 7.64 0.54## 7.23 0.45## 6.93 0.34# 4.91 0.20## 5.93 0.24*# Example 1 Pre-administration 5.68 0.31** 4.85 0.22** 7.85 0.28** 6.55 0.15** 6.95 0.29** Post-administration 7.95 0.30### 7.26 0.42## 6.90 0.37# 4.88 0.23## 5.60 0.19## Example 4 Pre-administration 5.74 0.27** 4.87 0.30** 7.84 0.43** 6.53 0.28** 6.98 0.36** Post-administration 7.89 0.25### 7.28 0.37## 6.87 0.41# 4.85 0.33## 5.63 0.25## Example 5 Pre-administration 5.73 0.21** 4.86 0.18** 7.82 0.16** 6.56 0.22** 7.03 0.26** Post-administration 7.87 0.27### 7.24 0.63## 6.82 0.19# 4.91 0.38## 5.66 0.21## Compared with the normal control group, *P < 0.05, **P < 0.01; Comparison between before and after administration, #P < 0.05, ##P < 0.01, ###p < 0.001

(69) The results of culture of the bacteria from the feces of mice (Table 6) show that, compared with the normal control group, the number of Lactobacillus and Bifidobacterium in the feces of antibiotics model mice were significantly decreased, and the number of Enterobacter, Enterococcus and Clostridium perfringens were significantly increased; Comparative Example 1 can significantly increase the number of Lactobacillus and Bifidobacterium in the feces of the model mice, and significantly reduce the number of Enterococcus and Clostridium perfringens, but had no significant effect on Enterobacter; Comparative Example 2 can significantly increase the number of Lactobacillus and Bifidobacterium in the feces of the model mice and significantly decrease the number of Enterobacter, Enterococcus and Clostridium perfringens; Example 1, Example 4 and Example 5 can significantly increase the number of Lactobacillus and Bifidobacterium in the feces of model mice and significantly decrease the number of Enterobacter, Enterococcus and Clostridium perfringens, and the effect was significantly superior to that of Comparative Examples 1 and 2 (P<0.01). The result shows that the probiotics composition provided by the invention has a synergistic effect, can improve the symptoms of antibiotic-induced diarrhea, and effectively improve the balance of intestinal flora.

(70) The foregoings are merely preferred embodiments of the present invention, and it should be noted that an ordinary skilled in the art can make a number of improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be deemed to be within the scope of the present invention.