Use of a composition for the preparation of a product for preventing or improving colon melanosis

Abstract

The present application relates to a kind of composition in the application of preparation preventing or improving colon melanosis product, the composition includes the following raw materials: pumpkin clothes 33.7wt%, pumpkin stem 20.8wt%, pumpkin 12.9wt%, 7.9wt% of fuzilu, 9.0wt% of silver fungus and xylo-oligosaccharide 15.7wt%.The composition of the present application, each component is reasonably matched, and it is proved by test that it can reduce lipofuscin accumulation in colon to prevent colon melanosis, and it can also remove the lipofuscin deposited in colon to improve colon melanosis.Therefore, the composition of the present application has the effect of preventing and improving colon melanosis, and can be used for preparing products for preventing or improving colon melanosis.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine composition technology, and relates to the application of a composition in the preparation of products for preventing or improving melanosis coli. Background Technology

[0002] Melanosis coli (MC) is a non-inflammatory bowel disease characterized primarily by melanin deposition in the colonic mucosa. Essentially, it is caused by the presence of large amounts of lipofuscin in macrophages within the lamina propria of the colonic mucosa. In recent years, the detection rate of melanosis coli has been increasing annually, showing a gradual upward trend in both men and women. The colonoscopic findings of melanosis coli mainly include: varying degrees and extent of pigmentation in the colonic mucosa, which may exhibit reticular or granular changes, diffusely distributed throughout the colonic mucosa or locally concentrated in a specific segment of the colon.

[0003] The impact of melanosis coli on patients' lives and mental well-being is undeniable, especially the frightening leopard-spot-like images on the colonoscopy report. Furthermore, related studies indicate that melanosis coli increases the incidence of colonic polyps. In 2009, the Department of Gastroenterology of Dali Bai Autonomous Prefecture People's Hospital and Tai'an Central Hospital published an article titled "Clinical Analysis of 56 Cases of Melanosis Coli" in the *China Medical Guide*. The article described 56 patients with melanosis coli admitted to the hospital, all of whom showed mucosal pigmentation under endoscopy, with 6 cases also exhibiting polyps. Therefore, developing products that can improve melanosis coli is of great significance.

[0004] However, there are currently no specific treatments or products for melanosis coli. Patients often need to improve their intestinal environment through dietary therapy to achieve the goal of improving melanosis coli. However, dietary therapy has a long cycle and the effect is not obvious. In addition, there are some traditional Chinese medicine compositions developed with constipation as the main symptom and melanosis coli as a secondary symptom. However, because their treatment principles are often to lubricate the intestines and promote bowel movements and improve intestinal motility, although they can effectively improve constipation, their effect on improving melanosis coli is not significant, and the treatment period is also long.

[0005] Therefore, developing a product that is highly effective, has a short treatment period, and can specifically improve melanosis coli has become an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides an application of the composition in the preparation of products for preventing or improving melanosis coli. The technical solution adopted by the present invention is as follows:

[0007] An application of a composition in the preparation of a product for the prevention or improvement of melanosis coli, the composition comprising the following raw materials: 33.7 wt% pumpkin skin, 20.8 wt% pumpkin stem, 12.9 wt% pumpkin fiber, 7.9 wt% Poria cocos, 9.0 wt% Tremella fuciformis, and 15.7 wt% xylooligosaccharides.

[0008] In a preferred embodiment of the present invention, the melanosis coli includes aloe-induced melanosis coli.

[0009] In a preferred embodiment of the invention, the prevention of melanosis coli includes reducing the accumulation of lipofuscin in the colon.

[0010] In a preferred embodiment of the invention, the improvement of melanosis coli includes the removal of lipofuscin deposited in the colon.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0012] The composition of this invention, with its components in a reasonable ratio, has been proven through experiments to both reduce the accumulation of lipofuscin in the colon, thereby preventing melanosis coli, and to remove existing lipofuscin deposits in the colon, thereby improving melanosis coli. Therefore, the composition provided by this invention has the effect of preventing and improving melanosis coli. Attached Figure Description

[0013] Figure 1 These are visual images of the colons of the five groups of test animals in this invention.

[0014] Figure 2 This is a schematic diagram showing the relative percentage of melanosis area in the colon of the five groups of test animals in this invention;

[0015] Figure 3 This is a schematic diagram of HE staining in the five groups of test animals of this invention;

[0016] Figure 4 This is a schematic diagram of melanin staining in the five groups of test animals of this invention;

[0017] Figure 5 This is a schematic diagram showing the relative percentage of melanin area in the five groups of test animals in this invention. Detailed Implementation

[0018] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the content of the present invention are still within the scope of protection of the present invention.

[0019] In this invention, the specific weight ratio of the composition is as follows: pumpkin skin 33.7 wt%, pumpkin stem 20.8 wt%, pumpkin fiber 12.9 wt%, Poria cocos 7.9 wt%, Tremella fuciformis 9.0 wt%, and xylooligosaccharides 15.7 wt%. The preparation method is as follows: Pumpkin skin, pumpkin stem, pumpkin fiber, Poria cocos, and Tremella fuciformis are selected according to the weight ratio, and after selection and impurity removal, they are soaked in water; after soaking, decoction is started, decocted 3 times, 30 minutes each time, and the decoctions from the three decoctions are combined and filtered; concentration is started until a thick paste is formed; then low-temperature drying is performed to ensure that the moisture content of the mixture is ≤8% and the ash content is ≤5%; after the requirements are met, it is ultrafinely pulverized using an air jet mill, finally pulverized to 500 mesh; after pulverization, it is thoroughly mixed with xylooligosaccharides to obtain the final product. It should be noted that the composition of the present invention is basically the same as the composition in the Chinese invention patent "Formula and preparation method of capsule for treating constipation prepared with pumpkin as the main raw material" (Publication No.: CN1533805A), the only difference being that aloe vera is not added. The other components and the corresponding preparation methods are exactly the same. It is actually Jianfang Tiantian Capsule, hereinafter referred to as Jianfang Tiantian Capsule.

[0020] All raw materials used in the examples were commercially available. The aloe vera powder was manufactured by Shaanxi Jiahe Biotechnology Co., Ltd., with batch number CLH-C-A108633.

[0021] All instruments used in the examples are commercially available instruments.

[0022] It should be noted that, since the diagrams of HE staining and melanin staining provide a clearer view of the colon tissue under color images, the applicant submitted the color illustrations of the instruction manual as part of other supporting documents. These illustrations are identical to the instruction manual illustrations, differing only in that they are color images.

[0023] Example 1

[0024] 1. Preparation of the test sample

[0025] 1.1 Sodium carboxymethyl cellulose solution: Weigh 3g of sodium carboxymethyl cellulose and dissolve it in 1000mL of water to obtain a sodium carboxymethyl cellulose solution with a concentration of 0.3% (or 3g / L). Sodium carboxymethyl cellulose is a common suspending agent, abbreviated as CMC-Na.

[0026] 1.2 Aloe vera powder solution: Weigh 1g of aloe vera powder and dissolve it in 10mL of the sodium carboxymethyl cellulose solution prepared above to obtain an aloe vera powder solution with a concentration of 1000mg / 10mL (i.e., 100mg / mL).

[0027] 1.3 Low-dose group test samples: Weigh 1 g of aloe vera powder and 0.7 g of Jianfang Tiantian capsules respectively, and dissolve them in 10 mL of the above-prepared sodium carboxymethylcellulose solution to obtain low-dose group test samples with an aloe vera concentration of 100 mg / mL and a Jianfang Tiantian capsule concentration of 70 mg / mL.

[0028] 1.4 Medium-dose group test samples: Weigh 1 g of aloe vera powder and 3 g of Jianfang Tiantian capsules respectively, and dissolve them in 10 mL of the above-prepared sodium carboxymethylcellulose solution to obtain medium-dose group test samples with an aloe vera concentration of 100 mg / mL and a Jianfang Tiantian capsule concentration of 300 mg / mL.

[0029] 1.5 High-dose group test samples: Weigh 1 g of aloe vera powder and 6 g of Jianfang Tiantian capsules respectively, and dissolve them in 10 mL of the above-prepared sodium carboxymethylcellulose solution to obtain medium-dose group test samples with an aloe vera concentration of 100 mg / mL and a Jianfang Tiantian capsule concentration of 600 mg / mL.

[0030] 2. Animal grouping, model establishment and drug administration

[0031] 2.1 Test animals and animal grouping

[0032] The test animals used are guinea pigs, and their strain is Hartley guinea pigs. All test animals are of clean grade, and their certificate number is SCXK (Su) 2024-0007. The test animals are all fed in cage racks, with 4 animals in each cage, and males and females are fed separately in cages. The room temperature is kept at about 20 °C, the relative humidity is 40-60%, and the light cycle is 12 h. During the whole test process, the test animals are given ordinary feed and free drinking water. The guinea pigs are adaptively fed for 1 week, weighed, and randomly divided into 5 groups according to their body weights. Then, the model is established and the drugs are administered.

[0033] Table 1 Test animals and animal grouping

[0034] Group test animals gender weight Number of animals Blank group Hartley Guinea Pig Half male and half female (400+30)g 12 Model group Hartley Guinea Pig Half male and half female (400+30)g 12 low-dose group Hartley Guinea Pig Half male and half female (400+30)g 12 medium dose group Hartley Guinea Pig Half male and half female (400+30)g 12 High-dose group Hartley Guinea Pig Half male and half female (400+30)g 12

[0035] 2.2 Test animal model establishment and drug administration

[0036] Table 2 Test animal model establishment and drug administration

[0037]

[0038]

[0039] 3 Test animal detection

[0040] After continuous drug administration for 4 weeks, the guinea pigs in each group are subjected to tissue collection, tissue storage and tissue detection. Among them, the method for collecting and storing colon tissue is: after taking blood from the abdominal aorta, immediately cut the colon part of each guinea pig and take pictures for record (such as Figure 1 Three guinea pigs were randomly selected from each group, and a section of about 0.5 cm was cut from the proximal colon (connecting to the cecum). The sections were rinsed and soaked in 10% formalin for staining.

[0041] 3.1 Degree of melanosis in intestinal tissue

[0042] Colonic tissue samples were collected from guinea pigs in each group and photographed. ImageJ software was used to quantify the melanosis area in the photographs, obtaining the relative percentage of melanosis area, calculated as melanosis area / total area * 100%. After obtaining the relative percentage of melanosis area for each group, statistical analysis was performed using GraphPad Prism 8.0 software. The final data are expressed as mean ± standard deviation. A t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups.

[0043] 3.2 Intestinal histopathological examination

[0044] The colon, preserved in formalin, was removed, embedded in paraffin, and sectioned. The specific embedding and sectioning methods were as follows: two sections were taken from each group, one for hematoxylin-eosin staining and the other for melanin staining. If pathological examination of the colon reveals varying degrees of macrophage deposition phagocytizing brownish pigment in the lamina propria of the mucosa, while the epithelial cell layer remains normal, this indicates melanosis coli.

[0045] 3.2.1 Hematoxylin-eosin (H&E) staining

[0046] a) Fixation and dehydration: After fixing the colon tissue with 4% paraformaldehyde for 24 hours, it was transferred to a fully automated tissue dehydrator for dehydration.

[0047] b) Embedding: The dehydrated colon tissue is embedded in paraffin to form a tissue wax block;

[0048] c) Sectioning: The paraffin block containing embedded colon tissue was cut into tissue sections with a thickness of 4 μm;

[0049] d) Dewaxing: Place the tissue sections in xylene solution and dewax for 10 minutes, then replace with fresh xylene solution and continue dewaxing for 10 minutes;

[0050] e) Hydration: The tissue sections were hydrated with ethanol solutions of different concentrations in the following order: anhydrous ethanol 5 min → 95% ethanol 5 min → 80% ethanol 5 min → 75% ethanol 5 min → distilled water 2 min.

[0051] f) Hematoxylin staining: Immerse the hydrated tissue sections in hematoxylin solution for 5-10 minutes, then differentiate with 1% hydrochloric acid alcohol for 10 seconds, and then rinse with tap water for 5-10 minutes.

[0052] g) Eosin staining of cytoplasm: Place the rinsed sections into an eosin solution and stain for 1-3 minutes;

[0053] h) Dehydration and mounting: After staining, dehydrate the sections with ethanol and xylene solutions of different concentrations. The specific order is 95% ethanol for 5 min → 95% ethanol for 5 min → anhydrous ethanol for 5 min → xylene I for 5 min → xylene II for 5 min. Then remove the sections and air dry them. Finally, mount them with neutral resin.

[0054] i) Place the slide on an inverted microscope, observe it, and take a photograph.

[0055] 3.2.2 Melanin staining

[0056] a) Guinea pig colon tissue was treated using the same fixation and dehydration, embedding, sectioning, dewaxing and hydration procedures as in section 3.2.1;

[0057] b) Immerse in ferrous sulfate solution and place in the dark for 18 hours;

[0058] c) Wash with distilled water for 2 minutes;

[0059] d) Immerse in acidic potassium ferricyanide solution;

[0060] e) Wash with distilled water;

[0061] f) Apply nuclear fixed red staining solution to counterstain the nucleus;

[0062] g) Rinse twice with distilled water;

[0063] h) Dehydrated, transparent, and sealed with synthetic resin.

[0064] After melanin staining, ImageJ software was used to quantitatively analyze the positive area of ​​the stained melanin. The relative percentage of melanin area represents the percentage of the positive area to the total area. After obtaining the relative percentage of melanin area for each group, GraphPad Prism 8.0 software was used for statistical analysis. The final data are expressed as mean ± standard deviation. A t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups.

[0065] 4 Experimental Results

[0066] 4.1 Effect of Jianfang Tiantian Capsules on the Degree of Melanosis in Guinea Pig Intestinal Tissue

[0067] Figure 1Photographs of the colons of the five groups of test animals are shown. Macroscopic observation reveals that the colon of the control group guinea pigs is light red, with no melanosis in the cecum or colon, and no obvious pigmentation. The colon of the model group guinea pigs is dark brown, with the cecum and its proximal colon showing distinctly dark brown discoloration; no melanosis is observed in the small intestine. Comparison of the colons of the model group and the control group shows that the model group exhibits significant colonic melanosis.

[0068] Although the colons of guinea pigs in the low-dose, medium-dose, and high-dose groups still exhibited varying degrees of melanosis, the overall color of their colons was lighter than that of the model group. Specifically, the colons of guinea pigs in the low-dose and medium-dose groups were brown, with melanosis appearing in the cecum and part of the proximal colon; no melanosis was observed in the remaining small intestine and other tissues. Figure 1 It can be seen that the color of the colon of guinea pigs in the low-dose group was slightly darker than that in the medium-dose group, especially in the cecum and proximal colon.

[0069] In the high-dose group, the colons of guinea pigs were light brown. Although melanosis was observed in the cecum and part of the proximal colon, the area of ​​melanosis was significantly reduced and the length of melanosis was noticeably shorter. No melanosis was observed in other tissues such as the small intestine. Figure 1 It can be seen that the colon color of the high-dose group guinea pigs was slightly lighter than that of the medium-dose group guinea pigs, especially in the cecum and proximal colon, and the length of colonic melanosis was shorter. More importantly, the colon color of the high-dose group guinea pigs was close to that of the control group guinea pigs.

[0070] It should be noted that, due to the large size of the cecal tissue, it was inconvenient to uniformly photograph and collect tissue samples. Furthermore, the cecum of guinea pigs in the low-dose, medium-dose, and high-dose groups all showed melanosis, and the degree of melanosis was similar. Therefore, in... Figure 1 Its cecum was not shown in the image.

[0071] The percentage of relative melanosis area in the colon of guinea pigs in five groups was obtained using ImageJ software, and statistical analysis of the percentage of relative melanosis area was performed using GraphPad Prism 8.0 software. The results are as follows: Figure 2 As shown. Figure 2 In the diagram, ### indicates a highly significant difference between the group and the control group (P < 0.001), * indicates a significant difference between the group and the model group (P < 0.05), and *** indicates a highly significant difference between the group and the model group (P < 0.001).

[0072] according to Figure 2It was found that, compared with the control group, the percentage of relative melanosis area in the colon of guinea pigs in the model group was significantly increased (P < 0.001), indicating that significant melanosis occurred in the model group. Compared with the model group, the percentage of relative melanosis area in the colon of guinea pigs in the low-dose group was significantly decreased (P < 0.05); compared with the model group, the percentage of relative melanosis area in the colon of guinea pigs in the medium-dose and high-dose groups was significantly decreased (P < 0.001). Therefore, it can be concluded that the low-dose, medium-dose, and high-dose groups of Jianfang Tiantian capsules can both reduce the accumulation of lipofuscin in the colon and clear the lipofuscin already deposited in the colon, thus having the effect of preventing and improving melanosis. Furthermore, from... Figure 2 It can be clearly seen that the percentage of relative melanosis in the colon of guinea pigs in the high-dose group is close to that in the control group.

[0073] 4.2 Effects of Jianfang Tiantian Capsules on Pathological Changes in Guinea Pig Intestinal Tissue

[0074] Figure 3 A schematic diagram of HE staining in five groups of test animals is shown. According to... Figure 3 It was found that the colonic mucosa of the control group guinea pigs was intact, with glands and crypts clearly visible. No inflammatory cell infiltration was observed in the lamina propria and submucosa, and no macrophages containing brownish granules were found in the lamina propria. In contrast, the model group showed partial shedding of colonic mucosal epithelial cells, significant neutrophil infiltration in the lamina propria and submucosa, reduced crypts and glands, disordered structure, and numerous brownish pigment granules deposited between the muscularis mucosae and lamina propria.

[0075] In the low-dose, medium-dose, and high-dose groups, only a small number of colonic mucosal epithelial cells were shed, and only a small number of neutrophils were observed infiltrating the lamina propria and submucosa. Scattered brownish-yellow pigment granules were present in the lamina propria. Compared with the model group, the shedding of colonic mucosal epithelial cells, neutrophil infiltration, and the presence of brownish-yellow pigment granules were significantly improved, with the medium-dose group showing better results than the low-dose group, and the high-dose group showing better results than the medium-dose group. Furthermore, the high-dose group had very few brownish-yellow pigment granules, essentially approaching the level of the control group.

[0076] Figure 4 A schematic diagram of melanin staining in five groups of test animals is shown. According to... Figure 4 It can be seen that no positive staining granules appeared in the control group, while a large number of positive staining granules appeared in the model group. Although the low-dose, medium-dose, and high-dose groups all showed varying degrees of positive staining granules, the degree of positivity in all three groups was milder compared to the model group. Furthermore, the medium-dose group had fewer positive staining granules than the low-dose group, and the high-dose group had fewer positive staining granules than the medium-dose group. In addition, the high-dose group had very few positive staining granules, almost approaching the level of the control group.

[0077] The positive area of ​​melanin staining was quantified, and the percentage of the relative area of ​​melanin region was as follows: Figure 5 As shown. Figure 5 The relative percentage of melanin area in the five groups of test animals is shown. Figure 5 In the diagram, ### indicates a highly significant difference between the group and the control group (P < 0.001), * indicates a significant difference between the group and the model group (P < 0.05), and *** indicates a highly significant difference between the group and the model group (P < 0.001).

[0078] according to Figure 5 It was found that, compared with the control group, the relative percentage of melanin area in the model group of guinea pigs was significantly increased (P < 0.001), indicating that the model group showed obvious melanosis coli. Compared with the model group, the relative percentage of melanin area in the medium-dose group was significantly decreased (P < 0.05), and the relative percentage of melanin area in the high-dose group of guinea pigs was also significantly decreased (P < 0.001). Therefore, it can be concluded that the medium-dose and high-dose groups of Jianfang Tiantian capsules can both reduce the accumulation of lipofuscin in the colon and clear the lipofuscin already deposited in the colon, thus having the effect of preventing and improving melanosis coli. Furthermore, from... Figure 5 It can be clearly seen that the percentage of the relative area of ​​melanin region in the high-dose group of guinea pigs is close to that in the control group.

Claims

1. The use of a composition in the preparation of a medicine for improving melanosis coli, said composition comprising the following raw materials: Pumpkin skin 33.7wt%, pumpkin stem 20.8wt%, pumpkin fiber 12.9wt%, Poria cocos 7.9wt%, Tremella fuciformis 9.0wt%, and xylooligosaccharides 15.7wt%; the melanosis coli was induced by aloe vera.

Images