A composition for moistening the intestines and relieving constipation, reducing blood sugar and losing weight, and anti-aging and promoting sleep

Abstract

The present invention provides a composition for promoting bowel movement, reducing blood sugar, losing weight, anti-aging and promoting sleep, which can be used in the fields of biology, medicine, etc. In some embodiments, the composition comprises an appropriate amount of water (optional), recombinant human-like collagen (FLM1.6PHPV fusion factor, hereinafter referred to as fusion factor), γ-aminobutyric acid, chia seeds, walnut kernels, carrots, and cassia seeds, and the amino acid sequence of the recombinant human-like collagen is as shown in SEQ ID NO:1. The present invention also provides the recombinant human-like collagen and a drug containing the composition. The composition and protein of the present invention unexpectedly have the functions of promoting bowel movement, reducing blood sugar, losing weight, anti-aging and promoting sleep to a certain extent, and have good market prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a composition for moistening the intestines and relieving constipation, reducing blood sugar and losing weight, and anti-aging and promoting sleep. Background Art

[0002] Lactoferrin is an iron-binding lactoprotein that can promote the growth of specific probiotic strains and can affect the growth and diversification of the gut microbiota, including (i) enhancing the permeability of the epithelial cell monolayer and (ii) providing signals to balance the anti-inflammatory and pro-inflammatory responses that lead to gut homeostasis. The effects of lactoferrin on the intestinal barrier are mainly reflected in the following aspects: by promoting the proliferation of intestinal epithelial cells, increasing the expression of tight junction proteins, and improving the intestinal tissue morphology to repair the damaged intestinal physical barrier; by activating intestinal immune cells and regulating the secretion of cytokines to protect the intestinal immune barrier; by positively regulating the distribution of the gut microbiota to protect the intestinal biological barrier; by enhancing the expression of mucin and regulating the secretion of antimicrobial peptides to protect the intestinal chemical barrier. Generally speaking, lactoferrin has a variety of biological functions, including antibacterial activity, anti-inflammatory activity, intestinal barrier protection, and immune cell regulation, and is involved in maintaining the intestinal mucosal immune homeostasis. It has been reported that lactoferrin can reduce cognitive impairment caused by a Western diet through the microbiota-gut-brain axis.

[0003] Intestinal collagen plays an important role in intestinal health, mainly reflected in the following aspects: enhancing the intestinal barrier, collagen helps to support and strengthen the protective layer of the digestive tract and prevent the occurrence of leaky gut syndrome; promoting tissue repair, when the intestine is injured, the accumulation of collagen can stimulate cell regeneration and help repair the damaged tissue; improving the intestinal inner wall, collagen is rich in glycine, which helps to rebuild the intestinal inner wall tissue and reduce intestinal inflammation; regulating intestinal function, the amino acids in collagen are considered to help regulate the production of gastric acid and prevent gastric ulcers. In summary, intestinal collagen is crucial for maintaining healthy intestinal function.

[0004] Interestingly, it has been reported that collagen peptides can exhibit probiotic activity by interacting with the gut microbiota, and the gut microbiota can reduce the symptoms associated with metabolic diseases by producing short-chain fatty acids and branched-chain fatty acids.

[0005] γ-Aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system. It has been confirmed that GABA has food safety and physiological effects such as improving sleep quality and lowering blood pressure. Modern pharmacological research shows that the main components of Cassia obtusifolia include anthraquinones, phenylpropyrrolidones, fatty acids, glycosides, etc., and have the effects of protecting the liver, improving eyesight, neuroprotection, and promoting sleep. The GI value of chia seeds is less than 55, belonging to low-GI foods; the GI value refers to the value of how quickly blood sugar rises after a food enters the body. Low-GI foods have a slower blood sugar-rising speed, so they help prevent excessive blood sugar fluctuations in the human body and maintain cardiovascular health. The latest research shows that carrots have the potential to improve blood sugar regulation and promote a healthier gut microbiota, and may provide a natural and possibly side-effect-free adjuvant diet therapy for diabetic patients. It is reported that ingesting 2-3 walnut kernels per day can reduce total blood cholesterol and low-density cholesterol. Summary of the Invention

[0006] In one aspect of the present invention, there is provided a composition for relieving constipation, reducing blood sugar and losing weight, anti-aging and promoting sleep, and the composition contains a protein with an amino acid sequence shown in SEQ ID NO:1. The applicant names the protein with an amino acid sequence shown in SEQ ID NO:1 as FLM1.6 PHPV fusion factor.

[0007] In another aspect of the present invention, there is provided a composition for relieving constipation, reducing blood sugar and losing weight, anti-aging and promoting sleep, and the composition contains recombinant human-like collagen, γ-aminobutyric acid, chia seeds, walnut kernels, carrots, and Cassia obtusifolia, and the amino acid sequence of the recombinant human-like collagen is shown in SEQ ID NO:1.

[0008] Walnut kernels are high in plant fat and protein, with 70% fat and 17%-27% protein. Walnut kernels not only contain elements such as calcium, iron, and phosphorus, but also contain minerals such as zinc, manganese, and chromium that are indispensable for the human body. A variety of vitamins, proteins, and minerals are all nutrients beneficial to the beauty of the appearance. The linoleic acid in the plant fat rich in walnut kernels is regarded as a beauty agent that can moisten the skin. It can make the skin moist, smooth and elastic.

[0009] In some specific embodiments of the present invention, the composition contains 0.05-0.2 parts by weight of recombinant human-like collagen, 0.05-0.2 parts by weight of γ-aminobutyric acid, 1.5-2.5 parts by weight of chia seeds, 0.5-2 parts by weight of walnut kernels, 0.5-2 parts by weight of carrots, and 0.5-2 parts by weight of Cassia obtusifolia.

[0010] In some specific embodiments of the present invention, the composition comprises 0.1 part by weight of recombinant collagen-like protein, 0.1 part by weight of γ-aminobutyric acid, 2 parts by weight of chia seeds, 1 part by weight of walnut kernels, 1 part by weight of carrots, and 1 part by weight of cassia seeds.

[0011] In some specific embodiments of the present invention, the chia seeds are chia seed powder. In some specific embodiments of the present invention, the walnut kernels are walnut kernel powder. In some specific embodiments of the present invention, the carrots are carrot powder. In some specific embodiments of the present invention, the cassia seeds are cassia seed powder.

[0012] In some specific embodiments of the present invention, the cassia seeds are the mature seeds of Cassia obtusifolia.

[0013] In some specific embodiments of the present invention, the composition further comprises water. In some specific embodiments of the present invention, the composition comprises an appropriate amount of water.

[0014] In some specific embodiments of the present invention, the composition further comprises 20 - 30 parts by weight of water. In some specific embodiments of the present invention, the composition further comprises 25 parts by weight of water.

[0015] In some specific embodiments of the present invention, the preparation method of the composition is as described in Example 2. In some specific embodiments of the present invention, the preparation method of the composition is as described in Example 3.

[0016] In some specific embodiments of the present invention, the composition comprises 0.1 part by weight of recombinant collagen-like protein, 0.1 part by weight of γ-aminobutyric acid, 2 parts by weight of chia seeds, 1 part by weight of walnut kernels, 1 part by weight of carrots, 1 part by weight of cassia seeds, and 20 - 30 parts by weight of water.

[0017] In some specific embodiments of the present invention, the composition comprises 0.1 part by weight of recombinant collagen-like protein, 0.1 part by weight of γ-aminobutyric acid, 2 parts by weight of chia seed powder, 1 part by weight of walnut kernel powder, 1 part by weight of carrot powder, 1 part by weight of cassia seed powder, and 20 - 30 parts by weight of water.

[0018] In some specific embodiments of the present invention, the composition comprises 0.1 part by weight of recombinant collagen-like protein, 0.1 part by weight of γ-aminobutyric acid, 2 parts by weight of chia seed powder, 1 part by weight of walnut kernel powder, 1 part by weight of carrot powder, 1 part by weight of cassia seed powder, and 25 parts by weight of water.

[0019] In some specific embodiments of the present invention, the composition is a composition for promoting bowel movement, reducing blood sugar and weight, and anti-aging and promoting sleep.

[0020] In another aspect of the present invention, there is provided a protein for moistening the intestines and relieving constipation, reducing blood sugar and losing weight, and anti-aging and promoting sleep, and the amino acid sequence of the protein is as shown in SEQ ID NO: 1 or 3.

[0021] In some specific embodiments of the present invention, the amino acid sequence of the protein is as shown in SEQ ID NO: 1. In some specific embodiments of the present invention, the amino acid sequence of the protein is as shown in SEQ ID NO: 3.

[0022] In another aspect of the present invention, there is provided a nucleic acid encoding the protein as described in the present invention.

[0023] In another aspect of the present invention, there is provided a drug for moistening the intestines and relieving constipation, reducing blood sugar and losing weight, and anti-aging and promoting sleep, and the drug comprises the composition as described in the present invention or the protein as described in the present invention.

[0024] In some specific embodiments of the present invention, the drug comprises the composition as described in the present invention. In some specific embodiments of the present invention, the drug comprises the protein as described in the present invention.

[0025] In another aspect of the present invention, there is provided a powder for moistening the intestines and relieving constipation, reducing blood sugar and losing weight, and anti-aging and promoting sleep, and the powder comprises the composition as described in the present invention or the protein as described in the present invention.

[0026] In another aspect of the present invention, there is provided a compressed tablet candy, and the compressed tablet candy comprises the composition as described in the present invention or the protein as described in the present invention.

[0027] In another aspect of the present invention, there is provided the use of the composition as described in the present invention or the protein as described in the present invention in moistening the intestines and relieving constipation, reducing blood sugar and losing weight, and anti-aging and promoting sleep.

[0028] In another aspect of the present invention, there is provided the use of the composition as described in the present invention or the protein as described in the present invention in the preparation of a drug for moistening the intestines and relieving constipation, reducing blood sugar and losing weight, and anti-aging and promoting sleep.

[0029] In some specific embodiments of the present invention, there is provided the use of the composition as described in the present invention in the preparation of a drug for relieving constipation, reducing blood sugar, and anti-aging.

[0030] In some specific embodiments of the present invention, there is provided the use of the composition as described in the present invention in the preparation of a drug for relieving constipation. In some specific embodiments of the present invention, there is provided the use of the composition as described in the present invention in the preparation of a drug for reducing blood sugar. In some specific embodiments of the present invention, there is provided the use of the composition as described in the present invention in the preparation of a drug for anti-aging.

[0031] In some specific embodiments of the present invention, there is provided the use of the protein as described in the present invention in the preparation of a drug for laxative, blood sugar lowering, and anti-aging effects.

[0032] In some specific embodiments of the present invention, there is provided the use of the protein as described in the present invention in the preparation of a drug for laxative effects. In some specific embodiments of the present invention, there is provided the use of the protein as described in the present invention in the preparation of a drug for blood sugar lowering effects. In some specific embodiments of the present invention, there is provided the use of the protein as described in the present invention in the preparation of a drug for anti-aging effects.

[0033] The composition and protein of the present invention have both strong effects of moistening the intestines and relieving constipation, as well as certain effects of reducing blood sugar, losing weight, anti-aging, and promoting sleep. It has good market prospects. Specific Embodiments

[0034] The following further illustrates the present disclosure by way of examples, but does not limit the present disclosure to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or according to the product instructions. Unless otherwise specified, the reagents and raw materials used in the present disclosure are all commercially available.

[0035] Example 1. Design and Preparation of Recombinant Collagen-like Protein (Fusion Factor)

[0036] Based on years of in-depth research in the field of collagen, the applicant designed a recombinant collagen-like protein (named FLM1.6 PHPV fusion factor, abbreviated as fusion factor), and its sequence is shown in SEQ ID NO:1. The protein architecture is PEP-1-linker-collagen-like sequence-linker-lactoferrin-like sequence-His tag. The fusion factor PHPV is prominent and contains a collagen fragment with optimized sequence, which can repair intestinal mucosal damage; it contains a bio-defense protein fragment with optimized sequence (a chimera of the conserved sequences of bovine and human lactoferricin), which can improve the intestinal microecology.

[0037] The coding nucleic acid fragment of the recombinant collagen-like protein (fusion factor) (codon-optimized for Escherichia coli) was cloned into the BlpI and XbaI restriction enzyme sites of the pET28a(+) vector. The reaction solution was transformed into Stbl3 competent cells and cultured for 15 h, and then positive clones were selected for sequencing to identify. Then the positive clones with correct sequencing were cultured, and the expression vector plasmid of the recombinant collagen-like protein (SEQ ID NO:2) was extracted.

[0038] The obtained vector plasmid was transformed into BL21(DE3) E. coli competent cells (NEB, C2527I). It was cultured in LB medium containing kanamycin at 37 °C for 16 h. IPTG was added to a concentration of 0.5 mM and induced at 16 °C for 19 h. The cells were centrifuged and collected, lysate was added and the cells were broken by sonication, and then centrifuged again to obtain the supernatant containing recombinant collagen-like protein. It was filtered using a 0.45 μm filter membrane, and then purified using a Ni-NTA affinity chromatography column and ion chromatography chromatography. The eluate was freeze-dried to obtain recombinant collagen-like protein (SEQ ID NO:1) with a purity of 96.1%. It showed only a single band in SDS-PAGE.

[0039] Collagen-like protein (SEQ ID NO:3) was prepared using the same method. The protein structure was PEP-1-linker-collagen sequence-linker-lactoferrin sequence-His tag.

[0040] Example 2. Preparation of the composition

[0041] Fresh and mature carrots were taken, peeled and cut into small pieces with a length, width and height of 2 - 3 mm. The carrot pieces were placed in a hot air dryer at 60 °C and dried for about 6 - 10 hours until the water content dropped to 6% - 10%. The dried carrot pieces were ground into carrot powder with an average particle size of about 100 microns using a grinder.

[0042] Dry and clean chia seeds were taken and ground into chia seed powder with an average particle size of about 100 microns using a grinder.

[0043] Dry and clean walnut kernels were taken and cryogenically ground into walnut kernel powder with an average particle size of about 200 microns using liquid nitrogen.

[0044] Dry and clean semen cassiae (mature seeds of Cassia obtusifolia) were taken and ground into semen cassiae powder with an average particle size of about 80 microns using a grinder.

[0045] 100 mg of recombinant collagen-like protein (fusion factor), 100 mg of γ-aminobutyric acid, 2 g of chia seed powder, 1 g of walnut kernel powder, 1 g of carrot powder, and 1 g of semen cassiae powder were taken and mixed evenly to obtain a solid powder, which was composition CTS-1.

[0046] 100 mg of collagen-like protein (SEQ ID NO:3), 100 mg of γ-aminobutyric acid, 2 g of chia seed powder, 1 g of walnut kernel powder, 1 g of carrot powder, and 1 g of semen cassiae powder were taken and mixed evenly to obtain a solid powder, which was composition CTS-2.

[0047] Take 100 mg of γ-aminobutyric acid, 2 g of chia seed powder, 1 g of walnut kernel powder, 1 g of carrot powder, and 1 g of cassia seed powder, mix them evenly to obtain a solid powder, which is composition CTS-3.

[0048] Example 3. Laxative function test

[0049] Take adult C57BL / 6 mice (female), weighing 19.7 - 20.3 g, randomly divide them into groups with 10 mice in each group. Intragastrically administer the modeling drug compound diphenoxylate to obtain a mouse constipation model. By intragastrically administering ink, measure the time for the first black stool to be excreted, the number of stools excreted within 5 hours, and the weight of the stools excreted by the mice, so as to reflect the defecation situation of the model mice.

[0050] Respectively stir each composition (CTS-n, n is the serial number) prepared by the method described in Example 2 in sterile water at room temperature for 5 minutes to obtain a uniform solid-liquid mixture with a concentration of 200 mg composition / mL (CTS-n-L, n is the serial number, corresponding one-to-one with the serial number n of CTS-n).

[0051] Stir the recombinant human-like collagen (fusion factor) prepared by the method described in the foregoing examples in sterile water at room temperature for 5 minutes to obtain a uniform suspension with a concentration of 20 mg / mL.

[0052] Group 1 mice were intragastrically administered the solid-liquid mixture CTS-1-L, group 2 mice were intragastrically administered the composition CTS-2-L, group 3 mice were intragastrically administered the composition CTS-3-L, and group 4 mice were intragastrically administered the recombinant human-like collagen suspension; administer once a day by gavage. The administered volume is 10 mL / kg body weight. Normal control group and model group mice were given 10 mL / kg body weight of sterile water.

[0053] After continuous gavage for 8 days, the mice in each group were fasted but allowed to drink water for 18 hours. The normal control group was given normal saline, and group 1 - group 4 and model group mice were intragastrically administered a suspension of compound diphenoxylate tablets in water at a dose of 25 mg diphenoxylate / kg body weight (diphenoxylate concentration 2.5 mg / mL). 30 minutes later, each group of mice was intragastrically administered an equal amount of ink. Starting from the administration of the ink, record the excretion time of the first black stool of each mouse and the weight of the black stools excreted during a specific time period. Specifically as shown in Table 1, each data is the average value of the mice within the group. The weight of the black stools excreted within 12 h in group 1 and group 4 was significantly higher than that in the model group, group 2, and group 3, with statistically significant differences (P < 0.05) respectively; in addition, the excretion time of the first black stool in group 1 and group 4 was significantly shorter than that in the model group, group 2, and group 3 (P < 0.05). It shows that both composition 1 and recombinant human-like collagen have strong laxative functions.

[0054] Table 1. Results of laxative function test

[0055]

[0056] Example 4. Hypoglycemic Function Test

[0057] Male healthy Kunming mice (weighing 19.0 - 22.3 g) were taken. After normal feeding for 3 days, they were fasted for 15 hours without water deprivation. 10 mice were randomly selected as the normal control group; the other mice were used for modeling. Each mouse was intraperitoneally injected with 160 mg / kg streptozotocin. After 48 hours, the fasting blood glucose of the mice was measured. Mice with a blood glucose concentration higher than 11.1 mmol / L were considered to have successfully established the diabetes model.

[0058] Then the model mice were randomly divided into groups of 10 each.

[0059] The test group was gavaged with the test substances once a day, namely recombinant collagen-like protein (SEQ ID NO: 1) and collagen-like protein (SEQ ID NO: 3). After being stirred in water at 50 mg / mL for 1 hour to disperse evenly, they were gavaged, and the administered volume was 10 mL / kg body weight.

[0060] Another group of mice was gavaged with the solid-liquid mixture CTS-1-L (200 mg composition CTS-1 / mL) prepared by the method of the previous example once a day, and the administered volume was 10 mL / kg body weight.

[0061] The normal control group and the model group were gavaged with an equal volume of normal saline once a day for 12 consecutive days. After fasting for 12 hours on the last day of gavage, the fasting blood glucose was measured (the results are expressed as the mean), and the results are shown in Table 2.

[0062] The fasting blood glucose of the mice in the recombinant collagen-like protein group after administration was significantly lower than that of the model group, with a statistically significant difference (P < 0.05), and was also significantly lower than that of the collagen-like protein group (P < 0.05). The fasting blood glucose of the mice in the solid-liquid mixture CTS-1-L group after administration was also significantly lower than that of the model group (P < 0.05), indicating that the recombinant collagen-like protein and the solid-liquid mixture CTS-1-L have a certain hypoglycemic function, and thus achieved unexpected technical effects.

[0063] Table 2. Fasting Blood Glucose of Mice

[0064]

[0065] Example 5. Anti-aging Function Test

[0066] Male C57BL / 6 mice at 8 weeks of age and weighing 19.1 - 21.2 g were taken and randomly divided into groups of 10 animals each.

[0067] An aging mouse model was obtained by intraperitoneal injection of 200 mg / kg of D-galactose, and the mice in the blank group were intraperitoneally injected with an equal volume of normal saline. The solid-liquid mixture CTS-n-L prepared by the method of the foregoing example was taken, and the solid-liquid mixture (0.2 mL / d) was administered by gavage once a day starting at the same time as the modeling, for 6 weeks. The blank group and the model group were given an equal amount of sterile water. On the second day after the end of the administration, the Morris water maze experiment was conducted for testing. The results are shown in Table 3, and the average values were taken for each group.

[0068] Table 3. Test results of the water maze experiment

[0069] Grouping Latency of crossing the platform (s) Number of times of crossing the platform (times) Blank group 27.0 5.1 Model group 57.4 1.4 CTS-1-L 41.3 2.8

[0070] Compared with the model group, the latency to cross the platform in the CTS-1-L group was significantly shortened (P < 0.05), and the number of times of crossing the platform was significantly increased (P < 0.05). It was proved that CTS-1-L could improve the learning and memory abilities of aging mice to a certain extent.

[0071] Although the embodiments of the present disclosure have been described above, it can be understood that the above embodiments are exemplary and do not constitute a limitation to the present disclosure. Those of ordinary skill in the art can make changes, modifications and variations to the above embodiments within the scope of the present disclosure.

Claims

1. A composition, characterized in that The composition comprises 0.05-0.2 parts by weight of recombinant collagen, 0.05-0.2 parts by weight of γ-aminobutyric acid, 1.5-2.5 parts by weight of chia seeds, 0.5-2 parts by weight of walnut kernels, 0.5-2 parts by weight of carrots, and 0.5-2 parts by weight of cassia seeds; the amino acid sequence of the recombinant collagen is shown in SEQ ID NO:

1.

2. The composition according to claim 1, characterized in that The composition comprises 0.1 parts by weight of recombinant collagen, 0.1 parts by weight of gamma-aminobutyric acid, 2 parts by weight of chia seeds, 1 part by weight of walnut kernels, 1 part by weight of carrots, and 1 part by weight of cassia seeds.

3. The composition according to claim 1, characterized in that The chia seeds are chia seed powder, the walnut kernels are walnut kernel powder, the carrots are carrot powder, and the cassia seeds are cassia seed powder.

4. The composition according to claim 1, characterized in that The composition comprises a suitable amount of water.

5. A protein, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO:

1.

6. A drug, characterized in that The medicine comprises the composition according to any one of claims 1 to 4.

7. Use of the composition according to any one of claims 1 to 4 in the preparation of medicaments for laxative, hypoglycemic and anti-aging effects.

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