Oligomeric agaro-oligosaccharide oral solution with constipation relieving function

Abstract

The invention discloses an agaro-oligosaccharide oral solution with a constipation relieving function, and belongs to the technical field of food biology. The invention provides a preparation method of agarotriose. Main components which mainly exert anti-inflammatory activity are analyzed through clinical evaluation. According to the invention, agarotriose is extracted by adopting an enzymolysis method to treat LOP-induced constipation mice, and the influence of main components on model mice is investigated from the aspects of excrement water content, small intestine propulsion rate, first-grain black excrement discharge time, colon pathological structure and the like. Experimental results show that compared with an LOP group, the agarotriose high-dose group can significantly improve constipation symptoms, relieve inflammatory response and promote colonic mucosa repair, so that agarotriose can be used as a good functional food for relieving constipation.

Description

TECHNICAL FIELD

[0001] The present application relates to a low oligosaccharide oral solution with the function of relieving constipation, belonging to the technical field of biological medicine. BACKGROUND

[0002] Constipation is a common intestinal functional disorder, and its exact pathogenesis has not been fully elucidated. Clinically, it is mainly characterized by difficult defecation, reduced defecation frequency (less than 3 times per week), dry and hard stool, and a sense of incomplete defecation. Due to its prolonged course, it can easily lead to abdominal distension, abdominal pain and other discomfort symptoms, and can also induce complications such as hemorrhoids and anal fissure, which significantly affects the quality of life of patients. Current clinical treatment relies on stimulant laxatives, osmotic laxatives and prokinetic drugs to promote defecation, which can achieve short-term effects, but long-term use can easily cause electrolyte imbalance, drug dependence and intestinal function degradation, and has a high risk of recurrence after drug withdrawal. Therefore, developing a mild and non-stimulating product that can long-term regulate the intestinal tract without side effects has become an important research direction to improve the current treatment of constipation. SUMMARY

[0003] In view of the deficiencies of the prior art, the present application provides a low oligosaccharide oral solution with the function of relieving constipation, aiming to solve the technical problem of the lack of natural active ingredients with anti-inflammatory activity that can effectively relieve or assist in improving constipation and have few side effects in the prior art.

[0004] The present application provides the use of low oligosaccharide in the preparation of a drug for relieving constipation.

[0005] In one embodiment, the drug has low oligosaccharide as the only active ingredient.

[0006] In one embodiment, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral solution.

[0007] In one embodiment, the relief of constipation includes but is not limited to at least one of the following effects: (1) increasing the water content of the stool of constipated individuals; (2) shortening the time of the first black stool of constipated individuals; (3) increasing the small intestine propulsion rate of constipated individuals; (4) reducing the number of inflammatory cell infiltrates in individuals with colitis and improving it; (5) inhibiting the expression of pro-inflammatory factors IL-6, IL-1β and TNF-α in colonic tissue of individuals with colitis.

[0008] In one embodiment, the content of low oligosaccharide in the drug is 30 mg / mL.

[0009] The present application also provides the use of low oligosaccharide in the preparation of health products that help to moisten the intestines and promote defecation.

[0010] In an embodiment, the health care product is an oral solution containing the gultriose and potassium sorbate.

[0011] In an embodiment, the gultriose is prepared by the following steps: (1) enzymatic hydrolysis of agar solution with α-agarase; (2) inactivation of the enzyme in the solution after step (1); (3) centrifugation of the solution after step (2) to remove the precipitate and obtain the supernatant, which is a mixed solution of agar oligosaccharides containing gultriose; (4) centrifugation of the mixed solution prepared in step (3) and purification by gel chromatography.

[0012] In an embodiment, the concentration of agar solution in step (1) is 12.5% (w / w); and the amount of α-agarase added is 130 U / g of agar gel.

[0013] In an embodiment, the α-agarase in step (1) is α-AGA derived from Catenovulum agarivorans The nucleotide sequence encoding the α-agarase is shown as SEQ ID NO. 1, and the degradation end product of the enzyme acting on agar contains gultriose and gultetraose.

[0014] In an embodiment, the temperature of the enzymatic hydrolysis is 35°C, and the enzymatic hydrolysis time is 30 h.

[0015] In an embodiment, the purification is performed by a Superdex 30 Increase 10 / 300 GL gel chromatography column.

[0016] The present application also provides an oral solution prepared from gultriose with a final concentration of 30 mg / mL and potassium sorbate with an addition amount of 0.02%.

[0017] Beneficial effects The present application provides a new use of gultriose for relieving constipation. The gultriose prepared by enzymatic hydrolysis is used to treat LOP-induced constipation mice, and the effects of the main components on the model mice are investigated from aspects of fecal characteristics and colonic pathological structure. The experimental results show that, compared with the model group, the high-dose gultriose group can significantly improve the symptoms of constipation, reduce inflammatory reactions, and promote the repair of colonic mucosa. Therefore, gultriose can be used as a good functional food for relieving constipation. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The ion chromatogram of the product obtained in Example 2.

[0019] Figure 2 The liquid phase result of the product obtained in Example 3 is shown in the figure.

[0020] Figure 3 The liquid phase mass spectrum result of the product obtained in Example 3 is shown in the figure.

[0021] Figure 4 The picture of the feces water content of each group of mice in Example 4 is shown; wherein, CON represents the blank control group, LOP represents the loperamide group, LAOS represents the low-dose jujube oligosaccharide group, MAOS represents the medium-dose jujube oligosaccharide group, HAOS represents the high-dose jujube oligosaccharide group, and LAC represents the positive drug control group lactulose group.

[0022] Figure 5 The picture of the small intestine propulsion rate of each group of mice in Example 4 is shown; wherein, CON represents the blank control group, LOP represents the loperamide group, LAOS represents the low-dose jujube oligosaccharide group, MAOS represents the medium-dose jujube oligosaccharide group, HAOS represents the high-dose jujube oligosaccharide group, and LAC represents the positive drug control group lactulose group.

[0023] Figure 6 The picture of the first black feces excretion time of each group of mice in Example 4 is shown; wherein, CON represents the blank control group, LOP represents the loperamide group, LAOS represents the low-dose jujube oligosaccharide group, MAOS represents the medium-dose jujube oligosaccharide group, HAOS represents the high-dose jujube oligosaccharide group, and LAC represents the positive drug control group lactulose group.

[0024] Figure 7 The picture of the colon tissue H&E staining of each group of mice in Example 4 is shown; wherein, CON represents the blank control group, LOP represents the loperamide group, HAOS represents the high-dose jujube oligosaccharide group, and LAC represents the positive drug control group lactulose group. DETAILED DESCRIPTION

[0025] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are for better explaining the present application and are not used to limit the present application.

[0026] (I) Test method: 1. DNS method for measuring Ca-AGA enzyme activity: The Ca-AGA hydrolytic activity was characterized by the change of reducing sugar content determined by 3,5-dinitrosalicylic acid (DNS) method. The 0.15% (w / w) agarose substrate solution was prepared by Tris-HCl buffer (20 mM, pH 8.0). 0.1 mL enzyme solution was directly taken and added into 0.9 mL agarose solution, and then 1 mL DNS was added to terminate the reaction after 30 min reaction at 35°C. After 5 min boiling water bath, the sample was quickly cooled, 2 mL deionized water was added for dilution, and then the absorbance value was determined at 540 nm. The inactivated enzyme was used as a control, and the standard curve was drawn by using galactose as a standard.

[0027] Enzyme activity definition: the enzyme activity per unit (U) was defined as the amount of enzyme required to produce 1 μmol of reducing sugar (calculated as galactose) per minute.

[0028] 2. Detection of trisaccharide or pentasaccharide The sample after agarase hydrolysis by Ca-AGA was boiled for 15 min to inactivate the enzyme, and then centrifuged, diluted, and filtered through a 0.22 μm water phase filter membrane. The product was analyzed by high performance anion exchange chromatography system (HPAEC-PAD) and ultra performance liquid chromatography-electrospray-quadrupole-time of flight mass spectrometer (UPLC-Q-TOF-MS). The conditions of HPAEC-PAD were as follows: Dionex CarboPac PA-200 anion exchange column, column temperature 35°C, mobile phase 100 mM NaOH, 100 mM NaOH & 500 mM NaAc, and flow rate 0.5 mL / min. The conditions of UPLC-Q-TOF-MS were as follows: ACQUITY UPLC BEH Amide chromatographic column, column temperature 40°C, mobile phase A 0.1% ammonia water-acetonitrile solution, B water-methanol (2:1, V / V) solution, flow rate 0.3 mL / min, and mass spectrometry analysis was performed in negative ionization mode by using electrospray ionization (ESI) source.

[0029] 3. Calculation of trisaccharide yield and yield The agarase hydrolysis solution had a mass fraction of Q (dry basis), a total mass of M g, an enzyme addition amount of N g, a sample amount after hydrolysis of m g, a total volume after dilution and constant volume of V mL. The sample was further diluted n times and filtered, and then ion chromatography detection was performed. The trisaccharide standard had a concentration of 10 ug / mL, and the peak area corresponding to the standard was S1. The total peak area corresponding to the hydrolysis sample was S2.

[0030] The mass of trisaccharide contained in the sample was Y (g): ; The total trisaccharide content in the agarase hydrolysis solution was Z (g): ; The yield of gulose in the product is P (g / g agar): ; The yield of gulose in the product is R (%): ; Therefore, the following can be obtained: ; .

[0031] (II) Reagents and materials: LB liquid medium: yeast powder (National Pharmaceutical Group) 5 g / L, tryptone (Shanghai Maierye) 10 g / L, NaCl (National Pharmaceutical Group) 10 g / L, pH 7.0.

[0032] LB solid medium: yeast powder (National Pharmaceutical Group) 5 g / L, tryptone (Shanghai Maierye) 10 g / L, NaCl (National Pharmaceutical Group) 10 g / L, pH 7.0, 1.5% (w / v) agar (National Pharmaceutical Group).

[0033] Fermentation liquid medium: yeast powder (National Pharmaceutical Group) 12 g / L, tryptone (Shanghai Maierye) 24 g / L, sucrose (National Pharmaceutical Group) 5 g / L, KH2PO4 (National Pharmaceutical Group) 17 mM, K2HPO4 (National Pharmaceutical Group) 72 mM, pH 9.0.

[0034] Plasmid pET-28a(+) and strain E. coli JM109 are commercial plasmids and strains.

[0035] Example 1 Expression of recombinant alpha-carrageenase (1) Strain construction: The restriction endonuclease Nco I and Xho I were used to clone the recombinant Ca-aga gene (nucleotide sequence as shown in SEQ ID NO. 1) into plasmid pET-28a(+), and the obtained expression vector pET-28a(+) / Ca-aga was transformed into E. coli JM109, and coated on plates containing kanamycin, and the transformants were picked for sequencing and double enzyme digestion verification. The expression vector with correct sequencing was transformed into competent cells E. coli BL21(DE3), and the recombinant strain E. coli BL21(DE3) / pET-28a(+) Ca-aga was obtained by culture.

[0036] (2) Plate streaking: Using a loop to dip the recombinant strain E. coli BL21(DE3) / pET-28a(+) Ca-aga The bacterial liquid was streaked on LB solid medium containing 100 μg / mL kanamycin and incubated in a 37℃ constant temperature incubator for 12 h.

[0037] (3) Activation: A single colony was picked into LB medium containing 100 μg / mL kanamycin and incubated at 37℃, 200 rpm for 8~12 h to prepare the seed liquid.

[0038] (4) Fermentation: The seed liquid obtained in step (3) was transferred to 50 mL fermentation medium containing 100 μg / mL kanamycin at a 4% (v / v) inoculation amount, and the OD 600 value reached 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG, final concentration 0.01 mM) was added, and the flask was incubated at 25℃, 200 rpm for 20 h; the fermentation broth was collected, centrifuged at 4℃, 10000 rpm for 20 min to obtain the bacterial cells, and the bacterial cells were resuspended with an equal volume of lysis buffer (20 mM Tris-HCl, pH 8.0) and treated with an ultrasonic cell disruptor for 15 min.

[0039] The supernatant was obtained by low-temperature high-speed centrifugation, and the Ca-AGA enzyme activity was measured by the DNS method to be 80.79 U / mL.

[0040] Example 2: Method for preparing gulose The specific steps are as follows: (1) 25 g of agar was weighed and added to 200 mL of ionized water, stirred at 60℃, 350 rpm for 10 min, and mixed uniformly to obtain agar gel with a concentration of 12.5% (w / w); (2) After the temperature of the water bath was reduced to 35℃, 130 U / g of Ca-AGA crude enzyme liquid was added, and after 30 h of enzymolysis at 35℃, the enzyme was inactivated by boiling for 15 min; (3) The enzyme solution obtained in step (2) was centrifuged in a centrifuge for 10 min, and the supernatant was taken and the precipitate was removed; (4) 1 g of the enzyme solution obtained in step (3) was taken and diluted to 10 mL, and after dilution 2000 times, it was detected by HPAEC-PAD through a 0.22 μm water phase filter membrane, and the results are shown in Figure 1 The main product was gulose; (5) The reaction system obtained in step (4) was first freeze-dried and then purified by a Superdex 30 Increase 10 / 300 GL gel chromatography column to obtain chitotriose with a purity of 98.3%; (6) The yield and yield rate of chitotriose were calculated.

[0041] The results show that the yield of chitotriose is 0.3638 g / g of agar, and the yield rate of chitotriose is 36.38%.

[0042] Example 3: Isolation and characterization of chitotriose The reaction system obtained in step (3) of Example 2 was centrifuged to obtain the supernatant, which was freeze-dried and concentrated and weighed. After re-dissolving, the sample was separated by size exclusion chromatography (SEC) using a Superdex 30 Increase 10 / 300 GL column. Milli-Q water was used as the eluent at a flow rate of 0.1 mL / min, and the sample was collected and analyzed by HPAEC-PAD and UPLC-Q-TOF-MS to verify the purity of chitotriose.

[0043] The ion chromatogram of the obtained product is shown in Figure 2 The purified chitotriose has no other saccharides and has a high purity of up to 98.3%.

[0044] The liquid chromatography-mass spectrometry result of the obtained product is shown in Figure 3 The component analysis is as follows: chitotriose [M-H] - The double-charge ion (m / z 242.0021) in the mass spectrum appears at 1 / 2 mass of the single-charge ion (m / z 485.0080) with the same mass unit, and the two peaks are the same substance. The mass spectrometry result further shows that high-purity chitotriose is obtained after separation and purification.

[0045] Example 4: Application of chitotriose in relieving LOP-induced constipation in mice (1) Animal experiments were carried out using chitotriose prepared in Example 3 as raw material at different dosages, and the preparation method of chitotriose referred to Examples 1-2.

[0046] 48 7-week-old male Balb / C mice (20~22 kg) purchased from Vantian Lihua were equally divided into 6 groups, 8 mice in each group, namely control group (CON group), loperamide group (LOP group), low-dose jujube tri-saccharide group (LAOS group, 50 mg / kg), medium-dose jujube tri-saccharide group (MAOS group, 100 mg / kg), high-dose jujube tri-saccharide group (HAOS group, 200 mg / kg) and positive drug group (LAC lactulose group). All mice were adaptively fed for 7 days, and the intervention period started from the first day. The blank control group was given PBS phosphate buffer by gavage every day. The LOP group was given PBS phosphate buffer by gavage for the first seven days, and then given loperamide (LOP) by gavage for the next seven days, with a dose of 10 mg / kg bw. The low-dose jujube tri-saccharide group (50 mg / kg) was given low-dose jujube tri-saccharide by gavage for the first seven days, and then given loperamide solution (LOP) by gavage for the first hour and low-dose jujube tri-saccharide by gavage for the next hour. The medium-dose jujube tri-saccharide group (100 mg / kg) was given medium-dose jujube tri-saccharide by gavage for the first seven days, and then given loperamide solution (LOP) by gavage for the first hour and medium-dose jujube tri-saccharide by gavage for the next hour. The high-dose jujube tri-saccharide group (200 mg / kg) was given high-dose jujube tri-saccharide by gavage for the first seven days, and then given loperamide solution (LOP) by gavage for the first hour and high-dose jujube tri-saccharide by gavage for the next hour. The lactulose group (200 mg / kg) was given high-dose lactulose by gavage for the first seven days, and then given loperamide solution by gavage for the first hour and high-dose lactulose by gavage for the next hour. The mice's body weight was measured every 2 days, and the relevant indicators were measured. The mice were sacrificed on the 7th day after modeling (14th day of the intervention period), and the distal part of the colon was taken and fixed with 4% neutral formaldehyde, paraffin-embedded, and then sectioned for HE staining. The sections were analyzed under a light microscope. (2) Experimental method: The mice's body weight was measured every 2 days during the experiment and the change curve was drawn. The daily weight change rate was calculated, and the fecal samples were collected for property evaluation. The time of the first black stool was measured before the mice were sacrificed, and the water content of the feces was measured. The mice were sacrificed at the end of the experiment, and the colon length and small intestine propulsion rate were measured. The colon tissue was routinely paraffin-embedded, sectioned, and then stained with hematoxylin-eosin (H&E). In the hematoxylin-eosin staining method, hematoxylin is an alkaline dye that stains the chromatin in the nucleus and the ribosomes in the cytoplasm purple-blue; eosin is an acidic dye that stains the components in the cytoplasm and extracellular matrix red. The colon tissue morphology, inflammatory cell infiltration and ulceration were observed by H&E staining. Enzyme-linked immunosorbent assay (ELISA) is a high-sensitivity immunological detection technology based on the specific binding of antigen-antibody. The concentration of target protein (such as cytokine) is quantified by enzyme-catalyzed substrate color reaction. The levels of IL-6, TNF-α and IL-1β in the intestinal tissue of mice were detected by enzyme-linked immunosorbent assay (ELISA) (Wuhan Sevier).

[0047] (3) Experimental results: As shown in Figures 4-7 , the experiment explored the protective effect of glycerol on LOP-induced constipation in mice, and three dose groups of 50 mg / kg, 100 mg / kg and 200 mg / kg were set. The experimental results fully confirmed the protective effect of A3: after determining the fecal water content, the time of the first black stool and the small intestine propulsion rate, it was found that the Loperamide-induced constipation model was more successful, which was manifested as a decrease in fecal water content, a significant increase in the time of the first black stool and a significant decrease in small intestine propulsion rate.

[0048] Table 1 Comparison of fecal water content of mice in each group (data expressed as mean ± standard error (SEM) (n = 8))

[0049] Table 2 Comparison of the time of the first black stool of mice in each group (data expressed as mean ± standard error (SEM) (n = 8))

[0050] Table 3 Comparison of small intestine propulsion rate of mice in each group (data expressed as mean ± standard error (SEM) (n = 8))

[0051] The effect of glycerol in high and medium doses in relieving constipation was better than that of lactulose, a common constipation treatment drug on the market. HE staining results showed that the control group had complete colon tissue structure and regular gland arrangement; the LOP model group showed mucosal erosion, ulcer, a large number of inflammatory cell infiltration and destruction of crypt structure; the colon tissue damage of each dose A3 intervention group was significantly reduced.

[0052] Table 4 Comparison of pro-inflammatory factors of mice in each group (unit: pg / mL protein; data expressed as mean ± standard error (SEM) (n = 8))

[0053] As shown in Table 4, each dose of A3 can significantly inhibit the release of pro-inflammatory factors IL-6, IL-1β and TNF-α, and the effect of the medium and high dose groups is the most significant, and the high dose group is better than the lactulose group. These results comprehensively show that A3 can improve clinical symptoms, repair intestinal barrier function and regulate the balance of inflammatory factors, and show obvious dose dependence.

[0054] Example 5: Application of glycerol in the preparation of oral solution An oral solution containing, by final concentration: 30 mg / mL of juncosaccharide, 0.02% (v / v) potassium sorbate. The effect of the oral solution on model mice was verified according to the method of Example 4, and the results showed that the oral solution prepared in this example had the effect of relieving constipation in mice.

[0055] Although the present application has been disclosed in its preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the scope of protection of the present application should be defined by the claims.

Claims

1. Use of chitotriose in the preparation of a medicament for relieving constipation.

2. Use according to claim 1, characterized in that, The medicament has chitotriose as the only active ingredient.

3. Use according to claim 1 or 2, characterized in that, The medicament is in the form of a powder, granules, capsules, tablets, pills or oral solution.

4. The use according to any one of claims 1 to 3, characterized in that, The relieving of constipation includes, but is not limited to, at least one of the following effects: (1) increasing the water content of the stool of a constipated individual; (2) shortening the time to the first black stool of a constipated individual; (3) increasing the small intestinal transit rate of a constipated individual; (4) reducing the number of inflammatory cell infiltrates and improving the condition of a colitis individual; (5) inhibiting the expression of pro-inflammatory factors IL-6, IL-1β and TNF-α in the colon tissue of a colitis individual.

5. The content of chitotriose in the medicament is 30 mg / mL.

6. Use of chitotriose in the preparation of a health product for lubricating the intestines and relieving constipation.

7. Use according to claim 6, characterized in that, The health product is an oral solution containing chitotriose and potassium sorbate.

8. The use according to any one of claims 1 to 7, characterized in that, The chitotriose is prepared by the following steps: (1) enzymatic hydrolysis of agar solution with α-chitosanase; (2) inactivation treatment of the solution after step (1); (3) centrifugation of the solution after step (2) to remove the precipitate and obtain the supernatant, which is a chitooligosaccharide mixture containing chitotriose; (4) centrifugation of the chitooligosaccharide mixture prepared in step (3) and purification by gel chromatography.

9. Use according to claim 8, characterized in that, The gene sequence of the α-chitosanase in step (1) is shown in SEQ ID NO.

1.

10. An oral solution characterized in that, An oral solution prepared by containing chitotriose at a final concentration of 30 mg / mL and 0.02 (v / v) potassium sorbate.

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