CN114989258B

CN114989258B - Application of plant extract composition in preparing product for treating constipation and reducing weight

Info

Publication number: CN114989258B
Application number: CN202210467601.8A
Authority: CN
Inventors: 钟姝凝; 朱亚贤; 徐双成
Original assignee: Qingfeng Chain Soda Beverage Jilin Co ltd
Current assignee: Qingfeng Chain Soda Beverage Jilin Co ltd
Priority date: 2022-04-29
Filing date: 2022-04-29
Publication date: 2023-03-24
Anticipated expiration: 2042-04-29
Also published as: CN114989258A

Abstract

The application discloses application of a plant extract composition in preparing a product for treating constipation and losing weight. The plant extract composition is prepared on the basis of eucheuma peptide of amino acid selected from any one of SEQ ID NO. 1-12, has the effects of treating constipation and losing weight, can excrete uric acid, dissolve tophus, relax bowel, induce diuresis to reduce edema, inhibit sugar absorption, control blood sugar, improve insulin resistance, tonify kidney and consolidate semen, tonify kidney and spleen, strengthen stomach and promote digestion, promote bifidobacterium growth, remove moisture, strengthen heart and tranquilize mind, clear heat and reduce internal heat, reduce blood sugar, blood fat and blood pressure, strengthen liver and promote gallbladder, decompose alcohol, dispel alcohol effects of alcoholic intoxication, beautify and lighten spots, delay aging, nourish yin and nourish blood, reduce cholesterol, resist thrombosis, resist atheroma, clear intestinal toxins and in vivo nutritional waste, improve sleep and promote intestinal excretion.

Description

Application of plant extract composition in preparing product for treating constipation and reducing weight

Technical Field

The application relates to the technical field of plant extraction, in particular to application of a plant extraction composition in preparing a product for treating constipation and losing weight.

Background

Obesity refers to a condition of excess accumulation of body fat, especially triglycerides, due to a degree of significant overweight and an excessively thick fat layer. Excessive accumulation of fat in the body due to excessive food intake or altered metabolism of the body causes excessive weight gain and causes pathological, physiological changes or latency in the human body. Obesity increases the risk of diabetes, cardiovascular disease, hypertension, hyperlipidemia, fatty liver, various tumors, metabolic disorders, and the like. The beverage is prepared from medicinal and edible traditional Chinese medicinal materials with lipid-lowering and weight-losing effects, such as lotus leaves, hawthorn, chrysanthemum, cassia seeds and the like, and can be daily drunk as common food. The lotus leaf mainly has the functions of regulating fat, losing weight, resisting oxidation, resisting aging and inhibiting bacteria. The fructus crataegi has effects in resisting myocardial ischemia and perfusion injury, promoting digestive enzyme secretion, and regulating gastrointestinal function. The flos Chrysanthemi has antibacterial, antiinflammatory, blood lipid reducing, antitumor, and plumbum expelling effects. The compatibility of hawthorn and chrysanthemum can promote blood circulation by removing blood stasis, dredge collaterals, reduce blood fat and improve microcirculation. The semen Cassiae extract has good antioxidant effect, and can improve lipid peroxidation injury caused by fatty liver. The hawthorn and the cassia seed and the compatibility thereof can reduce the deposition of fat in the liver and the kidney and lower the blood fat level, and the effect is better after the compatibility. The radish seed is clinically applied to treating hypertension, hyperlipidemia, eczema, phlegm asthma, abdominal distension and the like. Fructus Hippophae has effects in improving anoxia tolerance, reducing serum cholesterol, resisting aging, resisting radiation and virus, and enhancing immunity.

Constipation (constipation) is the reduction of stool frequency, generally less than 3 times per week, with difficulty in defecation and dry and hard stools. Constipation is a clinically common symptom, and is persistent for a long time, disturbing, affecting life quality, causing various causes, and most common intestinal diseases are intestinal diseases. "use medicine containing extracts of radish, tea, etc. to prevent and cure intestinal diseases and constipation [ J ] foreign medicine: botanical drug 2005, phase 3, "discloses the use of radish, tea, carrot, fig, onion, plum, apricot, and the like as raw materials for the prevention and treatment of intestinal diseases and constipation in mammals, and can also be made into health food.

However, no plant extract with obvious effects of losing weight and treating constipation exists in the prior art, and the development of the effects in the aspect is of great significance.

Disclosure of Invention

Eucheuma (Eucheuma) belongs to Solieriaceae (So-lieriaceae) of Rhodophyceae (Rhodophyceae) of Rhodophyceae of Rhodophyta, is a large-scale marine economic algae, is rich in polysaccharides, is carbohydrate and mineral elements in 76.99% and is a good calcium supplement with calcium content of 777.5mg/kg in coastal areas of southern province and southeast Asia of China, but is low in vitamin content. The research shows that the eucheuma polysaccharide has the physiological functions of immunoregulation, antivirus, anti-tumor and the like. The extraction of algal polysaccharides usually adopts hot water extraction method, physical crushing method, enzymolysis method, compounding method and the like.

The inventor creatively discovers that eucheuma peptide with the functions of losing weight and relieving constipation can be obtained by glycoprotein extracted from eucheuma through enzymolysis and deglycosylation, and can be used for preparing a plant extraction composition, so that the eucheuma peptide not only has the functions of treating constipation and losing weight, but also can excrete uric acid, dissolve tophus, relax bowels, induce diuresis to reduce edema, inhibit sugar absorption, control blood sugar, improve insulin resistance, tonify kidney and consolidate semen, tonify kidney and strengthen spleen, invigorate stomach and promote digestion, promote bifidobacterium growth, dispel moisture, calm nerves and strengthen heart, clear heat and remove fire, reduce blood sugar, reduce blood fat, reduce blood pressure, strengthen liver and promote gallbladder, decompose alcohol, dispel the effects of alcohol and sober up, protect liver, beautify spot, delay aging, nourish yin and blood, reduce cholesterol, resist thrombosis, resist atheroma, clear up intestinal toxins and in vivo nutritional waste, improve sleep and promote intestinal excretion.

In a first aspect, the embodiments of the present application disclose an eucheuma peptide selected from the group consisting of peptides, peptide salts and peptide derivatives of amino acids represented by any one of SEQ ID nos. 1 to 12.

In a second aspect, an Eucheuma peptide mixture comprises peptides, peptide salts and peptide derivatives of amino acids shown in SEQ ID No.1 and 2;

or peptides, peptide salts and peptide derivatives of the amino acids shown in SEQ ID NO.3, 4 and 5;

or peptides, peptide salts and peptide derivatives of the amino acids shown in SEQ ID NO.6, 7 and 8;

or peptides, peptide salts and peptide derivatives of the amino acids shown in SEQ ID NO.9 and 10;

or peptides, peptide salts and peptide derivatives of the amino acids shown in SEQ ID NO.11 and 12.

In a third aspect, the present application discloses a process for the preparation of the eucheuma peptides of the first aspect or the mixture of eucheuma peptides of the second aspect, which comprises:

making Eucheuma Gelatinosum powder;

and performing acid treatment and filtration on the powder, and collecting filtrate to obtain a first solid, a second solid, a third solid and a fourth solid in sequence.

In the application example, the preparation method comprises the steps of dissolving the second solid with PBS buffer solution with the pH value of 7.5, wherein the mass percent of the second solid is 2.5wt%, simultaneously adding a first enzyme preparation, wherein the adding amount of the first enzyme preparation is not less than 50000Unit/mL, carrying out reaction in a water bath at 25 ℃ for 2.5h, carrying out treatment at 105 ℃ for 10min, carrying out centrifugation at 8000 Xg 4 ℃ for 15min, taking supernatant, carrying out ultrafiltration concentration with the molecular weight cutoff of 10KD, taking filtrate, concentrating and drying to obtain a third solid; wherein the first enzyme preparation is lysostaphin.

In the embodiment of the application, the preparation method comprises the steps of dissolving the third solid with PBS (phosphate buffer solution) of p7.5, wherein the mass percent of the third solid is 1.25wt%, simultaneously adding a second enzyme preparation, wherein the adding amount of the second enzyme preparation is not less than 20000 units/mL, reacting in a water bath at 35 ℃ for more than 24 hours, treating at 105 ℃ for 10min, centrifuging at 8000 Xg 4 ℃ for 15min, taking the supernatant, performing ultrafiltration concentration with the molecular weight cutoff of 10KD, taking the filtrate, concentrating and drying to obtain a fourth solid; wherein the second enzyme preparation comprises cellulase with the concentration not less than 10000Unit/mL and endoglycosidase F with the concentration not less than 20 Unit/mL.

In the examples of the present application, the second enzyme preparation further comprises not less than 120Unit/mL of beta-galactosidase, not less than 12Unit/mL of beta-N-acetylglucosaminidase, and not less than 6000Unit/mL of fructosidase.

In a fourth aspect, the present application discloses a plant extract composition, which comprises eucheuma peptide 4.5-10.5 wt%, sour cherry juice 2-8.5 wt%, erythritol 0.3-1.5 wt%, L-arabinose 1-5.5 wt%, corn silk 0.1-2.5 wt%, mulberry leaf 0.15-1.5 wt%, gordon euryale seed 1-3.25 wt%, sunflower disc 0.05-1.5 wt%, hawthorn 0.5-2.5 wt%, papaya 4.5-10.5 wt%, white lotus 1-3.5 wt%, chicory 0.5-5.5 wt%, gardenia 0.1-0.5 wt%, kudzu root 0.1-2.5 wt%, poria 0.15-1.5 wt%, celery 2-6.5 wt%, eucommia ulmoides 0.05-2.5 wt% and male flower 0.5wt%, with the balance being acceptable auxiliary materials for food.

In a fourth aspect, the present application discloses a plant extract composition, which comprises the eucheuma peptide mixture of the second aspect 4.5-10.5 wt%, 2-8.5 wt% of sour cherry juice, 0.3-1.5 wt% of erythritol, 1-5.5 wt% of L-arabinose, 0.1-2.5 wt% of corn silk, 0.15-1.5 wt% of mulberry leaf, 1-3.25 wt% of gordon euryale seed, 0.05-1.5 wt% of enteromorpha, 0.5-2.5 wt% of hawthorn, 4.5-10.5 wt% of papaya, 1-3.5 wt% of white lotus, 0.5-5.5 wt% of eucommia, 0.1-0.5 wt% of gardenia, 0.1-2.5 wt% of kudzu root, 0.15-1.5 wt% of tuckahoe, 2-6.5 wt% of celery, 0.05-2.5 wt% of male flower and 0.5wt% of flower, and the balance acceptable auxiliary materials for food.

In the examples herein, the plant extract composition comprises 4.5wt% of the eucheuma peptide of the first aspect or the mixture of eucheuma peptides of the second aspect.

In a fifth aspect, the present application discloses the use of the eucheuma peptide of the first aspect or the eucheuma peptide mixture of the second aspect in the preparation of a plant extract composition or medicament for weight loss or constipation treatment.

Compared with the prior art, the application has at least one of the following beneficial effects:

the deglycosylated peptides extracted from eucheuma are proved to have antioxidant and dipeptidyl peptidase IV inhibiting activities by in vitro experiments; and the cell experiment explains the non-toxicity of the eucheuma peptide on cells and the action mechanism of inhibiting the activity of the dipeptidyl polypeptidase IV.

Furthermore, the application also proves that the plant extract composition consisting of the eucheuma peptide and more than ten components has the effects of losing weight and relieving constipation through animal experiments. In addition, long-term clinical tests show that the plant extract composition not only has the effects of treating constipation and losing weight, but also can excrete uric acid, dissolve tophus, relax bowels, induce diuresis to reduce edema, inhibit sugar absorption, control blood sugar, improve insulin resistance, tonify kidney and arrest spontaneous emission, tonify kidney and spleen, invigorate stomach and promote digestion, promote growth of bifidobacteria, dispel moisture, strengthen heart and tranquilize mind, clear heat and reduce fire, reduce blood sugar, reduce blood fat, reduce blood pressure, strengthen liver and promote gallbladder, decompose alcohol, relieve alcoholism, protect liver, beautify skin and lighten spots, delay senility, nourish yin and nourish blood, reduce blood, resist thrombosis, resist atheroma, clear up intestinal toxins and in-vivo nutritional waste, improve sleep and promote intestinal excretion.

Drawings

FIG. 1 is a diagram showing HPLC detection results of monosaccharides provided in examples 1 to 5 of the present application.

FIG. 2 is a graph showing the HPLC detection results of monosaccharides provided in comparative examples 1 to 5 of the present application.

FIG. 3 is a graph showing HE staining patterns of mouse liver tissues in a normal group (A), a model group (B), a control group (C) and a test group (test article provided in example 1) (D) provided in an animal experiment of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application. Reagents not individually specified in detail in this application are conventional and commercially available; methods not specifically described in detail are all routine experimental methods and are known from the prior art.

It should be noted that the terms "first", "second", and the like in the description and claims of the present invention and in the drawings are used for distinguishing similar objects, and do not necessarily have to be used for describing a specific order or sequence or have a substantial limitation on technical features thereafter. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

Preparation of Eucheuma peptide

The eucheuma peptide provided by the embodiment of the application is obtained by extracting glycoprotein from eucheuma, and then carrying out enzymolysis and deglycosylation. The specific process is described in detail below.

1. Preparation method of eucheuma peptide

In the embodiment of the step, the method comprises the steps of obtaining dried eucheuma powder, carrying out acid treatment on the eucheuma powder, sequentially obtaining a first solid, a second solid, a third solid and a fourth solid, and purifying the third solid to obtain the eucheuma peptide. 1. Obtaining dried Eucheuma Gelatinosum powder and subjecting it to acid treatment

In one embodiment of this step, clean and dry Eucheuma Gelatinosum (International Logistics Co., ltd., juzurich island) was ground into powder; soaking powdered Eucheuma Gelatinosum in hydrochloric acid solution with pH =3 for several times, filtering, and collecting the combined filtrate.

2. Sequentially obtaining a first solid, a second solid, a third solid and a fourth solid

In the embodiment of the step, the method comprises the steps of performing ammonium sulfate precipitation on the filtrate obtained in the step to obtain a first solid, performing alcohol dissolution and centrifugation on the first solid to obtain a second solid, performing first enzymolysis on the second solid to obtain a third solid, and performing second enzymolysis on the third solid to obtain a fourth solid.

In the step of obtaining the first solid in example 1, the pH of the filtrate was adjusted to 4.5 with sodium bicarbonate, while adding ammonium sulfate at a ratio of 1.

In the step of obtaining the first solid in comparative example 1, ammonium sulfate was added to the filtrate in a ratio of 1.

In the step of obtaining the second solid in example 1, the first solid obtained in example 1 was dissolved in a 35% ethanol solution and centrifuged at 15000 Xg 4 ℃ for 15min to obtain the second solid.

In the step of obtaining the second solid in comparative example 2, the first solid obtained in example 1 was dissolved in a 30% ethanol solution and centrifuged at 15000 Xg 4 ℃ for 15min to obtain the second solid.

In the step of obtaining the second solid in comparative example 3, the first solid obtained in example 1 was dissolved in 40% ethanol solution and centrifuged at 15000 Xg 4 ℃ for 15min to obtain the second solid.

In the step of obtaining the third solid substance in example 1, the second solid substance obtained in example 1 was dissolved in PBS buffer solution with ph7.5, wherein the mass percentage of the second solid substance was 2.5wt%, and the first enzyme preparation was added at the same time, the amount of the first enzyme preparation was not less than 50000Unit/mL, the reaction was performed in water bath at 25 ℃ for 2.5h, the treatment was performed at 105 ℃ for 10min, and the centrifugation was performed at 8000 × g 4 ℃ for 15min, and the supernatant was subjected to ultrafiltration concentration on the enzymatic hydrolysate using an ultrafiltration cup (DNC 300 home cup ultrafilter, saltroius 14639-63-D PES, molecular weight cut-off of 10 KD), and the third solid substance was obtained by taking the filtrate, concentrating and drying. Wherein the first enzyme preparation is lysostaphin (L9043, ≧ 3000unit/mg, sigma-Aldrich).

In the third solids obtaining step of example 2, the second solids obtained in example 1 were treated with the same treatment steps as in example 1 except that a different first enzyme preparation comprising endo- β -N-acetylglucosaminidase (A6805,. Gtoreq.80 units/mg, sigma-Aldrich) not less than 30000Unit/mL and N-acetylmuramyl-L-alanine amidase not less than 30000Unit/mL was used.

In the third solids procedure of example 3, the second solids from example 1 were treated with the same treatment procedure as in example 1, except that a different first enzyme preparation was used, which included endo- β -N-acetylglucosaminidase (A6805,. Gtoreq.80 units/mg, sigma-Aldrich) at no less than 30000Unit/mL and glycylglycine endopeptidase at no less than 15000 Unit/mL.

In the step of obtaining the third solid of comparative example 4, the second solid obtained in example 1 was treated by the same treatment steps as in example 1 except that a different first enzyme preparation comprising pepsin (10108057001, 2500units/mg, roche) not less than 50000Unit/mL and trypsin (T2600000, sigma-Aldrich) not less than 15000Unit/mL was used.

In the step of example 4 for obtaining the fourth solid, the third solid obtained in example 1 is dissolved in PBS buffer p7.5, wherein the mass percent of the third solid is 1.25wt%, and the second enzyme preparation is added at the same time, the addition amount of the second enzyme preparation is not less than 20000Unit/mL, the reaction is carried out in a water bath at 35 ℃ for more than 24h, the treatment is carried out at 105 ℃ for 10min, the centrifugation is carried out at 8000 Xg 4 ℃ for 15min, the enzymolysis liquid is ultrafiltered and concentrated by an ultrafiltration cup (DNC 300 cup type ultrafilter, SARTIUS 14639-63-D PES, molecular weight cut-off is 10 KD) by taking the supernatant, and the filtrate is concentrated and dried to obtain the fourth solid. Wherein the second enzyme preparation comprises cellulase (C0615 ≧ 5000units/g, sigma-Aldrich) no less than 10000 units/mL and endoglycosidase F (E2264 ≧ 30units/g, sigma-Aldrich) no less than 20 units/mL.

In the fourth solid-producing step of example 5, the third solid obtained in example 1 was treated by the same treatment steps as in example 1 except that a different second enzyme preparation was used, which included cellulase at not less than 10000 units/mL, endoglycosidase F at not less than 20 units/mL, beta-galactosidase at not less than 120 units/mL (G6920,. Gtoreq.140 units/mg, sigma-Aldrich), beta-N-acetylglucosaminidase at not less than 12 units/mL (. Gtoreq.15 units/mg protein, cat. HZM9197, shanghai province Biotechnology Co., ltd.), and fructosidase at not less than 6000 units/mL (200U/mg, CAS:9001-57-4, nanjing Dou Biotechnology Co., ltd.).

In the fourth solids-obtaining step of comparative example 5, the third solids obtained in example 1 were treated by the same treatment procedure as in example 1 except that a different second enzyme preparation comprising not less than 20Unit/mL of endoglycosidase F and not less than 6000Unit/mL of fructosidase was used.

3. Purifying the third solid

In the step, the step of purifying the fourth solid by gel chromatography and/or reverse phase chromatography is included.

In one example of the gel chromatography purification step, a fourth solid PolySe-GFC-P4000 column (7.8 mM × 300 mM) is purified by using a high performance gel chromatography column packed with Sephadex G-50, the gel column is equilibrated with 20mM Tris-HCL buffer solution of ph7.8, then the fourth solid is dissolved in the equilibration buffer solution to 2.5G/mL, the sample is loaded with 2mL, 0.15% NaCL salt solution water is used as mobile phase, the flow rate is 0.5mL/min, the detector is uv absorption chromatography, the eluate corresponding to the a260nm detection absorption elution peak is collected, 1.5mL per tube, tricine-glycerol SDS-PAGE detection is performed by using peptides of 20 100, 14, 400, 7 823, 856, 3 313Da as standard substances, and the electropherogram is analyzed by using a gel imaging system to obtain the content (mass percentage) of the component corresponding to each band in the fourth solid substance, specifically, refer to "yellow leaf et al; a comparative experiment for detecting the antigen content of the porcine circovirus vaccine by SDS-PAGE [ J ]. Contemporary livestock raising, published 12/25 in 2019.

In one example of the anion chromatography purification step, the eluate obtained from the above gel chromatography purification is collected, freeze-dried, dissolved in 20mM pH 8.0Tris-HCl buffer (A), filtered through a 0.22 μ M membrane, and subjected to anion chromatography (Hitrap Q), after which it is further washed with 20mM pH 8.0Tris-HCl buffer until the baseline becomes stable, and then eluted with a linear gradient of 20mM pH 8.0Tris-HCl buffer (B) containing 1.0M NaCl at a concentration of 0 to 7min,100% A; 7-12min, 0-25% of B; 12-42min, 65%; 42-47min, 65-100% B; 47-49min, 100% B. And (3) collecting elution peaks at a flow rate of 1mL/min and 3 min/tube, and freeze-drying to obtain the eucheuma peptide.

2. Identification of Eucheuma peptide

1. Amino acid sequence identification of Eucheuma glycopeptide

The eucheuma peptides prepared in the above examples 1 to 5 and comparative examples 1 to 5 were determined by a liquid chromatography-mass spectrometry combined method and analyzed and identified by a high performance liquid chromatography system. Analysis was performed by Thermo LTQ XL linear ion trap mass spectrometer using a C18 column (100. Mu. M.times.1 ℃ C.) with mobile phase A water (0.1% trifluoroacetic acid); mobile phase B, acetonitrile (0.1% trifluoroacetic acid), loading 2 uL, and elution flow rate 240 uL/min. Elution conditions of 0-10min and 98% A; for 10-45 min, 98-75% of A; 45-60min, 75-l 0% A; 60-69min, 10% A; 69-70 min,10% -95% A; 70-75min, 95-98% A; 75-100min, 98% A. Specific experimental setup parameters: fragment ion mass error; 0.5Da; parent ion tolerance of peptide fragment: plus or minus 20ppm; the protease is trypsin; the maximum non-enzyme-cutting site is 1; fixed modification, iodophthalein amination of cysteine (+ 57 Da); variable modifications methionine oxidation (+ 15.9 Da), G1cNAc tag at Asn (+ 203 Da), dephthalamido (+ 0.98 Da); and selecting b and y series ion search.

Mass spectrometry results were analyzed using SEQUEST software, which output two primary scores Xcorr and DeltCn, respectively, with filter conditions where Xcorr (+ 1, +2, + 3) is greater than 1.9,2.5,3.75, respectively, and De1tCn is greater than 0.1 to remove incorrect identification results. And performing Mascot database retrieval on the obtained MS/MS spectrum, and performing auxiliary analysis by combining with an NCBI database.

2. Sugar residue detection of Eucheuma peptide

(1) Hydrolysis

Taking 5mL of eucheuma peptide obtained in the examples 1-5 and the comparative examples 1-5 respectively, mixing the eucheuma peptide with 2M trifluoroacetic acid, fully stirring, sealing, hydrolyzing at 120 ℃ for 2h, cooling, concentrating the solution at 40 ℃ under reduced pressure until the solution is dried, adding 3mL of methanol, evaporating to dryness, and repeating the operation for 4-5 times to remove the trifluoroacetic acid completely.

(2) Pre-column derivatization and HPLC detection

Respectively taking 2mL of mixed monosaccharide standard solution (the mass concentration of each monosaccharide is 0.5g/L, glucose (glu), fructose (fru), mannose (man), galactose (gala), rhamnose (rha), xylose (xyl), arabinose (arab), ribose (rib) and lyx (lyx), sigma) and 2mL of 0.6M NaOH solution, and uniformly mixing the mixed monosaccharide standard solution and the 2mL of 0.6M NaOH solution in a 10mL test tube with a plug; then 1mL of the mixed solution is taken to be put into a 25mL round-bottom flask with a stopper, and 1mL of 0.5M 1-phenyl-3-methyl-5-pyrazolone PMP methanol solution is added and mixed evenly; reacting in an oven at 70 ℃ for 100min; taking out and standing for 10min to cool to room temperature; adding 1mL of 0.3mol/L HCl for neutralization; adding water to 20mL, adding equal volume of chloroform, shaking, standing, discarding the chloroform phase, and extracting repeatedly for 3 times. The aqueous phase was filtered through a 0.45 μm microporous membrane and analyzed by HPLC injection. The hydrolyzed sample was dissolved in 2mL of water, and the derivatization step was performed with a standard solution of mixed monosaccharides.

Performing HPLC detection on the derivatized mixed monosaccharide standard solution and the sample solution respectively, wherein the HPLC conditions are as follows: water 2695-2996 liquid chromatography systems (Waters, USA); sunfire C18 chromatography column (Waters, usa); the column temperature is 25 ℃; the mobile phase is NaOH-KH2PO4 buffer solution (pH6.7) (phase A) and acetonitrile (phase B); elution procedure: 0-30 min, 15% B; 30-60 min, 15-32% B; 60-65min, 32-20% B; the sample loading amount is 10 mu L; the flow rate is 1mL/min; the detection wavelength is 245nm.

3. Results

The fourth solid obtained in each of examples 1 to 5 and comparative examples 1 to 5 was purified by gel chromatography, and the eluate corresponding to the peak of the A260nm absorbance measurement was collected and analyzed by SDS-PACE. It is clear that the molecular weights of examples 1 to 5 are all below 5000Da, and the bands are clear; whereas the peptides of 5000Da or less were obtained in comparative examples 1 to 3 with a low content (the bands are blurred in the figure), comparative example 4 and comparative example 5 showed a large number of bands of 5000Da or more.

Further purification by anion chromatography as described above, and analysis of the purified single-component mass spectrum and content measurement, the results are shown in Table 1, to obtain the amino acid sequence of eucheuma peptide in the fourth solid, its reference sequence in NCBI database and its mass percentage in the fourth solid, respectively.

TABLE 1

And the fourth solid matters of examples 1 to 5 and comparative examples 1 to 5 were hydrolyzed, and the monosaccharide composition analysis in the hydrolysate was determined by 1-phenyl-3 methyl-5-pyrazolone (PMP) pre-column derivatization HPLC after the hydrolysis, as shown in FIGS. 1 to 2, no detection of saccharide components was observed in the fourth solid matters provided in examples 1 to 5, while comparative examples 4 and 5 had different contents of saccharide components.

In vitro Performance of Eucheuma peptides

1. In vitro antioxidant capacity determination

1. Determination of DPPH radical scavenging Rate

Preparing 0.2mM DPPH solution by 95v/v% ethanol solution, placing 2mL into a test tube, adding 2mL of aqueous solution of eucheuma peptide provided in examples 1-5 and comparative examples 1-5 with different mass fractions as samples, mixing uniformly by vortex, reacting for 30min at room temperature, and measuring the absorbance at the wavelength of 517 nm. Calculating DPPH free radical clearance rate: DPPH clearance% = (1- (A1-A2)/A0 x 100%, wherein A1 is the absorbance of a sample to be tested, A2 is the absorbance of a 95% ethanol solution instead of DPPH solution, and A0 is the absorbance of a 95% ethanol solution instead of the sample, and the formula is used for calculating the concentration of eucheuma peptide, namely the IC50 value, of which the DPPH clearance reaches 50% in examples 1-5 and comparative examples 1-5 respectively.

2. Determination of OH clearance

2mL of the aqueous solution samples of eucheuma peptides provided in examples 1 to 5 and comparative examples 1 to 5, respectively, at different concentrations were added with 2mL of 6mM FeSO ₄ And 2mL of 6mM H ₂ O ₂ And after vortex mixing reaction for 10min, adding 2mL of 6mM salicylic acid, mixing uniformly, standing at room temperature for 30min, and detecting the absorbance at 510 nm. Calculating OH clearance% = (1- (A1-A2)/A0) × 100%, wherein A1 is absorbance of the sample to be tested, and A2 is double distilled water instead of H ₂ O ₂ The obtained light absorption, A0 is the absorbance measured by replacing the sample with water; and the concentration of Eucheuma peptide provided by examples 1-5 and comparative examples 1-5 respectively reaching 50% in OH clearance, i.e. IC50 value, is calculated according to the formula. 2. In vitro dipeptidyl polypeptidase IV inhibitory Activity

The detection was divided into 3 groups, each group was repeated 3 times, and the test group (kylin peptide + enzyme + substrate + buffer), the positive group (sildendin A + enzyme + substrate + buffer), and the negative group (enzyme + substrate + buffer) were identified. 2500U/. Mu.L of dipeptidyl-polypeptidase IV (DPPIV, D4943-1VL, sigma-Aldrich) and 0.2mM of glycyl-prolyl-p-nitroaniline as a substrate (Gly-Pro-PNA, CAS103213-34-9, cat # XY-SH-SJ-1446, shanghai Xuan Yao) and eucin peptide aqueous solutions of different concentrations were first subjected to water bath at 37 ℃ for 30min, and then added to 96-well plates in the order of enzyme, buffer and substrate in the following order in amounts as shown in Table 2 below, with a total volume of 100. Mu.L per well. Wherein the positive group of control standard substance is statin A (CAS: 90614-48-5, bailingwei science and technology Co., ltd., beijing) with a concentration of 100 μ M.

TABLE 2 dipeptidyl-polypeptidase IV catalyzed reaction System (. Mu.L)

Calculating the inhibition rate of the dipeptidyl polypeptidase IV (% = (A1-A0)/(A2-A0) × 100%, wherein A1 is the absorbance of the test group, A2 is the absorbance of the positive group, and A0 is the absorbance of the negative group; and the concentration of the eucheuma peptides provided in examples 1 to 5 and comparative examples 1 to 5 respectively reaching 50% inhibition rate of dipeptidyl polypeptidase IV, i.e. IC50 value, is calculated according to the formula.

3. Results

TABLE 3

IC50 of DPPH clearance, IC50 of OH clearance and IC50 of DPPIV enzyme inhibition of eucheuma peptide provided by examples 1-5 and comparative examples 1-5, respectively, are shown in Table 3, all data tested are expressed as mean values and standard deviations, data were processed using SPSS13.0 software, and multiple comparisons and marked for significant differences were made for each line of data; and wherein "-" indicates no detection. As shown in Table 3, the inhibitory effects on DPPH and OH scavenging effect and on DPPIV enzyme were significantly superior to those of comparative examples 1 to 3 for the eucheuma peptides provided in examples 1 to 5, while those of comparative examples 4 and 5 were not examined.

Eucheuma peptide cell assay

1. Materials and methods

1. Cytotoxicity assays

Culturing to obtain Caco-2 cells (purocity) of 10 th to 25 th generations, wherein the cell concentration is 10 ⁵ cells/cm ² The Caco-2 cell viability was determined by MTT colorimetric assay. Caco-2 cells were cultured at 5X 10 ⁴ cells/well Density seeded in 96-well plates at 5% CO ₂ Incubation at 37 ℃. After 21 days of culture, the medium was removed and 200. Mu.L of the eucheuma peptide solution containing 10.0 mg/mL) as provided in examples 1 to 5 and comparative examples 1 to 5, respectively, was added and incubated for 24h. Wells with DMEM alone served as controls. After additional 200. Mu.L of MTT (5.0 mg/mL) was added to each well, the incubation of Caco-2 cells was continued at 37 ℃ for 4h. The medium containing MTT was then removed and 150 μ L of DMSO solution was added to each well. The 96-well plate was placed on a bench top shaker and shaken for 0.5h. Finally, the absorbance of the 96-well plate was measured at 570nm in a microplate reader. Sample concentrations with more than a 10% reduction in cell viability compared to the control were considered cytotoxic.

2. DPP-IV enzyme inhibition activity assay of Caco-2 cell monolayer

Caco-2 cells were cultured at 5X 10 ⁴ cells/well density were seeded in 12-well Transwell plates and Caco-2 cells were treated with 10mg/mL Eucheuma peptides as provided in examples 1-5 and comparative examples 1-5, respectively, for 24h. Protein content of each sample was then assessed using BCA protein assay kit (Nanjing Senega), final volume adjusted using pH 8.0Tris-HCl buffer, protein concentration in Caco-2 cell extracts and supernatants normalized. The DPP-IV enzyme content was measured using a mixture of the supernatant culture solution and the cell extract. The relative absorbance units (RLU) signal, which is proportional to the DPP-IV enzyme activity, is measured within 10min using a microplate reader. The treated samples were compared to control samples comprising cell lysate and cell supernatant supplemented with DMEM only.

Protein concentration was measured by BCA method, in which 0.5mg/mL of a protein standard solution was prepared, diluted to 0.1, 0.5, 2, 5 and 10mg/mL of standards, and then added to a 96-well plate at 20. Mu.L per well. mu.L of each sample was added to a 96-well plate, and PBS was added to make up to 20. Mu.L. 200 μ LBCA solution was added to each well and left at 37 ℃ for 30min. The absorbance (detection wavelength: 562 nm) was measured by a microplate reader, and a standard curve was plotted.

3. RNA extraction and DPP-IV enzyme gene expression analysis in Caco-2 cells

(1) siRNA interference control group

A si-RNA-Lipofectamine2000 mixture was prepared using Lipofectamine2000 and serum-free DMEM medium, containing DPP-IV siRNA (Thermo Fisher Co.) in an amount of 4pmol. The 6-well plates inoculated with Caco-2 cells were removed, washed twice with PBS and once with serum-free optimized DMEM medium. Removing the cleaning solution, adding serum-free DMEM medium into each well, adding 0.5mL of si-RNA-lipofectamine2000 mixed solution, shaking, stirring at 37 deg.C and 5% CO ₂ And putting the cells into a cell culture box for incubation for 6h to 6h, then sucking liquid in the pore plate, replacing normal serum-containing culture solution for continuous incubation for 24h, and taking the obtained Caco-2 cells as siRNA-interfered cells as an interference control group. And 10mg/mL of eucheuma peptide provided in examples 1-5 and comparative examples 1-5 was added to treat Caco-2 cells for 24h to serve as an inhibition experiment group.

(2) Analysis of Gene expression

1 mu L of Caco-2 cell RNA in an interference control group and an inhibition experiment group is extracted by a TRIZOL kit (Thermo Fisher company), 1 mu L of Random primer and 4 mu L of RNA-free enzyme water are added, after water bath at 65 ℃ is carried out for 5min, 1 mu L of RT Mix and 10 mu L of 2 × Reaction Buffer are continuously added into a Reaction tube after ice water is used for cooling for 2min, the total Reaction system is ensured to be 20 mu L for reverse transcription Reaction, the Reaction program is that the total Reaction system is incubated at 25 ℃ for 10min, then incubated at 42 ℃ for 30min, and finally enzyme is inactivated at 85 ℃. The obtained reverse transcription product cDNA is subjected to PCR amplification, beta-actin is used as an internal reference, and an amplification primer (synthesized by Shanghai Biotechnology Co., ltd.) comprises a forward direction of ttgtggatagcaagcgattg and a reverse direction of cacagctattccgcacttgaa. The reaction system comprises 5 mu L

Premix Ex TaqTMII, 0.2. Mu.L of each of the upstream and downstream primers, 0.2. Mu.L of Rox Reference Dye II, 1.0. Mu.L of cDNA, and 10. Mu.L of double distilled water. The reaction procedure comprises: pre-deformation 95 ℃ 5min,95 ℃ 15s and 60 1min (35 cycles total)) Then sequentially carrying out the processes of 95 ℃ for 15s, 60 ℃ for 30s, 95 ℃ for 30s and 60 ℃ for 1min. After the reaction, beta-actin was used as an internal reference gene, and then the relative expression level of the target gene was calculated using normalized 2-DELTA-CT.

4. Data analysis

All test data are expressed as mean and standard deviation, data were processed using SPSS13.0 software, and multiple comparisons and marked for significant differences for each column of data.

2. As a result, the

TABLE 4

Detailed description of the preferred embodiments	Cell viability%	DPP-IV enzyme Activity	DPP-IV enzyme mRNA levels
				Example 1	92.4±3.5b	35.7±1.8d	98.5±1.2b
Example 2	93.2±2.3b	34.8±1.9d	98.1±1.3b
				Example 3	93.0±1.9b	32.2±1.7d	98.2±1.1b
Example 4	93.6±1.8b	30.8±1.6d	98.3±1.4b
				Example 5	92.5±1.7b	28.4±1.4d	97.8±1.5b
Comparative example 1	93.1±2.3b	79.6±3.5b	94.2±3.9b
				Comparative example 2	92.3±1.6b	81.7±2.8b	94.8±3.2b
Comparative example 3	91.9±2.2b	80.2±3.4b	95.2±4.1b
				Comparative example 4	67.4±1.8c	53.6±2.5c	94.9±4.3b
Comparative example 5	63.5±1.8c	51.7±2.4c	95.5±3.2b
				Control group	100％a	100％a	100％a
Interference control group	－	－	46.2±1.2c

The viability of Caco-2 cell monolayers after treatment with 10mg/mL of eucheuma peptide provided in examples 1-5 and comparative examples 1-5, respectively, was determined using the MTT assay and the results are shown in Table 4, where the viability of eucheuma peptide monolayers in Caco-2 cells was higher in examples 1-5 and comparative examples 1-3, but weaker in comparative examples 4 and 5. This suggests that the eucheuma peptide provided in the examples of the present application has no significant cytotoxicity.

Table 4 also shows DPP-IV enzyme activity in Eucheuma peptide treated monolayers of Caco-2 cells provided in examples 1-5 and comparative examples 1-5 at 10mg/mL, respectively. As can be seen from Table 4, the DPP-IV enzyme activities in the eucheuma peptide treated single-layer Caco-2 cells respectively provided in examples 1 to 5 are significantly reduced compared with the control group and are significantly lower than those in comparative examples 1 to 5, thereby demonstrating that the eucheuma peptides provided in the examples of the present application have stronger inhibition effects on the DPP-IV enzyme activities in the single-layer Caco-2 cells.

Table 4 also shows the level of DPP-IV enzyme transcription in single-layer Caco-2 cells treated with interference and with eucheuma peptide provided in examples 1 to 5 and comparative examples 1 to 3. As a result, the transcriptional level of DPP-IV enzyme in the cells of the interference treated group is significantly reduced compared with the control group, but the Eucheuma peptides provided in examples 1 to 5 and comparative examples 1 to 3 have no limit inhibition effect on the transcriptional level of DPP-IV enzyme in the cells. Thus, the eucheuma peptides provided in the examples of the present application have no effect on cellular DPP-IV enzyme transcription, and may act on the post-transcriptional enzyme functional stage.

Plant extract composition

Based on the eucheuma peptide provided by the above examples, the eucheuma peptide is found to have obvious antioxidant activity and inhibitory effect on DPPIV enzyme, and is suggested to have application in reducing blood sugar, losing weight and treating constipation.

Therefore, the embodiment of the application also provides a composition for constipation and weight loss, which comprises the eucheuma peptide, sour cherry juice, erythritol, L-arabinose, corn stigma, mulberry leaf, gorgon fruit, sunflower disc, hawthorn, pawpaw, white lotus, chicory, gardenia jasminoides, kudzu root, poria cocos, celery seed, eucommia male flower and enteromorpha provided in the above embodiments 1-5.

In the examples of the present application, the preparation of sour cherry juice generally comprises: steaming dried and pitted fructus Pruni Pseudocerasi and white sugar to obtain syrup, filtering the mixture, separating all fruit juice, further decocting and stewing the fruit juice to obtain maple syrup, cooling to room temperature, transferring into a sealed container, and refrigerating in refrigerator.

In the embodiment of the application, erythritol, L-arabinose, corn stigma, mulberry leaf, gorgon fruit, sunflower disc, hawthorn, papaya, white lotus, chicory, gardenia, kudzu root, poria cocos, celery seed, eucommia male flower and enteromorpha are all commercially available and are solid raw materials or powdery raw materials.

Further, an embodiment of the present application provides a plant extract composition, which includes 4.5 to 10.5wt% of eucheuma peptide, 2 to 8.5wt% of sour cherry juice, 0.3 to 1.5wt% of erythritol, 1 to 5.5wt% of l-arabinose, 0.1 to 2.5wt% of corn silk, 0.15 to 1.5wt% of mulberry leaf, 1 to 3.25wt% of gordon euryale seed, 0.05 to 1.5wt% of sunflower disc, 0.5 to 2.5wt% of hawthorn, 4.5 to 10.5wt% of papaya, 1 to 3.5wt% of white lotus, 0.5 to 5.5wt% of eucommia, 0.1 to 0.5wt% of gardenia, 0.1 to 2.5wt% of kudzu root, 0.15 to 1.5wt% of poria, 2 to 6.5wt% of celery, 0.05 to 2.5wt% of male flower, and the balance acceptable food adjuvants.

In the present examples, "food acceptable excipients" include sweeteners, flavors, excipients, and water.

In an embodiment of the application, the sweetener is selected from rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, dulcoside a, dulcoside B, rubusoside, stevia, stevioside, mogroside IV, mogroside V, luo han guo sweetener, and combinations thereof;

in the embodiment of the present application, said flavoring agent is selected from the group consisting of alpha-ionone, allyl-alpha-ionone, cyclic ionone, dehydrodihydroionone, dihydro-alpha-ionone, dihydro-beta-ionone, dihydromethyl-alpha-ionone, dimethylionone, (E) -6, 10-dimethylundec-5, 9-dien-2-one, gamma-ionone, gamma-methylionone, ionone, alpha-ionone, beta-ionone, trans-beta-ionone, beta-ionone epoxide, gamma-ionone, alpha-ionone, isobutyl ionone, and mixtures thereof alpha-isomethyl ionone, beta-isomethyl ionone, methyl-alpha-ionone, alpha-methyl ionone, methyl-beta-ionone, methyl-delta-ionone, methyl-alpha-ionone dehydroglycerate, beta-methyl ionone diethyl ketal, methyl isopseudoionone, pseudomethyl ionone, 3,4,5,6-tetrahydropseudoionone, pseudoionone raspberry essence, raspberry seed extract, jasmine absolute, bronto absolute, and mixtures thereof.

In the examples of the present application, the excipient refers to an additive added to a solid preparation such as a tablet, a powder (powder), or a granule for the purpose of forming, increment, or dilution, and for example, carrageenan, dextrin, lactose, crystalline cellulose, and the like.

The embodiment of the application also provides a preparation method of the plant extract composition, which comprises the steps of adding a proper amount of distilled water into the components according to the formula, fully homogenizing, keeping the temperature at 98 ℃ for 10 minutes, cooling to 65 ℃, adjusting the pH value to about 7.0 by using sodium bicarbonate, keeping the temperature, stirring for about 3 hours, cooling to room temperature, and spray-drying to obtain the plant extract composition.

Animal experiments

1. Materials and methods

1. Laboratory animal and test article

Kunming mouse, SPF grade, 9 weeks old, male, beijing Wittingli laboratory animal technology, inc. The experimental animals were acclimatized for 1 week prior to the experiment.

To this end, the examples of the present application also provide a plant extract composition prepared based on the above formulation, wherein each of 4.5wt% eucheuma peptide is provided by examples 1 to 5 and comparative examples 1 to 5, respectively, and the ratio of the other components is 8.5wt% sour cherry juice, 1.25wt% erythritol, 3.5wt% L-arabinose, 2.2wt% corn silk, 0.6wt% mulberry leaf, 1.75wt% gorgon fruit, 0.12wt% sunflower disc, 1.65wt% hawthorn, 7.8wt% papaya, 2.6wt% white lotus, 3.6wt% chicory, 0.4wt% gardenia, 0.8wt% kudzu, 0.85wt% poria, 4.5wt% celery seed, 1.6wt% male flower, 1.72wt% enteromorpha, 1.35wt% stevia, 1.75wt% alpha-iso-methyl ionone, 1.42wt% beta-methyl ionone, 1.5wt% and 1.24 wt% methyl ionone, 1.5wt% orcein water. To be used as a test sample of the animal experiment.

2. Constipation model and grouping experiment

(1) Molding die

The weight of the mice is accurately weighed 1 time every 2 days, and the real time is used as the basis of the administration dosage. The compound diphenoidol suspension is infused while normal diet of a normal KM mouse is performed, the dosage is 10mg/kg of body weight, the infusion is performed for 1 time every day for 20 days, and according to the water content of excrement of the mouse and the water content of ink propulsion test results, the time (ink propulsion test) required for discharging the first black excrement after the carbon powder liquid is infused into the stomach and the water content of the excrement can be observed so as to qualitatively judge the enterokinesia function, the water absorption function and the excrement difficulty degree of the mouse.

(2) Grouping experiment

After 20 days of continuous drenching, the KM mice are subjected to random model control group, experimental group and positive control group, and the mice which are not drenched are taken as normal groups. The experimental group was administered with 50mg/kg body weight of the test product half-at 9 o 'clock in the morning, while administering the compound diphenoxylate suspension at 9 o' clock per day. The positive control group was given lactulose at the same administration time and administration dose. The administration was carried out for a total of 28 days.

(3) Sample collection and processing

Fecal samples were collected from mice at 28 days of dosing.

The materials are taken 28 days after administration, after mice are anesthetized, abdominal aorta is taken out by operation, blood is collected by 10mL by a heparin vacuum blood collection tube, centrifuged at 4 ℃ and 3000rpm for 10min, and the blood is preserved at minus 80 ℃ to be used as a blood sample of the mice.

Materials were taken 28 days after administration, after anesthetizing the mice, the mice were surgically harvested for cecum, the cecum was trimmed, rinsed, and the adipose tissue around the intestinal wall was removed. Wherein 2 cm of colon tissue near the cecum is preserved in a pathology bottle containing 10% formaldehyde solution. And putting the rest colon tissue of 4cm into a freezing tube, putting into liquid nitrogen for preservation, and then transferring to a refrigerator at the temperature of-80 ℃ for refrigeration.

(4) Extraction of RNA

Taking out the above colon tissue sample frozen in liquid nitrogen tank, shearing about 10mg tissue, adding into EP tube containing 200 μ L Tridol, grinding with grinder, adding 800 μ L Tridol, and lysing on ice for 30min. Add 200. Mu.L of chloroform to each EP tube, shake vigorously for L seconds, and mix well. Centrifuge at 12000g \4 deg.C for 15min. The supernatant was decanted, the liquid was aspirated, 1000. Mu.L of 7daro DEPC alcohol was added, and the mixture was centrifuged at 7500g at 4 ℃ for 5min. After centrifugation, the supernatant was decanted, the white precipitate was retained, the RNA pellet was vacuum-dried, and 10. Mu.L of DEPC water was added to dissolve the RNA. Blood samples were referenced to this method for RNA extraction.

(5) Reverse transcription of mRNA into cDNA

After extracting RNA, taking 5 mu L for reverse transcription; RNA/primer mix,10 μ L reaction: 5 μ L template RNA and 1 μ LRandom primer; procedure, 70 ℃,2min water bath for at least 2min; preparing reverse transcription mix,10 microliter reaction system, 2 microliter 5 Mmlv buffer; 0.5. Mu.L dNTP mix (10 mm); 0.25 μ LRNase inhibitor (40 u/. Mu.L); 0.25. Mu.L M-mlv (200 u/. Mu.L); 1 μ L of DEPC water; adding into the above reaction solution, shaking, and processing at 30 deg.C, 10min, 42 deg.C, 60min, 70 deg.C, and 15min; water bath at 4 ℃.

RT-PCR: preparing a quantitative PCR mix, 20. Mu.L reaction (PCR primer reference PCR primer list) 10. Mu.L SYBR Green (2X); 0.8. Mu.L of PCR Forward Primers (10. Mu.M); 0.8. Mu.LPCR Reverse Primers (10. Mu.M); 0.4 μ LROX Reference Dye II (50 ×); 2 μ template (cDNA); 6 μ L of RNase-free water. Mixing, and placing in 7500 fluorescent quantitative PCR instrument with the program of 50 deg.C, 2min → 95 deg.C, 10min → 95 deg.C, 15s, 60 deg.C, 1min (40 cycles) → 95 deg.C, 15s → 60 deg.C, 1min → 95 deg.C, 30 → 60 deg.C, 15s. Beta-actin was used as an internal reference gene, and the normalized 2-DELTA Delta CT was used to calculate the relative expression level of the target gene.

The primer sequence is beta-actin-F, cctcactgtccacccttcca, beta-actin-R, gggtgtaaacgccagctca;

zo-1-F:ctttgaccagtacccacga,zo-1-R:tcagaggaggaacaactgc；

Occludin-F:ctactcctccaacggcaa,Occludin-R:agtcatccacggacaagg；

Aqp3-F:ggaccctcatccttgtga,Aqp3-R:gtgacagcgaactccaaa；

Aqp8-F:tgagatcaaggggaagga,Aqp3-R:ccgatagacacccaatgaa.

(6) Fecal flora 16SrDNA sequencing analysis

Genomic DNA of mouse stool samples was extracted using a hexadecaltrimethylalaminium bromide (CTAB). Sequencing was performed by using the Solexa sequencing technique, and the obtained off-machine data (CRaw PE) were subjected to splicing, quality control, and chimera removal, and the purity and concentration of DNA were measured by agarose gel electrophoresis (gel concentration: 2%, voltage: 80V, electrophoresis time: 40 min). The selected V3-V4 variable region was PCR amplified using specific primers with Barcode and high fidelity DNA polymerase depending on the choice of sequencing region. And detecting the PCR product by using 2% agarose gel electrophoresis, and cutting and recovering the target fragment, wherein the gel recovery adopts a gel recovery kit. And (3) detecting and quantifying the PCR amplification recovery product by using a blue fluorescence quantification system according to the electrophoresis preliminary quantification result, and mixing according to the corresponding proportion according to the sequencing quantity requirement of each sample. Library construction was performed using MEB kext. Ultra DKA Library Prep Kit. The constructed library is subjected to quality inspection through Agilent Bioanalyzer 2100 and Qubit, and the library is subjected to on-machine sequencing after being qualified.

According to the characteristics of the amplified 16S region and a sequencing platform, a small fragment library is constructed by using a double-ended sequencing (PE) method, and then double-ended sequencing is carried out. After Reads mosaic filtering, classification Units (OTUs) were clustered, and the Venn chart was used to analyze OTUs specific to each group species and overlapping OTUs. In the context of diversity analysis, use was made. Diversity was evaluated for the diversity of the flora organization within a single sample, using the Shannon index.

(7) Short chain fatty acid detection

100 μ L of water (20% aqueous phosphate) and 500 μ L of 4-methylvaleric acid (IS) were added to a 2mL glass centrifuge tube. The fecal sample suspension was homogenized and centrifuged at 14000 Xg for 20 minutes. mu.L of the supernatant was collected and subjected to gas chromatography-mass spectrometry using Agilent 7890-5977GC-MS system. In order to quantitatively detect the short-chain fatty acid, a calibration curve with the concentration range of 0.1-100 ug/ml is constructed. IS used to correct for injection variations between samples and minor variations in instrument response. Samples were separated using an Agilent FFAP capillary gas chromatography column (30 m. Times.0.25 mm ID. Times.0.25 μm). The initial temperature was 100 deg.C, increased to 160 deg.C at 5 deg.C/min, further increased to 150 deg.C at 5 deg.C/min, increased to 240 deg.C at 80 deg.C/min, and held for 6min. The carrier gas was helium (1.0 mL/min). Under the SIM model, the injection inlet temperature is 260 ℃ and the ion source temperature is 230 ℃.

3. Obesity model and weight loss experiment

(1) Obesity model establishment

The experiment is divided into a normal group, a model group, a control group and an experimental group. Except for the normal group and the control group, the mice in the other groups were fed with high-fat feed (63.8% basal feed + +15% lard + +10% sucrose +0.2% sodium cholate +10% yolk + +1% cholesterol) for 7 weeks, and obesity model mice were established, and weighed 1 time per week, and the weight of the obesity mice exceeded the weight of the mice fed with the normal feed by more than 20%, which indicated successful modeling.

The normal group was given the same volume of saline lavage as the dosing group: in the control group, obese mice were administered 2g/kg orlistat dispersed in 2mL of physiological saline for intragastric administration. In the model group, obese mice are subjected to intragastric gavage by the same volume of physiological saline as the administration group. The experimental group is that obese mice are administrated with 2g/kg of test sample dissolved in 2mL of normal saline for intragastric administration. And continuously performing intragastric administration for 8 weeks, feeding with high-fat feed in the period, weighing after 8 weeks, taking blood from abdominal aorta, separating serum, and weighing liver, heart, kidney and body fat (white adipose tissues around the kidney and around the kidney) immediately after conventional sacrifice, and performing subsequent experiments.

(2) Observation index and measurement

The kit (Abcam China) is adopted to detect the contents of Total Cholesterol (TCH), triglyceride (TG), low-density lipoprotein (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in the serum of the mouse, and the Lee's index is calculated according to the body mass and the body length.

The organ coefficient can visually represent the quality of the mouse adipose tissues, the adipose tissues can be divided into intra-abdominal fat, subcutaneous fat, interstitial adipose tissues and the like according to fat storage parts, and the adipose tissues mainly play a role in energy storage in an organism and are necessary tissues for the organism to maintain survival, but when the fat is excessively accumulated, the organism is sub-healthy.

Total Cholesterol (TCH) includes free cholesterol and cholesterol esters, and refers to the total amount of cholesterol contained in lipoproteins in the blood, which is mainly synthesized in the liver and stored for later use, and the amount of total cholesterol in the serum is used as a measure of the degree of lipid metabolism.

4. Data analysis

All test data are expressed as mean and standard deviation, processed using SPSS13.0 software, and multiple comparisons and marked significant differences were performed for each column of data.

2. Results

TABLE 5

Table 5 shows the water content of the stool samples of each group of the KM mouse constipation test and the time required for the first black stool to be discharged after intragastric administration with carbon powder liquid. As can be seen from Table 5, the water content of the model group mice was significantly lower than that of the normal group, and the time required for the first black stool to be excreted was significantly higher than that of the normal group, indicating that the molding was successful. In the experimental group, the moisture content in the feces of the mice is obviously higher than that of the model group after the test articles provided by examples 1 to 5 are administered, and the time required for discharging the first black feces is obviously lower than that of the normal group, so that the feces of the constipation-relieved mice are improved after the constipation-relieved mice are administered with the plant extract composition consisting of eucheuma peptide and more than ten components in examples 1 to 5 in the application of the book, which indicates that the constipation-relieved mice have the function.

Table 5 also shows the transcript levels of zo-1, occludin, aqp3 and Aqp8 genes in colon tissues of various groups of KM mice constipation experiments. As can be seen from Table 5, the transcript levels of the zo-1, occludin, aqp3 and Aqp8 genes in the colon tissues of the mice in the model group were all significantly higher than those in the normal group, while the transcript levels of the zo-1, occludin, aqp3 and Aqp8 genes in the colon samples of the mice after the test articles provided in examples 1 to 5 in the experimental group were administered were all significantly reduced to be equivalent to those in the normal group. It is suggested that the plant extract composition comprising eucheuma peptide and more than ten components in examples 1 to 5 of the present application can down-regulate two proteins of ZO-1 and occludin to increase aqueous electrolytes entering intestinal lumens and down-regulate the expression of AQP3 and AQP8 to reduce the association of water absorption of intestinal epithelial cells from intestinal contents after being administered to constipation mice, thereby achieving the effects of increasing the water content of enteric substances and relieving constipation.

TABLE 6

Table 6 shows the shannon index, F/B ratio and short-chain fatty acid content (mg/g) in the stool samples of each group of the KM mice constipation test. As can be seen from Table 6, the shannon index, F/ratio and short-chain fatty acid content in the stool sample of the model group mice were all significantly lower than those of the normal group, while the shannon index, F/B ratio and short-chain fatty acid content in the colon sample of the mice given the test article provided in examples 1-5 of the experimental group were all significantly increased to be equivalent to those of the normal group, even higher than those of the normal group. It is suggested that the plant extract composition comprising eucheuma peptide and more than ten components in examples 1 to 5 of the present application can regulate the diversity of intestinal flora of mice, provide the distribution of beneficial flora, increase the content of short chain fatty acid of enteric-coated substances, and suggest the action mechanism of the enteric-coated substances in improving constipation of mice after being administered to the mice with constipation.

Table 7 shows the blood lipid levels in blood samples of each group of KM mice weight loss experiments. As can be seen from Table 7, the blood lipid levels TCH, TG and LDL-C in the fecal samples of the mice in the model group were all significantly higher than those in the normal group, while HDL-C was significantly lower than that in the normal group; in contrast, the test articles provided in examples 1-5 in the experimental group significantly decreased TCH, TG and LDL-C, while HDL-C was significantly increased in the blood samples of the mice after administration. It is suggested that the plant extract composition comprising eucheuma peptide and more than ten components in examples 1 to 5 of the present application can regulate the accumulation of blood fat and fat in the mouse, control the contents and the ratio of LDL-C and HDL-C in the mouse, have a significant weight-reducing effect, and improve the health status of the mouse when administered to an obese mouse.

In addition, the results of HE staining of liver tissue slices of each group of mice revealed that the plant extract composition comprising eucheuma peptide and up to ten components of example 1 to 5 of the present application was effective in preventing fatty liver and improving liver function when administered to obese mice.

TABLE 7

In summary, the deglycosylated peptides extracted from Eucheuma, which have antioxidant and dipeptidyl peptidase IV inhibiting activities, are proved by in vitro experiments; and the cell experiment explains the non-toxicity of the eucheuma peptide on cells and the action mechanism of inhibiting the activity of the dipeptidyl polypeptidase IV.

The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.

Sequence listing

<110> Qing maple Ling soda drink (Jilin) Co Ltd

Application of plant extract composition in preparation of products for treating constipation and losing weight

<141> 2022-04-29

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<210> 12

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 12

Met Ser Gln Tyr Pro Ile Cys Ile Ser Ala Thr Ser Ser Gln Ile Leu

1 5 10 15

<210> 13

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 13

Gly Thr Met Gln Tyr Lys Gly Glu Gly Pro Ile Gly Phe Arg Gly Lys

1 5 10 15

Leu Ser Gly Ala Asn Asn Tyr Ala

20

<210> 14

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 14

Gly Val Asp His Ile His Ala Gly Thr Val Val

1 5 10

<210> 15

<211> 14

<212> PRT

<213> Artificial Sequence

<400> 15

Gly Thr Val Val Gly Lys Leu Glu Gly Asp Pro Leu Met Ile

1 5 10

<210> 16

<211> 29

<212> PRT

<213> Artificial Sequence

<400> 16

Gly Arg Val Val Tyr Glu Gly Leu Lys Gly Gly Leu Asp Phe Leu Lys

1 5 10 15

Asp Asp Glu Asn Ile Asn Ser Gln Pro Phe Met Arg Trp

20 25

<210> 17

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 17

Leu Glu Gly Asp Pro Leu Met Ile Arg Gly Phe Tyr Asn Thr Leu Leu

1 5 10 15

Leu Pro Tyr

<210> 18

<211> 30

<212> PRT

<213> Artificial Sequence

<400> 18

Asn Ile Asn Ser Gln Pro Phe Met Arg Trp Lys Glu Arg Phe Leu Tyr

1 5 10 15

Ala Met Glu Gly Val Asn Arg Ser Ile Ala Ala Thr Gly Glu

20 25 30

Claims

1. Eucheuma peptide is a peptide of amino acid selected from any one of SEQ ID No. 9-12.

2. A process for the preparation of eucheuma peptide according to claim 1, comprising:

(1) Making Eucheuma Gelatinosum powder;

(2) Soaking with hydrochloric acid solution with pH =3 for multiple times, filtering, and collecting combined filtrate;

(3) Adjusting the pH value of the combined filtrate to 4.5 by using sodium bicarbonate, adding ammonium sulfate according to a feed-liquid ratio of 1 (g/mL), stirring overnight, standing, removing a supernatant, filtering a lower layer, and collecting a first solid;

(4) Dissolving the obtained first solid with 35% ethanol solution, and centrifuging at 15000 Xg 4 deg.C for 15min to obtain second solid;

(5) A step of obtaining a third solid comprising:

dissolving the second solid with PBS buffer solution with pH7.5, wherein the mass percent of the second solid is 2.5wt%, simultaneously adding a first enzyme preparation with the mass percent of not less than 50000Unit/mL, reacting for 2.5h at 25 ℃, treating for 1min at 105 ℃, centrifuging for 15min at 8000 Xg 4 ℃, taking supernatant, performing ultrafiltration concentration with the molecular weight cutoff of 10KD, taking filtrate, concentrating and drying to obtain a third solid; wherein the first enzyme preparation is lysostaphin;

(6) A step of obtaining a fourth solid comprising:

dissolving the third solid with PBS buffer solution with pH7.5, wherein the mass percent of the third solid is 1.25wt%, simultaneously adding a second enzyme preparation with the mass percent of not less than 20000Unit/mL, reacting for more than 24h at 35 ℃ in a water bath, treating for 10min at 105 ℃, centrifuging for 15min at 8000 Xg 4 ℃, taking supernatant, carrying out ultrafiltration and concentration on the enzymolysis solution by adopting an ultrafiltration cup with the cut-off molecular weight of 10KD, taking filtrate, concentrating and drying to obtain a fourth solid; wherein the second enzyme preparation comprises cellulase not less than 10000Unit/mL and endoglycosidase F not less than 20 Unit/mL;

or

Dissolving the third solid substance by using PBS buffer solution with the pH value of 7.5, wherein the mass percent of the third solid substance is 1.25wt%, simultaneously adding a second enzyme preparation with the mass percent of not less than 20000Unit/mL, reacting for more than 24h in a water bath at the temperature of 35 ℃, treating for 10min at the temperature of 105 ℃, centrifuging for 15min at the temperature of 8000 Xg 4 ℃, taking supernate, carrying out ultrafiltration and concentration on the enzymatic hydrolysate by using an ultrafiltration cup with the molecular weight cut-off of 10KD, taking filtrate, concentrating and drying to obtain a fourth solid substance; wherein the second enzyme preparation comprises cellulase not less than 10000Unit/mL, endoglycosidase F not less than 20Unit/mL, beta-galactosidase not less than 120Unit/mL, beta-N-acetylglucosaminidase not less than 12Unit/mL and fructosidase not less than 6000 Unit/mL;

(7) And (3) carrying out gel chromatography purification and/or reverse phase chromatography purification on the fourth solid substance.

3. A plant extract composition, which comprises the eucheuma peptide of claim 1, wherein the weight percentage of the eucheuma peptide is 4.5 to 10.5wt%, 2 to 8.5wt% of sour cherry juice, 0.3 to 1.5wt% of erythritol, 1 to 5.5wt% of L-arabinose, 0.1 to 2.5wt% of corn stigma, 0.15 to 1.5wt% of mulberry leaves, 1 to 3.25wt% of gordon euryale seeds, 0.05 to 1.5wt% of sunflower discs, 0.5 to 2.5wt% of hawthorn, 4.5 to 10.5wt% of pawpaw, 1 to 3.5wt% of white lotus, 0.5 to 5.5wt% of chicory, 0.1 to 0.5wt% of gardenia, 0.1 to 2.5wt% of kudzu root, 0.15 to 1.5wt% of tuckahoe, 2 to 6.5wt% of celery, 0.05 to 2.5wt% of eucommia ulmoides, and 2.5wt% of auxiliary materials, and the balance of acceptable food.

4. The plant extract composition of claim 3, comprising the eucheuma peptide of claim 1 in an amount of 4.5wt%.