A method for enzymatic hydrolysis of Sipunculus quadripunctatus, enzymatic hydrolysis product and application thereof

Abstract

The invention discloses an enzymatic hydrolysis method, enzymatic hydrolysis product and application of a Sipuncula spp., and belongs to the field of biomedical technology. The Sipuncula spp. enzymatic hydrolysis product provided by the invention has good water solubility and no toxicity. The Sipuncula spp. enzymatic hydrolysis product can significantly reduce the content of creatinine and urea nitrogen in the blood, significantly downregulate serum insulin levels, reduce insulin resistance index (HOMA-IR), increase insulin sensitivity (HOMA-IS), and significantly reduce the expression of inflammatory factors IL-1β, TNF-α, IL-6, and CRP in renal tissue, and improve renal tissue pathological damage caused by diabetes.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an enzymatic hydrolysis method of Sipunculus quadripunctatus, an enzymatic hydrolysis product and an application thereof. Background Art

[0002] Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes and an important reason why the mortality rate of diabetic patients has remained high. In the early stage of DN, the main manifestations are increased glomerular filtration rate and microalbuminuria. As the disease progresses, symptoms such as glomerular sclerosis and renal interstitial fibrosis appear, and eventually the kidney's compensatory ability is weakened, leading to renal failure. The occurrence and development of DN is the result of multiple factors and pathways. It is currently believed that the occurrence and development of DN is closely related to the following factors: oxidative stress, inflammation, and autophagy.

[0003] The design of treatment strategies for DN should focus on how to prevent or delay its occurrence and development. In previous treatments, anti-inflammatory drugs such as retinoic acid can improve limb damage in diabetic mice. However, these drugs often have certain side effects. Therefore, it is of great significance to find safe, non-toxic and natural food-derived peptides to alleviate the occurrence and development of DN.

[0004] Sipunculus sphaeroides, commonly known as sandworms, are marine organisms native to the Beibu Gulf region of China. They have traditionally been used to treat carbuncle, tuberculosis, nocturia, regulate the functions of the stomach and spleen, and restore the body's weakness caused by various pathogens and aging. Modern pharmacological studies have shown that Sipunculus sphaeroides has antioxidant, anti-inflammatory, anti-radiation damage, immunity-enhancing, and anti-aging effects. There are currently no reports on the use of Sipunculus sphaeroides in the treatment of diabetic nephropathy. Summary of the invention

[0005] The purpose of the present invention is to provide an enzymatic hydrolysis method, enzymatic hydrolysis product and application of Sipunculus quadripunctatus to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides an enzymatic hydrolysis method of Siegespidae, comprising the following steps: using Siegespidae body wall as raw material, performing enzymatic hydrolysis by using trypsin, and separating enzymatic hydrolysis products.

[0008] Preferably, the enzymatic hydrolysis reaction condition is 45° C. for 4 hours.

[0009] Preferably, the separation of the enzymatic hydrolysis product comprises: centrifuging the enzyme product to collect the supernatant, and separating the supernatant by ultrafiltration membrane to obtain the enzymatic hydrolysis product.

[0010] Preferably, the centrifugal condition is 9000 rpm, 20 min.

[0011] Preferably, the ultrafiltration membrane separation is specifically as follows: the supernatant is passed through 2.5 kDa and 50 kDa ultrafiltration membranes respectively, and the filtrate is collected.

[0012] The invention also provides a Sipunculus enzymatic hydrolysis product prepared by the enzymatic hydrolysis method.

[0013] The present invention also provides application of the Sipunculus quadrilateral enzymatic hydrolysis product in preparing medicine for treating diabetic nephropathy.

[0014] The invention also provides a medicine for treating diabetic nephropathy, comprising the Sipunculus quadrilateral enzymatic hydrolysis product.

[0015] The present invention also provides a pharmaceutical preparation for treating diabetic nephropathy, comprising the Sipunculus quadrilateral enzymatic hydrolysis product or the drug and a pharmaceutically acceptable carrier.

[0016] Preferably, the pharmaceutically acceptable carrier is suitable for solid dosage form, liquid dosage form, paste dosage form or emulsion dosage form.

[0017] Based on the above technical solution, the present invention has the following technical effects:

[0018] The invention provides a Sipuncula enzymatic hydrolysis product, which has good water solubility and no toxicity. The Sipuncula enzymatic hydrolysis product can significantly reduce the content of creatinine and urea nitrogen in the blood, significantly downregulate the serum insulin level, reduce the insulin resistance index (HOMA-IR), increase the insulin sensitivity (HOMA-IS), and significantly reduce the expression of inflammatory factors IL-1β, TNF-α, IL-6, and CRP in kidney tissue, and improve the kidney tissue pathological damage caused by diabetes, wherein the medium dose has the best effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 Effects of Siempuri spp. enzymatic hydrolysate on body weight, fasting blood glucose, food intake and drinking water of DN mice, including (a) body weight, (b) fasting blood glucose, (c) water intake, (d) food intake, all values ​​are expressed as x±SEM;

[0021] Figure 2Effects of Sipunculus quadratus enzymatic hydrolysate on renal function of DN mice, including (a) serum creatinine, (b) blood urea nitrogen, (c) urine protein, and (d) renal organ index; all values ​​are expressed as x±SEM; *p<0.05, **p<0.01, compared with the blank group; #p<0.05, ##p<0.01, compared with the model group;

[0022] Figure 3 The effect of Sipuncula enzymatic hydrolysis products on blood lipid levels, including (a) high-density lipoprotein, (b) low-density lipoprotein, (c) triglycerides, and (d) total cholesterol; all values ​​are expressed as **p<0.01, compared with the blank group; #p<0.05, ##p<0.01, $p<0.05, $$p<0.01, compared with the model group;

[0023] Figure 4 Effects of Sipuncula truncatula enzymatic hydrolysate on insulin and insulin resistance, including (a) serum insulin, (b) HOMA-IR, (c) HOMA-IS; all values ​​are expressed as *p<0.05, **p<0.01, compared with the blank group; ##p<0.01, compared with the model group;

[0024] Figure 5 The effect of Siemprenus enzymatic hydrolysate on the pathological sections of kidney tissues of DN mice, including (A) normal control, (B) model group, (C) model group + A1 (100 mg / kg), (D) model group + A1 (400 mg / kg), (E) model group + A1 (800 mg / kg), (F) model group + A2 (100 mg / kg), (G) model group + A2 (400 mg / kg), (H) model group + A2 (800 mg / kg), 200 times magnification observation;

[0025] Figure 6 Effects of Sipunculus quadrilateral enzymatic hydrolysate on inflammatory factors IL-1β, TNF-α, IL-6 and CRP, including (a) TNF-α, (b) IL-1β, (c) IL-6, (d) CRP; all values ​​are expressed as x±SEM; **p<0.01, compared with the blank group; #p<0.05, ##p<0.01, $p<0.05, $$p<0.01, compared with the model group. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0032] Example 1 Preparation of marine Sipunculus enzymatic hydrolysate

[0033] After the body wall of the siphonophore was washed and crushed, water 10 times the mass of the siphonophore was added, and trypsin was added. The reaction was hydrolyzed at 45°C for 4 hours, and the reaction was terminated at 60°C for 10 minutes. After the sample was cooled, the supernatant was collected and centrifuged at 9000 rpm for 20 minutes to obtain an enzymatic hydrolyzate. The supernatant was passed through 2.5 kDa and 50 kDa ultrafiltration membranes respectively, and the filtrate was collected, concentrated and dried to obtain siphonophore enzymatic hydrolyzates A1 (<2.5 kDa) and A2 (2.5 kDa-50 kDa).

[0034] Example 2

[0035] ①Establishment of DN mouse model and animal grouping

[0036] To explore the role of Sipunculus quadratus enzymatic hydrolysates in DN, we established a type 2 DN (T2D) mouse model by combining a high-fat diet (HFD) with a small dose of intraperitoneal injection of streptozotocin (STZ). After 1 week of adaptation, the mice were randomly divided into a control group and a diabetic group. The normal control group (NC) mice were fed a standard diet, and the diabetic group (DN) was given a high-fat diet to induce insulin resistance until the end of the experiment. After 5 weeks of dietary control, the HFD-fed mice were intraperitoneally injected with a single dose of streptozotocin solution (30 mg / kg body weight, citrate buffer pH 4.5) once a week for three consecutive weeks, and the control group mice were injected with an equal amount of citrate buffer. Five days after the last injection of STZ, the blood glucose concentration of the model group mice was measured on an empty stomach for 12 hours, and 11-18mM confirmed T2D. Subsequently, the mice were randomly divided into the following groups (10 in each group), specifically:

[0037] Blank control group: An equal amount of physiological saline was used for gavage instead of the enzymatic hydrolysate of Sipunculus truncatula;

[0038] Model group: The same amount of saline was used instead of the enzymatic hydrolysate of Sipunculus truncatula for gavage;

[0039] A1-L group: daily oral administration of A1 component (100 mg / kg);

[0040] A1-M group: daily oral administration of A1 component (400 mg / kg);

[0041] Group A1-H: daily intragastric administration of component A1 (800 mg / kg);

[0042] A2-L group: daily intragastric administration of component A2 (100 mg / kg);

[0043] A2-M group: daily oral administration of A2 component (400 mg / kg);

[0044] Group A2-H: daily oral administration of component A2 (800 mg / kg).

[0045] Each group was given oral administration for 6 consecutive weeks.

[0046] ②Effects of Sipunculus quadrilateral enzymatic hydrolysis products on body weight, fasting blood glucose, food intake and drinking water intake of DN mice

[0047] During the experiment, the weight changes, diet, and water intake of the mice were recorded weekly, and the blood glucose changes of the mice were tested weekly using a blood glucose tester kit.

[0048] The results are as follows Figure 1As shown in the figure, in the constructed DN mouse model, the body weight of mice in the DN group was always lower than that in the blank control group. During the drug administration period, compared with the A2 dose groups, the weight loss trend of mice in the A1 low, medium and high dose groups slowed down. After HFD / STZ combined induction, the blood glucose level of mice in the diabetic group gradually increased and was ≥11.1mmol / L, and was always higher than that in the blank control group. There was no obvious downward trend in blood glucose levels in each drug administration group after 6 weeks of intervention. Compared with the blank control group, the amount of water consumed by DN mice increased, and the water intake of DN mice was significantly reduced after treatment with medium and high doses of A1. However, each drug administration group had no effect on food intake.

[0049] ③Effects of Sipunculus quadratus enzymatic hydrolysis products on creatinine and urea nitrogen in kidney tissue of DN mice

[0050] After 6 weeks, mice were fasted for 12 hours but not water, blood was collected from the eyeballs, and the collected blood was centrifuged at 3000rpm for 20 minutes to separate serum, and the levels of serum creatinine (SCr) and urea nitrogen (BUN) of mice were tested according to the instructions of the reagent manufacturer. Urine was centrifuged at 3000rpm for 20 minutes to collect the supernatant, and the urine protein level was tested according to the instructions of the kit and the renal organ index was calculated: renal organ index = bilateral kidney weight / body weight (%).

[0051] Test results such as Figure 2 As shown in the results, in the DN model, the levels of blood creatinine, urea nitrogen, and urine microalbumin reflect the severity of renal damage to a certain extent. In this experiment, the levels of serum creatinine, urea nitrogen, and urine microalbumin in DN mice were significantly higher than those in the blank control group. After intervention, it was found that the levels of serum creatinine and urea nitrogen in group A1 decreased significantly (P < 0.05), and the blood creatinine and urea nitrogen in the low- and medium-dose groups A2 decreased to a certain extent, but the difference was not significant. Similarly, the urine microalbumin index in the low- and medium-dose drug-treated groups A1 and A2 decreased to a certain extent. The renal organ index of diabetic mice increased significantly (P < 0.05), and A1 and A2 treatments restored the ratio of renal mass to body weight to varying degrees, making it close to normal ( Figure 2 d), and the effects of the A1 treatment groups at various concentrations were better than those of the A1 treatment group.

[0052] ④Detection of blood lipid levels

[0053] The separated and collected serum was used to detect the contents of high-density lipoprotein (H-DLC), low-density lipoprotein (L-DLC), triglycerides (TG), and total cholesterol (TC) in mouse serum according to the kit instructions.

[0054] Test results such as Figure 3As shown in the results, compared with the normal group, the TG, LDL-C, and TC levels of mice in the DN group were significantly increased (P < 0.05), while the HDL-C level decreased (P < 0.05). After A1 treatment, TG and LDL-C in each administration group decreased significantly (P < 0.05), almost reaching normal levels, and the TC level in the medium-dose group decreased significantly. After A2 treatment, the TG levels in the medium-dose and high-dose groups and the LDL-C levels in the medium-dose group decreased significantly. The HDL-C level in the A1 high-dose group increased significantly (P < 0.05). The results showed that compared with A2, A1 was able to improve the four blood lipids of DN mice.

[0055] ⑤Detect FBG, insulin resistance and insulin sensitivity indicators

[0056] One day before the end of the experiment, the blood glucose changes of mice were detected using a blood glucose tester kit, and the serum insulin level of mice was detected using a test kit. The specific operation was carried out according to the instructions of the kit. And the following were calculated respectively:

[0057] Insulin resistance index (HOMA-IR) = fasting serum insulin (mU / ml) fasting blood glucose (mmol / L) / 22.5;

[0058] Insulin sensitivity (HOMA-IS) = 1 / [fasting blood glucose (mmol / L) fasting serum insulin (mU / ml)].

[0059] The results are as follows Figure 4 As shown in the figure, compared with the NC group, the serum insulin and insulin resistance index of the mice in the DN group increased, and the insulin sensitivity decreased significantly (P < 0.05). After 6 weeks of intervention with Sipunculus quadratus enzymatic hydrolysate, the serum insulin level was significantly reduced in the A1 group at all concentrations (P < 0.05), and there was no significant change in the A2 groups. The insulin resistance index of the A1-M, A2-M and A2-H treatment groups decreased to varying degrees, but the difference was not significant. The insulin sensitivity of the A1-M and A2-M groups increased, but the difference was not significant. A1 has a better effect than A2 in improving serum insulin and insulin tolerance in DN mice.

[0060] ⑥ Renal tissue pathological observation

[0061] At the end of the experiment, one kidney was fixed in 4% paraformaldehyde, dehydrated, paraffin-embedded, and sliced. The slice thickness was 4 μm. The steps were as follows: the slices were placed in xylene I for 10 min-xylene II for 10 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-75% alcohol for 5 min, and washed with tap water. Hematoxylin staining: the slices were placed in hematoxylin staining solution for 3-5 min, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue solution, and rinsed with running water; eosin staining: the slices were placed in 85% and 95% gradient alcohol for dehydration for 5 min each, and stained in eosin staining solution for 5 min. After dehydration and sealing, the severity of renal injury was assessed using an optical microscope with 200 times magnification.

[0062] like Figure 5 As shown in the figure, the renal tubular epithelial cells in the blank group had clear outlines, no obvious degeneration, no tubular expansion, no proliferation of glomerular mesangial cells, and the size of the spheres was relatively uniform. In the model group, a large number of renal tubular epithelial cells were degenerated, the cell boundaries were unclear, and the nuclear vacuolation was severe, as shown by the blue arrow; protein casts were visible in some renal tubules, and the lumen was filled with protein mucus, as shown by the yellow arrow; a large number of inflammatory cells were infiltrated in the interstitium, as shown by the black arrow; the glomeruli were slightly hypertrophic, and the mesangial cells were proliferated, as shown by the red arrow. In the renal tissue of each treatment group of A1, some renal tubular degeneration, epithelial cell nuclear vacuolation, and interstitial inflammatory cell infiltration were alleviated compared with the model group. Compared with the model group, the degree of inflammatory cell infiltration and tubular degeneration in each treatment group of A2 were improved. This shows that A1 and A2 can alleviate the pathological damage of renal tissue caused by diabetes.

[0063] ⑦ ELISA

[0064] After grinding an appropriate amount of frozen kidney tissue, centrifuge at 3000 rpm for 20 min and take the supernatant. Use BCA kit to determine the protein concentration, and use ELISA kit to detect IL-1β, TNF-α, IL-6 and CRP levels. Specific operations were carried out according to the kit instructions.

[0065] Test results such as Figure 6 As shown in the figure, compared with the NC group, the expression of four inflammatory factors IL-1β, TNF-α, IL-6 and CRP in the renal tissue of the DN group mice was significantly increased (P < 0.05). After 6 weeks of intervention, the results showed that the enzymatic hydrolysate A1 of Siempre punctatus could significantly reduce the levels of the four inflammatory factors (P < 0.05), indicating that the enzymatic hydrolysate A1 of Siempre punctatus has good activity in inhibiting inflammatory response. Although the enzymatic hydrolysate A2 of Siempre punctatus can also improve the level of inflammatory factors in renal tissue, the effect of A1 is more significant.

[0066] In summary, the present invention provides a Sipuncula enzymatic hydrolysis product, which has good water solubility and no toxic effects. Compared with the Sipuncula enzymatic hydrolysis product A2, the A1 enzymatic hydrolysis product can significantly reduce the content of creatinine and urea nitrogen in the blood. A1 and A2 treatments restored the kidney mass to body weight ratio to varying degrees, making it close to a normal state, and the effect of the A1 treatment group at each concentration was better than that of the A2 treatment group. After the intervention of the A1 enzymatic hydrolysis product, the A1 group significantly downregulated the serum insulin level at each concentration treatment, the insulin resistance index (HOMA-IR) decreased, and the insulin sensitivity (HOMA-IS) increased. It can significantly reduce the expression of inflammatory factors IL-1β, TNF-α, IL-6, and CRP in renal tissue, and improve the renal tissue pathological damage caused by diabetes, among which the medium dose has the best effect.

[0067] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. The use of Sipunculus quadrilateral enzymatic hydrolysis products in the preparation of a drug for treating diabetic nephropathy, characterized in that: The preparation method of the enzymatic hydrolysis product of Sipunculus sphenanthera comprises: using the body wall of Sipunculus sphenanthera as raw material, performing enzymatic hydrolysis by using trypsin, and separating the enzymatic hydrolysis product; The reaction conditions of the enzymatic hydrolysis are 45°C for 4h; The separation of the enzymatic hydrolysis product specifically comprises: centrifuging the enzymatic hydrolysis product to collect the supernatant, and separating the supernatant by ultrafiltration membrane to obtain the Sipunculus quadripunctatus enzymatic hydrolysis product; The centrifugation condition is 9000 rpm, 20 min; the ultrafiltration membrane separation is specifically: the supernatant passes through 2.5 kDa and 50 kDa ultrafiltration membranes respectively, and the filtrate is collected.

Images