阅读说明：本技术 一种胰蛋白酶激活剂及其在胰腺外分泌不足方面的应用 (Trypsin activator and application thereof in aspect of pancreatic exocrine insufficiency ) 是由 赵玉男 李瑞梅 陈飞燕 陈琳 程瑶 高健 于 2020-12-22 设计创作，主要内容包括：偶然发现达玛烷-24-烯-3,12,20-三醇或其可药用盐可激活胰蛋白酶,提高胰蛋白酶的活性,特别适用于肠道内胰蛋白酶活性低下的病理生理过程,可应用于各种病因导致的胰腺外分泌功能不足的预防和/或治疗,具体而言,它具有胰蛋白酶激活作用。(It is discovered that dammarane-24-alkene-3, 12,20-triol or its medicinal salt can activate trypsin, improve the activity of trypsin, is especially suitable for the pathophysiological process of low activity of trypsin in intestinal tract, can be used for preventing and/or treating pancreatic exocrine hypofunction caused by various pathogenies, and specifically has the function of activating trypsin.)

1. A trypsin activator comprising the formula) Dammarane-24-alkene-3, 12,20-triol or medicinal salt thereof

——（）。

2. The trypsin activator according to claim 1, wherein dammarane-24-en-3, 12,20-triol or a pharmaceutically acceptable salt thereof is used for the preparation of a medicament for the treatment of diseases associated with pancreatic hyposecretion.

3. The trypsin activator according to claim 1, wherein the amount of dammarane-24-en-3, 12,20-triol administered to a rat in a pancreatic exocrine insufficiency model is from 25mg/kg to 100 mg/kg.

4. The trypsin activator according to claim 1, wherein dammarane-24-en-3, 12,20-triol acts by increasing the enzymatic hydrolysis of trypsin.

5. The trypsin activator according to claim 1, wherein dammarane-24-en-3, 12,20-triol affects the activity of trypsin by forming hydrogen bonds at the S2 site with the hydroxyl group at C-3 with the amino acid residues ASN74 and ILE73 of trypsin.

6. The trypsin activator according to claim 1, wherein dammarane-24-en-3, 12,20-triol increases its ability to cleave protein substrates in a dose-dependent manner after incubation with trypsin in vitro at concentrations ranging from 12.5 μ M and 50 μ M.

7. The trypsin activator according to claim 6, wherein the protein substrate for enzymatic hydrolysis of dammarane-24-ene-3, 12,20-triol is brain-derived creatine kinase.

8. The trypsin activator according to claim 1, wherein the dammarane-24-ene-3, 12,20-triol affects the trypsin activity in an inverse "U" form in a dose-effect relationship therebetween, and the enzyme activity gradually increases and reaches a maximum value as the concentration of dammarane-24-ene-3, 12,20-triol gradually increases; however, when the concentration is increased, the enzyme activity starts to decrease.

9. The trypsin activator according to claim 1, wherein dammarane-24-en-3, 12,20-triol is used for preparing drugs for reversing weight loss, food consumption reduction and diarrhea of animals caused by pancreatic exocrine insufficiency.

10. The trypsin activator according to claim 1, wherein dammarane-24-en-3, 12,20-triol is used for the preparation of a medicament for treating anorexia, weight loss, diarrhea associated with pancreatic exocrine insufficiency.

Technical Field

The present invention belongs to the field of biomedical technology, with the aim of finding compounds having valuable properties, in particular those which can be used for the preparation of medicaments.

We unexpectedly discover from natural products that individual dammarane compounds can activate trypsin, improve the activity of the trypsin, are particularly suitable for pathophysiological processes of pancreatic enzyme activity low in intestinal tracts, and can be applied to prevention and/or treatment of pancreatic exocrine insufficiency caused by various causes.

In particular, it has trypsin activating action.

Background

Trypsin (Trypsin) is one of the proteases. In vertebrates, it functions as a digestive enzyme. After synthesis in the pancreas, trypsinogen is secreted into the intestine as a component of pancreatic juice, and is decomposed by enterokinase and/or "self-activation" to become activated trypsin. It not only acts as a protein digest, but also activates other digestive zymogens in pancreatic juice, such as: chymotrypsinogen, procarboxypeptidase, phospholipase, etc., play an important role in protein and lipolysis [ Wangchui, et al, high school science 2016,36(12):39-44 ].

Pancreatic Exocrine Insufficiency (PEI) refers to the condition that the secretion of pancreatic juice in human body is insufficient due to various reasons, which causes the activity of various pancreatic enzymes in intestinal digestive juice to be reduced, and further causes the digestive malabsorption of patients, and causes the symptoms of malnutrition such as inappetence, weight loss, and steatorrhea. PEI is common in primary pancreatic diseases, such as: acute and chronic pancreatitis, pancreatic cancer, pancreatectomy, diabetes, etc., and correction of PEI is an important strategy and method to improve the nutritional status and prognosis of patients with pancreatic disease [ maligen and royal jelly. J.Chinese clinicians 2014,8(2):265-269 ].

Pancreatic juice secreted by the pancreas contains about 17 digestive enzymes, such as amylase, lipase, protease, and hydrolase, and although trypsin is only 1 of them, its specificity has led to a reasonable expectation that enhancing the activity of trypsin would be an adjunct treatment for PEI. Trypsin activators are expected to increase the activity of various digestive enzymes in the intestinal tract of PEI patients, increasing the digestive capacity of the patients, and thereby improving the malnourished state of the patients.

The following structures, which are known in the art and enter the national drug standards catalog (Chinese food and drug laboratory), are described in more detail in this specification and are commercially available from reagent companies. However they have never been described as trypsin activators.

Dammarane-24-ene-3, 12,20-triol (Dammar-24-ene-3,12,20-triol)

Disclosure of Invention

The invention relates to dammarane compounds shown in formula (I), wherein R1, R2 and R3 are all hydroxyl groups, and R4 is hydrogen group [ formula (II) ]. Chemical designation of formula (II): dammarane-24-ene-3, 12, 20-triol. Analysis by ForteBio's intermolecular interactor Ocet RED96 showed: formula (II) has a direct interaction with trypsin.

Biofilm layer interference technique (BLI): affinity of formula (II) and Trypsin (K)_D) The value is (2.15. + -. 0.11). times.10^-5M。

Molecular docking analysis shows that the formula (II) has two potential binding sites with trypsin, namely enzyme activity center adjacent sites (S1 and S2). Formula (II) tends to hydrogen bond at the S2 site through the hydroxyl group at C-3 with the amino acid residues ASN74 and ILE73 of trypsin, thereby affecting the activity of trypsin.

After in vitro incubation with trypsin, the ability of the compound of formula (II) to cleave protein substrates can be increased dose-dependently, as measured by protein gel electrophoresis: the 12.5 mu M and 50 mu M of the formula (II) can respectively improve the enzymolysis capacity of the compound by 15.6 percent and 29.8 percent.

After the in vitro incubation of the formula (II) with trypsin, the activity of the trypsin can be increased, and the maximum increase amplitude is about 13.91%. In addition, it was found that as the concentration of formula (II) gradually increased, the enzyme activity gradually increased and reached a maximum; however, when the concentration is increased, the enzyme activity begins to decrease, and the dose-effect relationship between the two is in an inverted U shape.

The formula (II) is administered by intragastric administration, which can improve the activity of trypsin and lipase in duodenum of rat with pancreas exocrine insufficiency, and increase the content of elastase (FE-1) in feces of animal model, thereby improving dyspepsia symptoms such as weight loss, food intake reduction and diarrhea of rat model.

Drawings

FIG. 1 binding dissociation curves for trypsin at different concentrations of formula (II).

FIG. 2 potential S1 site for trypsin binding of formula (II).

FIG. 3 potential S2 site for trypsin binding of formula (II).

FIG. 4 optimal binding conformation of formula (II) at position S2 of trypsin.

Figure 5 effect of formula (II) on Trypsin (Trypsin) enzymatic activity in vitro (mean ± sd, n ═ 3). Trypsin and substrate brain-type creatine kinase were incubated for 25min, samples were separated on SDS-PAGE gels, and bands were analyzed for grayscale after silver staining (a). The influence of the formula (II) or the solvent DMSO in the enzymolysis system on the substrate enzymolysis capability of Trypsin is observed, wherein the influence of the formula (II) of 12.5 mu M (B) or 50 mu M (C) is observed. Two-factor analysis of variance<0.01vs.Trypsin(－)DMSO，^# P<0.05 vs. Trypsin(+)DMSO。

Detailed Description

The invention is further illustrated below with reference to examples and figures.

Example 1

Affinity (K) was calculated using the BLI technique to calculate the binding and dissociation constants between formula (II) and trypsin_D) Judging the interaction strength between the two, comprising the following scheme:

ForteBio Octet Red96 assay detection: (1) SA sensor (ForteBio) pre-wet: putting the new SA sensor into PBS for prewetting for 5-10 min; (2) balancing the instrument: equilibrating the SA sensor in a ligand buffer; (3) and (3) curing: solidifying the protein of interest (trypsin) onto the SA sensor; (4) balancing: equilibrating the sensor immobilized with the protein of interest in an assay buffer; (5) combining: respectively immersing the sensor with the target protein into solutions with different concentrations in the formula (II) for combination; (6) dissociation: dissociating the analyte bound to the target protein; (7) and (3) data analysis: data collected in real time were scored using ForteBio Data Analysis softwareAnalysis, calculation of the affinity (K) of formula (II) for Trypsin_D)。

The results of the interaction of formula (II) with trypsin at various concentrations are shown in fig. 1, and the detailed binding and dissociation kinetics data are shown in table 1, based on the BLI technique to calculate the affinity between formula (II) and trypsin. The results show that: the binding constant (Kon) of formula (II) to trypsin was 5.89X 10³1/Ms, dissociation constant (Koff) of 1.26X 10^-11/s, affinity (K)_D) Is 2.15 multiplied by 10^-5M。

TABLE 1 affinity constants of formula (II) for trypsin

Example 2

Molecular docking of formula (II) with trypsin: based on Yin endow science and technology cloud computing platform molecule docking service modulehttp://www.yinfotek.com/dock) The flexible docking mechanism is adopted, the conformation of the formula (II) and the trypsin is allowed to change freely, the docking result is accurately calculated, and the binding site and the binding mode of the formula (II) and the trypsin are discussed, and the scheme comprises the following steps:

study subjects: crystal structure 4i8h with the highest resolution of bovine trypsin.

Molecular docking simulation: the potential binding pocket for trypsin was analyzed systematically and S1 and S2 in fig. 2 and 3, respectively, represent the 2 potential binding sites detected by the system in 4i8h crystals. The binding energy of formula (II) at the S1 and S2 sites was-43.352474 and-49.774826 kcal/mol, respectively, and the docking conformation score was-48.811569 and-50.864765, respectively. A larger absolute value of the score value indicates a stronger binding ability of formula (II) to the site. The score at the S1 site was lower than the corresponding score at the S2 site, suggesting that formula (II) is prone to binding at the S2 site, with moderate binding. This binding is closer to the active center of trypsin, presumably having an allosteric effect on trypsin activity.

The optimal binding conformation at S2 of formula (II) is shown in fig. 4. The hydroxyl group at C-3 of formula (II) can form hydrogen bonds with the tryptic amino acid residues ASN74 and ILE 73. In addition, formula (II) can form van der Waals and hydrophobic interactions with the trypsin amino acid residues TYR151, HIS40 and TYR 39.

Example 3

The effect of formula (II) on the enzymatic ability of trypsin in vitro comprises the following steps.

Selecting brain-derived creatine kinase (CK-BB) as a substrate, and determining the enzymolysis capacity of protease by adopting a gel electrophoresis technology: (1) preparing 15 mu g/ml CK-BB solution by using lysis buffer, and adding the mother liquor of the formula (II) or a solvent DMSO for preparing the mother liquor so that the concentration of the formula (II) is 12.5 mu M or 50 mu M and the concentration of the solvent is 0.6 percent; (2) mu.l of the above solution was pipetted into a PCR tube, 4. mu.l of Trypsin solution (Trypsin: CK-MM ═ 1:25) was added, and incubated in a PCR apparatus (Biosafer) for 25min (25 ℃ C.), and for the indigestible sample 4. mu.l of TNC buffer (50mM Tris-HCl, 50mM NaCl,10mM CaCl)₂pH 8.0); (3) after the incubation is finished, adding 20X protease inhibitor cocktail (Roche) immediately, and continuing to incubate on ice for 10 min; (4) adding a proper amount of sample buffer solution into the sample, denaturing at 90 ℃ for 10min, and then carrying out SDS-PAGE gel electrophoresis separation; (5) after the gel was silver stained, the protein bands were subjected to grayscale analysis using a Tanon 4100 gel electrophoresis image analysis system.

The results show that: under the condition of not adding trypsin, the gray values of the solvent DMSO group and the histone substrate of the formula (II) have no obvious difference, which indicates that the adding of the histone substrates is kept uniform; under the condition of adding trypsin, the protein substrate can be obviously enzymolyzed, and the relative abundance of the gray value of the strip is reduced by more than half, which indicates that the enzymolysis function of the trypsin is normally exerted; the relative abundance of the substrate protein bands in group (II) was significantly reduced compared to the DMSO group, indicating that it could increase the enzymatic capacity of trypsin (fig. 5A). 12.5. mu.M and 50. mu.M of formula (II) increased the enzymatic activity by 15.6% (FIG. 5B) and 29.8% (FIG. 5C), respectively.

Example 4

The effect of formula (II) on trypsin activity in vitro comprising the following steps.

Experimental materials: formula (II) (Shanghai leaf Biotech Co., Ltd.), trypsin test kit (Nanjing institute of bioengineering), and bovine trypsin (Jiangsu Kai-ji Biotech Co., Ltd.).

Drawing a trypsin enzyme activity standard curve: (1) using diluted 5 trypsins (200mg/ml, 100mg/ml, 50mg/ml, 25mg/ml and 12.5mg/ml) with different concentrations as samples to be detected, and adding, mixing and incubating according to the steps provided by the kit specification, see table 2; (2) zero adjustment is carried out on the reaction liquid in the blank tube; (3) and reading an absorbance value A1 of the measuring tube 30s after uniformly mixing in a 1cm quartz cuvette at 253nm, reading an absorbance value A2 again after 20min, and solving the difference of absorbance twice (delta A is A2-A1). The values were recorded and a standard curve of trypsin was plotted.

TABLE 2 plotting of creatine kinase enzyme activity standard curve

The effect of formula (II) on trypsin enzyme activity in vitro: (1) taking 100mg/ml trypsin as a sample to be detected, taking 5 different concentrations of the trypsin in the formula (II) as liquid medicine (the control liquid medicine is a 50% DMSO aqueous solution), and adding, uniformly mixing and incubating according to the table 3; (2) zero adjustment is carried out on the reaction liquid in the blank tube; (3) reading the absorbance value A1 of the measuring tube and the contrast tube 30s after uniformly mixing in a 1cm quartz cuvette at 253nm, reading the absorbance value A2 again after 20min, and solving the difference of absorbance for two times (delta A is A2-A1); (4) the concentration of trypsin was calculated according to a standard curve of trypsin.

TABLE 3 Effect of Compounds on creatine kinase enzyme Activity

Standard curve of trypsin concentration: the trypsin presents better linearity in the interval of 12.5mg/ml to 200mg/ml, and the linear regression equation is as follows: y ═ 0.002X-0.0193 (R)²0.9979, n-3), where X is the enzyme concentration (mg/ml) and Y represents the absorbance OD₂₅₃The value is obtained.

As shown in table 4, formula (II) can increase the concentration of trypsin, i.e. increase the activity of trypsin, dose-dependently; compared with a solvent control group (0.3% DMSO), the trypsin activity can be remarkably increased by 25 mu M (P <0.05), 50 mu M (P <0.05) and 100 mu M (P <0.05) of the formula (II), and the increase range is respectively 7.17%, 13.91% and 9.03%; however, at concentrations of formula (II) above 50. mu.M, the trypsin-activating capacity begins to decrease, with an inverse "U" relationship between the two.

Table 4 effect of formula (II) on trypsin activity (mean ± standard deviation, n ═ 5)

Note: one-way anova,. P <0.05 vs.0.3% DMSO,. P <0.01 vs.0.3% DMSO.

Example 5

The effect of formula (II) on exocrine pancreatic dysfunction in vivo, comprising the following steps.

Experimental animals: wistar rats 50, male, 180-: SCXK (Shanghai) 2013 and 0006, which are raised in standard animal houses.

Experimental materials and instruments: formula (II) (Nanjing Xin thick Biotech Co., Ltd.), pancreatin capsule (Solvay Pharma), dibutyltin Dichloride (DBTC) (Sigma), trypsin and pancrelipase test kit (Solarbio), fecal elastase (FE-1) ELISA kit (Wuhan Huamei Biotech Co., Ltd.), and other reagents are all domestic analytical purifications. Multifunctional microplate readers (Spectramax M5, Molecular Devices, USA).

Experimental grouping, modeling and dosing: all rats were fed free diet for 7 days at 18-24 ℃ in the laboratory and acclimatized for 1 week. The groups were randomized into 6 groups of 10 individuals. Namely: control group, model group, pancreatin group (150mg/kg), low dose group of formula (II) (25mg/kg), medium dose group of formula (II) (50mg/kg), and high dose group of formula (II) (100 mg/kg). The rat tail vein of the model group and the administration group was slowly injected with a DBTC solution (7mg/kg, DBTC dissolved in a solution containing 20% ethanol, 40% glycerol and 40% dimethyl sulfoxide), and the tail vein of the control group was injected with a solvent of the same volume. After 2 weeks of molding, the formula (II) and the positive drug pancreatin are administered by gavage 1 time a day for 4 weeks; the control group was administered 1% sodium carboxymethylcellulose aqueous solution by gavage.

Experimental material taking and index analysis: the rats of each group were weighed for the last 3 days of the experiment and food intake, and were visually observed for hair color and fecal status, and feces were collected. Rat feces were scored using the following scoring criteria: shaping excrement, hardening and drying the surface (1 point is counted); the feces are shaped, soft and have more water on the surface (2 points is counted); loose stool, soft quality and easy adhesion (3 points). After weighing the collected fresh feces, determining the content of FE-1 in the feces according to the specification of an ELISA kit. Finally, the anesthetized rats were sacrificed, the duodenum was dissected open, the intestinal tract was flushed with 2ml of the extract, the extracts were collected and combined, centrifuged at 10000rpm for 10min at 4 ℃, and the supernatant was taken to measure the activity of trypsin and pancreatic lipase in the duodenum according to the kit instructions.

The results show (table 5): compared with the control group, the animals in the model group had dull, yellow and sparse hair, and had significantly reduced body weight (P <0.01) and food intake (P <0.01), and significant diarrhea symptoms (P < 0.01). Compared with the model group, the weight (P <0.01) and food intake (P <0.01) of the positive pancreatin group animals are obviously increased, and the diarrhea degree is obviously reduced (P <0.01), and the state of the positive pancreatin group animals is close to that of the control group animals. The middle dose (P <0.05) and the high dose group (P <0.01) of the formula (II) can obviously reverse the reduction of the body weight and the food consumption of model animals and obviously improve the condition of loose stools. Compared with the model group, the low dose group of the formula (II) has no statistical difference in each index.

TABLE 5 Effect of formula (II) on weight, food intake and diarrhea in rats with exocrine pancreatic insufficiency model (mean. + -. standard deviation, n ═ 10)

Note: the analysis of the one-way variance is carried out,^#P<0.05，^##P<0.01vs. control; p<0.05，**P<0.01vs. model set.

Pancreatic exocrine insufficiency (table 6): the model animal adopts a DBTC injection method, which can cause the destruction of bile duct epithelial cells to cause the blockage of pancreatic and bile ducts, and chronic inflammation can cause pancreatic fibrosis to cause the exocrine pancreatic gland to be insufficient, so that the animal has dyspepsia. Compared with the control group, the activity of trypsin (P <0.01) and lipase (P <0.01) in duodenum of the model group animals is obviously reduced, and the content of FE-1(P <0.01) in excrement is obviously reduced. Compared with the model group, the activities of the positive drug group animal duodenum trypsin (P <0.01) and lipase (P <0.01) are obviously improved, and the content of FE-1(P <0.01) in the excrement is obviously increased. The high dose of the formula (II) can obviously reverse the reduction of the activity of trypsin (P <0.01) and lipase (P <0.05) in duodenum of model animals, and improve the content of FE-1(P <0.05) in excrement of the model animals; in addition, the formula (II) with medium dosage has obvious improvement effect on the activity of duodenal trypsin (P <0.05) of a model animal; compared with the model group, the low dose group of the formula (II) has no statistical difference in each index.

TABLE 6 Effect of formula (II) on model rat intraduodenal trypsin, lipase and FE-1 (mean. + -. standard deviation, n ═ 10)

Note: the analysis of the one-way variance is carried out,^#P<0.05，^##P<0.01vs. control; p<0.05，**P<0.01vs. model set.

Definition of lipase activity: hydrolyzing olive oil at 37 deg.C per minute to generate 1 μmol fatty acid as an enzyme activity unit; definition of trypsin activity: the increase of 0.001 in absorbance at 253nm catalyzed per minute at 37 ℃ is one unit of enzyme activity.