CN117683087A - Eupolyphaga Seu Steleophaga peptide with effect of resisting hyperlipidemia and application thereof - Google Patents

Abstract

The invention belongs to the field of bioactive peptides, and in particular relates to an ground beetle peptide with anti-hyperlipidemia function and application thereof. The ground beetle peptide consists of KR-14 and/or VR-13, wherein the KR-14 peptide structure sequence KYPDDKPLGFPFDR is shown as SEQ ID NO. 1, and the VR-13 peptide structure sequence VAAPVAVAAPVAR is shown as SEQ ID NO. 2.

Description

Eupolyphaga Seu Steleophaga peptide with effect of resisting hyperlipidemia and application thereof

Technical Field

The invention belongs to the field of bioactive peptides, and in particular relates to an ground beetle peptide with anti-hyperlipidemia function and application thereof.

Background

Hyperlipidemia, also known as lipid metabolism disorder or abnormality, is a high risk factor for atherosclerosis of important target organs such as heart and brain, and is liable to cause serious complications such as cardiovascular diseases, thrombosis, fatty liver, etc. The hyperlipidemia in China has a tendency of 'younger', and how to better prevent and treat the hyperlipidemia is important to reduce the occurrence rate of cardiovascular and cerebrovascular events. At present, although the pathogenesis of diabetes is deeply studied in medicine, the most widely applied drugs for treating hyperlipidemia are statin drugs, fibrate drugs and nicotinic acid drugs, and adverse reactions such as liver damage and gastrointestinal tract reaction are often reported although the curative effect is positive. The culture of the traditional Chinese medicine is profound, the advantages of the traditional Chinese medicine in the treatment of the hyperlipidemia are fully exerted, and the traditional Chinese medicine has very positive significance.

Hyperlipidemia is a metabolic disease in which one or more of total cholesterol, triacylglycerol and low-density lipoprotein cholesterol in plasma is increased or high-density lipoprotein cholesterol is decreased due to various causes, and is mainly characterized by an increase in blood lipid index, and clinical manifestations are mainly xanthomas caused by deposition of lipids in dermis and arteriosclerosis caused by deposition of lipids in vascular endothelium. Although hyperlipidemia may cause xanthomas, its incidence is not very high; the occurrence and development of atherosclerosis is a slowly progressive process. Thus, in general, most patients are free of obvious symptoms and abnormal signs. Hyperlipidemia can be classified into primary and secondary. Primary is related to congenital and genetic, either due to single or multiple gene defects, abnormalities in receptors, enzymes or apolipoproteins involved in lipoprotein transport and metabolism, or due to environmental factors (diet, nutrition, drugs) and by unknown mechanisms. Secondary to metabolic disorders (diabetes, hypertension, myxoedema, hypothyroidism, obesity, liver and kidney diseases, adrenocortical hyperactivity) or other factors of age, sex, season, drinking, smoking, eating, physical activity, mental stress, emotional activity, etc.

Currently, known pharmaceutical preparations for improving hyperlipidemia state include statins, fibrates, nicotinic acid drugs, and the like. Statin drugs, i.e., 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors, act by blocking the intracellular hydroxymethylvalerate metabolic pathway by competitively inhibiting the endogenous cholesterol synthesis rate-limiting enzyme HMG-CoA reductase, thereby reducing intracellular cholesterol synthesis, thereby feedback stimulating the increase in the number and activity of Low Density Lipoprotein (LDL) receptors on the cell membrane surface, increasing serum cholesterol clearance, and reducing levels of serum cholesterol, and are clinically mainly used for reducing cholesterol, particularly low density lipoprotein-cholesterol (LDL-C), and treating atherosclerosis, and have become the most effective drugs for the prevention and treatment of coronary heart disease. Statin-related myopathy clinical manifestations include myalgia, myositis, and rhabdomyolysis, which can cause acute renal failure and impaired liver function. Fibrates (fibrids), also known as phenoxy acids, have not yet been fully defined as a mechanism of action, possibly by activating LPL or acting on peroxisome proliferator-activated receptors (PPARs) to exert lipid-lowering effects. However, fibrates can cause gastrointestinal reactions such as abdominal pain, diarrhea, nausea, etc., and can severely lead to myositis. The nicotinic acid medicine has broad-spectrum lipid regulating effect, can be used as single or auxiliary therapeutic medicine for patients with hypertriglyceridemia and mixed hyperlipidemia, and is especially suitable for patients with HDL-C reduction or combined triglyceride increase. It can inhibit glycerol esterase activity in adipose tissue and inhibit adipose tissue mobilization, thereby reducing liver VLDL synthesis; enhancing lipoprotein lipase (LPL) activity, promoting hydrolysis of plasma TG, reducing Very Low Density Lipoprotein (VLDL) concentration, and reducing VLDL to LDL conversion, thereby reducing total cholesterol and LDL-C. However, the toxic and side effects of these drugs have not been properly treated, and thus, effective strategies for effectively treating or preventing hyperlipidemia and complications of hyperlipidemia have yet to be established.

Eupolyphaga Seu Steleophaga, etc. The Eupolyphaga Seu Steleophaga is dry female body of Eupolyphaga Seu Steleophaga and Eupolyphaga Seu Steleophaga of Eupolyphaga Seu Steleophaga, and comprises amino acids, alkaloids, volatile oil and various lipid components. Is traditional Chinese medicine for promoting blood circulation and removing blood stasis. The main component of the ground beetle is protein, which accounts for 60% of the total body. Modern researches have found that Eupolyphaga Seu Steleophaga has pharmacological activities of inhibiting platelet aggregation, inhibiting thrombin activity, promoting fibrinolysis, and reducing blood lipid.

Currently, a number of natural hypolipidemic active ingredients have been found, such as: curcumin, paeoniflorin, notoginsenoside, and the like. However, the treatment of hyperlipidemia with biologically active polypeptides has been less studied.

Disclosure of Invention

The invention is based on the therapeutic effect of ground beetles on hyperlipidemia and the application of a bionic enzymatic hydrolysis process, and discovers that active peptides KYPDDKPLGFPFDR and VAAPVAVAAPVAR derived from ground beetles have the effect of reducing blood fat by regulating ACC and HMGCR signals in a body.

In a first aspect of the invention, there is provided an anti-hyperlipidemia ground beetle peptide, which consists of KR-14 and/or VR-13, wherein the KR-14 peptide structural sequence KYPDDKPLGFPFDR is shown as SEQ ID NO. 1, and the VR-13 peptide structural sequence VAAPVAVAAPVAR is shown as SEQ ID NO. 2.

In a second aspect of the present invention, there is provided a method for preparing the above KR-14 or VR-13 two ground beetle peptides, comprising the steps of: (1) resin treatment; (2) amino acid coupling reaction; (3) And (3) performing trifluoroacetic acid cleavage to obtain solid-phase synthesized peptides KYPDDKPLGFPFDR and VAAPVAVAAPVAR.

In another aspect of the invention, there is provided the use of two ground beetle peptides as described above for the treatment and/or prevention of hyperlipidemia.

In another aspect, the invention provides application of the two ground beetle peptides in anti-hyperlipidemia and complications thereof.

The hyperlipidemia complications are specifically hyperlipidemia liver injury, hyperlipidemia cardiomyopathy, hyperlipidemia amyotrophy, diabetic neuropathy, and diabetic coronary heart disease.

In another aspect of the invention, there is provided a medicament comprising two ground beetle peptides KR-14 and/or VR-13 having anti-hyperlipidemic properties.

Further, the medicine is one of injection, granule, capsule or pill.

A pharmaceutically acceptable carrier and/or additive comprising two ground beetle peptides KR-14 and/or VR-13.

The invention has the beneficial effects that:

(1) The ground beetle peptide has high purity, is safe and effective, eliminates the immunogen reaction caused by taking the heterologous protein and the like, and reduces the incidence rate of adverse reaction;

(2) In the prior art, no report of using KYPDDKPLGFPFDR and VAAPVAVAAPVAR ground beetle peptides for treating and/or preventing hyperlipidemia is disclosed, and the invention provides a reference for researching and developing a novel anti-hyperlipidemia and complications thereof therapeutic drug;

(3) According to the invention, KYPDDKPLGFPFDR and VAAPVAVAAPVAR ground beetle peptides are synthesized for the first time, and the inhibitory activity of the synthesized polypeptide on the hyperlipidemia rat is detected, wherein the synthesized polypeptide has potential protection effect on the hyperlipidemia;

(4) KYPDDKPLGFPFDR and VAAPVAVAAPVAR, the 2 kinds of peptide structures are high-content and high-activity small molecule bioactive peptide components obtained by refining, purifying and analyzing the small molecule peptides by the inventor in the bionic enzymolysis of ground beetles, and the 2 kinds of small molecule bioactive peptide components are discovered for the first time by the inventor and are not reported;

(5) The polypeptide sequence adopts a standard Fmoc scheme, and a reasonable polypeptide synthesis method is realized through resin screening. The C-terminal carboxyl of the target polypeptide is connected with an insoluble high molecular resin in a covalent bond form, and then the amino group of the amino acid is taken as a starting point to react with the carboxyl of another molecular amino acid to form a peptide bond. The process is repeated continuously, and the target polypeptide product can be obtained. Removing the protecting group after the synthesis reaction is finished, and separating the peptide chain from the resin to obtain a target product, wherein the polypeptide synthesis is a process of repeatedly adding amino acid, and the solid phase synthesis sequence is synthesized from the C end to the N end;

(6) The invention reveals the effect of ground beetle peptide in improving hyperlipidemia, is expected to provide new theoretical basis and new medicine treating target for treating hyperlipidemia and its complications, and provides reference for researching animal Chinese medicine polypeptide components.

Drawings

Fig. 1: KR-14 structure diagram;

fig. 2: VR-13 structural diagram;

FIG. 3 schematic representation of serum and liver TG and TC levels in groups of rats

Wherein, the abscissa is in proper order: a normal group, a model group, a positive medicine group, an ground beetle medicinal material group, an ground beetle protein group, a KR-14 group and a VR-13 group;

FIG. 4 rat liver ACC and HMGCR mRNA expression;

wherein, the abscissa is in proper order: a normal group, a model group, a positive medicine group, an ground beetle medicinal material group, an ground beetle protein group, a KR-14 group and a VR-13 group;

Detailed Description

EXAMPLE 1 preparation of ground beetle peptide

Resin treatment: fmoc-Leu-Wang resin (molar substitution coefficient 0.6 mmol/g) was selected as the starting resin, placed in a 3L reaction column, soaked in Dichloromethane (DCM), and drained to complete the swelling of the resin. Then adding 20% piperidine DMF solution, introducing nitrogen, stirring for 30min, filtering to dry the solvent, and circularly washing the resin for 6 times by using N, N-Dimethylformamide (DMF), and carrying out suction filtration to complete deprotection of the resin.

Amino acid coupling reaction: weighing corresponding amounts of O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate (TBTU) and protected amino acid in a beaker, and adding DMF for dissolution; then, the reaction solution was added to the resin, and then N, N-Diisopropylethylamine (DIEA) was added thereto, followed by bubbling with nitrogen for 90 minutes, followed by detection by ninhydrin reaction. After the reaction was completed, the solvent was removed, and the resin was washed 3 times with DMF. And adding 20% piperidine DMF solution into the resin, introducing nitrogen, continuously blowing for 30min, removing the solvent, and washing the resin for 6 times by using DMF to finish the coupling of the amino acid. Repeating the above reaction procedures until the condensation reaction of all the protected amino acids is completed, coupling to the last amino acid, connecting fluorescein in the last step, draining, loading into a centrifuge tube, adding FITC, HATU, DIEA, DMF and DCM, shaking in a shaker for 8-12h, avoiding light, and centrifuging. After centrifugation, the polypeptide was contracted, washed 3 times sequentially with DMF/DCM/MEOH, and then weighed after draining.

Trifluoroacetic acid cleavage: the resin was added to a previously prepared lysate (86% TFA/5% EDT/5% phenyl sulfide/3% phenol/2% pure water) and stirred for 150min. And then pumping the resin and the lysate, and adding diethyl ether to fully separate out the polypeptide. Suction filtration and thorough washing with diethyl ether for 6 times gave the solid phase synthetic peptides KYPDDKPLGFPFDR and VAAPVAVAAPVAR.

The obtained solid phase synthesis of tetradecapeptide KYPDDKPLGFPFDR is named: KR-14, sequence: lys-Tyr-Pro-Asp-Asp-Lys-Pro-Leu-Gly-Phe-Pro-Phe-Asp-Arg, i.e. lysine-tyrosine-proline-aspartic acid-lysine-proline-leucine-glycine-phenylalanine-proline-phenylalanine-aspartic acid-arginine, molecular weight 1693.84Da, purity 95.3%, isoelectric point 6.67, hydrophobicity index 22.42 Kcal. MoL ^-1 The water solubility is better, and the structural diagram is shown in figure 1.

The obtained solid phase synthesis decapeptide VAAPVAVAAPVAR is named as: VR-13, sequence: val-Ala-Ala-Pro-Val-Ala-Val-Ala-Ala-Pro-Val-Ala-Arg, i.e., valine-alanine-proline-valine-alanine-arginine, having a molecular weight of 1190.71Da, a purity of 96.0%, an isoelectric point of 11.11, a hydrophobicity index of 11.15 Kcal. MoL ^-1 The water solubility is better, and the structural diagram is shown in figure 2.

EXAMPLE 2 study of ground beetle peptide for the treatment of hyperlipidemia

2.1 experimental content:

2.1.1 laboratory animals

Male SD rats weighing 180-200 g were bred in SPF animal houses of the medical college of Japan, supplied by the laboratory animal breeding Limited of Jinan Pengyue. The environmental conditions are controlled at 23-25 ℃, the relative humidity is 55-65%, and the illumination/darkness is respectively carried out for 12 hours, so that the water is not limited.

2.1.2 grouping of animals, modeling and administration

Rats were randomly divided into normal groups and model building groups, the normal groups were given standard feed, the model building groups were given high fat diet (3% cholesterol, 0.5% cholic acid, 5% lard, 0.2% propylthiouracil were added to standard feed) and kept for 4 weeks. The high-fat feed is provided by Shandong Jinan He Yue laboratory animals Limited company, and consists of 65% of basic feed, 15% of lard, 5% of cholesterol, 10% of egg yolk and 5% of sodium bile acid. The model group, the positive medicine group, the ground beetle medicinal material group, the ground beetle protein group, the KR-14 group and the VR-13 group are continuously administered with the high-fat feed, and simultaneously, the high-fat feed is administered by stomach irrigation. The administration dosage of rats is calculated according to the body surface area method, the administration dosage of ground beetle medicinal material group is 3.00 g.kg-1, the administration dosage of ground beetle protein group is 1.50 g.kg-1, the administration dosage of positive medicine group is 20.00 mg.kg-1, the administration dosage of KR-14 group and VR-13 group is 25.00 mg.kg-1, and the blank group and the model group are administered with the same volume of physiological saline every day. After 4 weeks of administration, the change in body mass of each group of rats was recorded while detecting the change in blood lipid, and the feces of each group of rats were collected and liver tissue was collected.

2.1.3 biological sample collection and preservation

The biological sample of the rat is fasted for 12 hours before taking materials, water is not forbidden, and the feces in 12 hours after the last administration are collected. Naturally airing the manure sample at a ventilation position, and crushing for later use; after the rats of each group are anesthetized by 10% chloral hydrate, the abdominal aorta is taken and filled in an anticoagulation test tube, the anticoagulation test tube stands for 30min at room temperature, 3 500 r.min < -1 > is centrifuged for 25min, serum is taken and split charging is carried out, and the blood is preserved at-20 ℃ for standby; rapidly taking down liver tissue, and quenching in liquid nitrogen at-80deg.C.

2.1.4 Biochemical index determination

And (3) biochemical index measurement: the TC, TG and LDL contents of the serum samples of each group of rats were detected by a full-automatic biochemical analyzer. The liver was added with a mixed solvent of methanol and chloroform (2:1) to prepare a 5% homogenate, and the TG and TC contents were measured. Rat feces were homogenized in the same manner to determine TG content. The ATP and AMP contents in the liver tissue homogenate are measured by an ELISA method, and the measuring process is strictly carried out according to the specification of experimental operation and the instruction of a kit.

2.1.5qRT-PCR method for detecting rat liver acetyl-CoA carboxylase (ACC) and carboxymethylglutaryl-CoA reductase (HMGCR) mRNA expression

TRIzol lyses liver tissue, extracts total RNA, and determines its concentration and purity by ultra-micro spectrophotometry, and reverse transcribes cDNA. The primers used were ACC: upstream 5'-GGAGTAGTTGCTGTAGAAACCCG-3', downstream 5'-CATTAGAGGTAGCCCTTCACGG-3', amplification product length 174bp; ACTIN: upstream 5'-TGCTATGTTGCCCTAGACTTCG-3', downstream 5'-GTTGGCATAGAGGTCTTTACGG-3', amplification product length 240bp. The reaction system: cDNA 2.5. Mu.L, 1. Mu.L of each of the upstream and downstream primers, 12.5. Mu.L of 2 XqPCR Mix, and 8.0. Mu.L of ddH 2O. Reaction conditions: the sample was subjected to 40 cycles of 10min pre-denaturation at 95 ℃, 15s denaturation at 95 ℃, 1min annealing at 60 ℃, and 2min extension at 72 ℃.

2.1.6 statistical analysis

Data processing is carried out by adopting SPSS20.0 and Graphpad Prism, the results are expressed as mean ± standard deviation, and LSD test is carried out for comparison every two; if the variance is uneven, the difference is tested by adopting H-G test, and the difference is statistically significant when P is less than 0.05.

2.2 experimental results:

2.2.1KR-14 and VR-13 Effect on the body Mass of hyperlipidemic rats

There was no difference in initial mass for each group of rats; after 3 weeks of modeling, the mass of the rats in the other groups is increased compared with that of the rats in the blank group, and the difference has statistical significance, which indicates that the mass of the rats can be obviously increased by high-fat feeding; the body mass of rats was reduced in the positive drug group, the ground beetle protein group, the KR-14 group and the VR-13 group, and the differences were statistically significant as compared with the model group after 4 weeks of administration, as shown in Table 1.

TABLE 1 rat mass detection results

Note that compared to the blank group ¹⁾ P<0.01， ²⁾ P<0.05; compared with the model group ³⁾ P<0.01， ⁴⁾ P<0.05

2.2.2KR-14 and VR-13 Effect on hyperlipidemia rat blood lipid factor

Compared with a blank group, the serum and liver TG and TC contents of rats in the model group are increased, and the difference has statistical significance; compared with the model group, the positive medicine group, the ground beetle medicinal material group, the ground beetle protein group, the KR-14 group and the VR-13 group are reduced in serum and liver TG and TC contents, and the difference has statistical significance, as shown in figure 3.

2.2.3KR-14 and VR-13 Effect on rat liver ACC and HMGCR mRNA expression

Compared with the blank group, the expression of liver ACC and HMGCR mRNA of the model group rats is up-regulated, and the difference has statistical significance (P < 0.01); compared with the model group, the expression of the mRNA of the liver ACC and HMGCR of rats in the positive drug group, the ground beetle medicinal material group, the ground beetle protein group, the KR-14 group and the VR-13 group is down-regulated, and the difference has statistical significance (P <0.05 and P < 0.01), as shown in figure 4.

In summary, the hyperlipoidemia model rats induced by the hyperlipoidemia feed and emulsion show hyperlipoidemia and inflammatory reactions, and the abnormal expression of ACC, HMGCR and mTORC1 genes exists in liver tissues, so that the KR-14 peptide and VR-13 peptide can reduce the blood lipid factor index and improve endoplasmic reticulum stress by reducing ACC and HMGCR mRNA expression, AMPK phosphorylation and mTORC1 protein expression, thereby playing a role in treating hyperlipoidemia and complications thereof.

The polypeptide of the present invention may be suitably purified according to a method commonly used in the field of peptide chemistry, such as ion exchange resin method, partition chromatography, gel chromatography, affinity chromatography, high Performance Liquid Chromatography (HPLC), and countercurrent distribution method, in addition to the Fmoc-solid phase synthesis process used in example 1.

The polypeptides of the invention may also be produced by enzymatic degradation or hydrolysis of polypeptides or proteins comprising the above amino acid sequences.

Claims (8)

1. An ground beetle peptide with anti-hyperlipidemia function, which is characterized in that: the ground beetle peptide consists of KR-14 and/or VR-13, wherein the KR-14 peptide structure sequence KYPDDKPLGFPFDR is shown as SEQ ID NO. 1, and the VR-13 peptide structure sequence VAAPVAVAAPVAR is shown as SEQ ID NO. 2.

2. An woodlouse peptide having hyperlipidemia resistance as defined in claim 1, wherein: the preparation method of the KR-14 or VR-13 two ground beetle peptides comprises the following steps: (1) resin treatment; (2) amino acid coupling reaction; (3) And (3) performing trifluoroacetic acid cleavage to obtain solid-phase synthesized peptides KYPDDKPLGFPFDR and VAAPVAVAAPVAR.

3. An woodlouse peptide having hyperlipidemia resistance as defined in claim 1, wherein: use of KR-14 and/or VR-13 for the treatment and/or prevention of hyperlipidemia.

4. An woodlouse peptide having hyperlipidemia resistance as defined in claim 1, wherein: use of KR-14 and/or VR-13 in anti-hyperlipidemic and complications thereof.

5. An anti-hyperlipidemia ground beetle peptide as defined in claim 4, wherein: the hyperlipidemia complications are specifically hyperlipidemia liver injury, hyperlipidemia cardiomyopathy, hyperlipidemia amyotrophy, diabetic neuropathy and diabetic coronary heart disease.

6. A medicament having anti-hyperlipidemia comprising two ground beetle peptides of KR-14 and/or VR-13 of claim 1.

7. The medicament as claimed in claim 6, wherein: the medicine is one of injection, granule, capsule or pill.

8. A pharmaceutically acceptable carrier and/or additive, characterized in that: the carrier and/or additive comprises two ground beetle peptides of KR-14 and/or VR-13 of claim 1.