阅读说明：本技术 一种降低血压的豌豆寡肽及其制备方法和应用 (Blood pressure lowering pea oligopeptide and preparation method and application thereof ) 是由 张树成 周泉城 杨进洁 杨国栋 臧庆佳 刘长宝 武世敏 房万才 孙钊 于 2020-08-24 设计创作，主要内容包括：本发明公开了一种降低血压的豌豆寡肽及其制备方法和应用,其中豌豆寡肽的分子式：C<Sub>20</Sub>H<Sub>33</Sub>N<Sub>5</Sub>O<Sub>8</Sub>；分子量：471.50；命名为：(S)-5-氨基-2-((S)-1-((S)-2-((S)-2-氨基-3-甲基丁胺基)-4-羧基丁酰基)吡咯烷-2-甲酰胺基)-5-氧代戊烷酸。其具有通过抑制ACE活性,阻断肾素-血管紧张素系统,调节肠道菌群多样性、丰富度,以及特定菌群比例,从而发挥显著降压的作用,所述豌豆寡肽可用于功能性食品、补剂中。(The invention discloses a pea oligopeptide capable of reducing blood pressure as well as a preparation method and application thereof, wherein the molecular formula of the pea oligopeptide is as follows: c 20 H 33 N 5 O 8 (ii) a Molecular weight: 471.50, respectively; is named as: (S) -5-amino-2- ((S) -1- ((S) -2- ((S) -2-amino-3-methylbutanamido) -4-carboxybutanoyl) pyrrolidine-2-carboxamido) -5-oxopentanoic acid. It has effects in blocking kidney by inhibiting ACE activityThe vitamin-angiotensin system regulates the diversity and abundance of intestinal flora and the proportion of specific flora, thereby playing a role in remarkably reducing blood pressure, and the pea oligopeptide can be used in functional foods and supplements.)

1. A blood pressure lowering pea oligopeptide, wherein the pea oligopeptide has the following structural formula:

。

2. the method of preparing a pea oligopeptide according to claim 1, comprising the steps of:

firstly, using Wang Resin as a carrier to combine Fmoc-Gln (Trt) -OH for reaction to generate Fmoc-Gln (Trt) -Resin, and then introducing Fmoc-Pro-OH, Fmoc-Glu (otBu) -OH and Fmoc-Val-OH in three steps to combine the Fmoc-Gln (Trt) -Resin, the Fmoc-Glu (otBu) -OH and the Fmoc-Val-OH with the reaction product of the previous step respectively;

the products generated in each step in the process are respectively as follows:

Fmoc-Pro-Gln(Trt)-Resin、Fmoc-Glu(otBu)-Pro-Gln(Trt)-Resin、Fmoc-Val-Glu(otBu)-Pro-Gln(Trt)-Resin；

and then cracking the generated Fmoc-Val-Glu (otBu) -Pro-Gln (Trt) -Resin molecules to obtain a mixture of the Val-Glu-Pro-Gln oligopeptide and other short peptide molecular fragments, and finally purifying to obtain a pure Val-Glu-Pro-Gln pea oligopeptide product with the purity of more than 98%.

3. The use of pea oligopeptides according to claim 1, wherein said pea oligopeptides are applied in a dose of 100 to 150 mg/kg.

4. Use of a pea oligopeptide according to claim 1, wherein: the pea oligopeptide can be combined with Ala356, Pro407, Val518, Arg522 and Tyr523 amino acid residues of angiotensin converting enzyme and Zn ions, wherein the pea oligopeptide forms hydrogen bond with Ala356 and Arg522, and C-terminal carboxyl of the pea oligopeptide forms metal coordination bond with Zn ions; the pea oligopeptide is combined with the active site of the angiotensin converting enzyme mainly through the action of hydrogen bonds and the coordination with Zn ions, and plays a competitive inhibition role on the angiotensin converting enzyme, thereby inhibiting the activity of the angiotensin converting enzyme.

5. Use of a pea oligopeptide according to claim 1, wherein: the pea oligopeptide blocks a renin-angiotensin system by inhibiting the activity of angiotensin converting enzyme, thereby playing a role in remarkably reducing blood pressure.

6. Use of a pea oligopeptide according to claim 1, wherein: the pea oligopeptide maintains the abundance and diversity of flora within a normal range by adjusting the intestinal microenvironment, and maintains the proportion of the flora of Prevoteriaceae and Lachnospiraceae, so that the intestinal flora can normally utilize in-vivo substances and the function regulation function of flora metabolites is ensured, and the normal digestion, absorption and metabolism of nutrients are ensured.

7. Use of a pea oligopeptide according to claim 1, wherein: the pea oligopeptides reduce adverse effects on blood pressure caused by intestinal microbial disorders by improving the ability of the Lachnospiraceae to decompose carbohydrates.

8. Use of a pea oligopeptide according to claim 1, wherein: the pea oligopeptide has the regulation effect on kidney and spleen organs, so that the spleen index and the kidney index are kept in a normal range, the damage to visceral organs due to hypertension is avoided, and the blood pressure is regulated to a normal range.

9. Use of a pea oligopeptide according to claim 1, wherein: the pea oligopeptide is applied to functional foods and supplements.

10. Use of a pea oligopeptide according to claim 1, wherein: the pea oligopeptide is applied to preparing the medicine for treating hypertension.

Technical Field

The invention belongs to the technical field of oligopeptide extraction and application, and particularly relates to a pea oligopeptide capable of reducing blood pressure as well as a preparation method and application thereof.

Background

In recent years, more than one third of adults worldwide have symptoms of high blood pressure, and the statistical data of 'Chinese cardiovascular disease report 2019' shows that the total disease rate of hypertension in China has obvious trend of increasing year by year. In addition, hypertension can also cause other complications, more than half of cardiovascular diseases are related to hypertension, and stroke and myocardial infarction are the most common, so that the health and life of residents in China are seriously threatened.

However, the treatment of hypertension by using drugs alone causes various complications and side effects, and many people are only prone to hypertension and do not develop serious hypertension at present, and under the combined action of other factors such as the aging trend is strengthened and the medical insurance pressure is increased, the national policy pays more and more attention to the prevention of diseases. The compendium of the State "healthy China 2030" emphasizes the promotion of healthy Chinese construction, mainly aims at preventing and strengthening early diagnosis, early treatment and early rehabilitation.

The functional food has the main functions of enhancing immunity, promoting cell regeneration, promoting rehabilitation of patients and the like, can improve the sub-health state and the cure degree of patients with sub-health and chronic diseases, and also plays a role in prevention and health care for healthy residents. Therefore, under the conditions of falling of various policies and improvement of health consciousness, the related policies of functional foods are continuously perfected.

The oligopeptide has small molecular weight and good solubility; has good anticoagulation effect and good stability under heating or acid-base conditions. The direct supplement of oligopeptide can accelerate the absorption process of amino acid in vivo, improve the utilization rate of nitrogen by the body and provide nutrient substances required by the growth and development of human body.

Meanwhile, the amino acid sequences of some oligopeptides are similar to certain in vivo physiological activity regulators, and directly participate in the regulation of the physiological activities of organisms, or have the function of stimulating the secretion of intestinal receptor hormones or enzymes to play a plurality of very important physiological roles in human bodies. Such as regulating blood pressure, resisting free radicals, improving immunity, relieving fatigue, delaying aging, preventing and treating hyperlipemia, which are not possessed by raw material food protein or other amino acids.

At present, the research on the pea oligopeptides is less, and meanwhile, the application report of the pea oligopeptides in the aspect of reducing blood pressure is not seen, so that the development of the application of the pea oligopeptides in the aspects of reducing blood pressure and functional foods has important significance.

Disclosure of Invention

The invention provides a pea oligopeptide capable of reducing blood pressure, a preparation method and an application thereof, the pea oligopeptide has the effect of reducing blood pressure and can be applied to the fields of functional foods, medicines and foods with homology of medicine and food, and the pea oligopeptide is named as (S) -5-amino-2- ((S) -1- ((S) -2- ((S) -2-amino-3-methylbutanamido) -4-carboxybutyryl) pyrrolidine-2-formamido) -5-oxopentanoic acid.

The specific technical scheme is as follows:

a pea oligopeptide capable of reducing blood pressure, wherein the pea oligopeptide has a structural formula shown in figure 1, and the preparation method of the pea oligopeptide comprises the following steps:

firstly, using Wang Resin as a carrier to combine Fmoc-Gln (Trt) -OH for reaction to generate Fmoc-Gln (Trt) -Resin, and then introducing Fmoc-Pro-OH, Fmoc-Glu (otBu) -OH and Fmoc-Val-OH in three steps to combine the Fmoc-Gln (Trt) -Resin, the Fmoc-Glu (otBu) -OH and the Fmoc-Val-OH with the reaction product of the previous step respectively;

the products generated in each step in the process are respectively as follows:

Fmoc-Pro-Gln(Trt)-Resin、Fmoc-Glu(otBu)-Pro-Gln(Trt)-Resin、Fmoc-Val-Glu(otBu)-Pro-Gln(Trt)-Resin；

and then cracking the generated Fmoc-Val-Glu (otBu) -Pro-Gln (Trt) -Resin molecules to obtain a mixture of the Val-Glu-Pro-Gln oligopeptide and other short peptide molecular fragments, and finally purifying to obtain a pure Val-Glu-Pro-Gln pea oligopeptide product with the purity of more than 98%.

Preferably, the application dose of the pea oligopeptide is 100-150 mg/kg.

Preferably, the pea oligopeptide can be combined with Ala356, Pro407, Val518, Arg522, Tyr523 amino acid residues of angiotensin converting enzyme and Zn ions, wherein the pea oligopeptide forms hydrogen bonds with Ala356 and Arg522, and the C-terminal carboxyl group of the pea oligopeptide forms metal coordination bonds with the Zn ions; the pea oligopeptide is combined with the active site of the angiotensin converting enzyme mainly through the action of hydrogen bonds and the coordination with Zn ions, and plays a competitive inhibition role on the ACE enzyme, thereby inhibiting the activity of the angiotensin converting enzyme.

Preferably, the pea oligopeptides exert a significant blood pressure lowering effect by blocking the renin-angiotensin system through inhibition of angiotensin converting enzyme activity.

Preferably, the pea oligopeptides maintain the abundance and diversity of flora within a normal range by adjusting the intestinal microenvironment, and maintain the flora proportion of Prevoteriaceae and Lachnospiraceae, thereby ensuring the normal utilization of in vivo substances by the intestinal flora and the function regulation of flora metabolites and ensuring the normal digestion, absorption and metabolism of nutrients.

Preferably, the pea oligopeptides reduce adverse effects on blood pressure due to intestinal microbial disturbances by increasing the ability of the Lachnospiraceae family to break down carbohydrates.

Preferably, the pea oligopeptides have a regulating effect on kidney and spleen organs, so that spleen indexes and kidney indexes are kept in a normal range, damage to organs due to hypertension is avoided, and blood pressure is regulated to a normal range.

Preferably, the pea oligopeptide is applied to functional foods and supplements.

Preferably, the pea oligopeptide is applied to preparing the medicine for treating hypertension.

Has the advantages that:

1. the invention provides a pea oligopeptide capable of reducing blood pressure, which can be combined with Ala356, Pro407, Val518, Arg522 and Tyr523 amino acid residues of angiotensin converting enzyme and Zn ions, wherein the pea oligopeptide, Ala356 and Arg522 form hydrogen bond, and C-terminal carboxyl of the pea oligopeptide and the Zn ions form metal coordination bonds; the pea oligopeptide is mainly combined with the active site of ACE through the hydrogen bond effect and the coordination effect with Zn ions, and has a competitive inhibition effect on ACE enzyme, so that the activity of ACE is inhibited, a renin-angiotensin system is blocked, and the effect of remarkably reducing blood pressure is achieved.

2. According to the pea oligopeptide, the abundance and diversity of flora are maintained within a normal range by adjusting the intestinal microenvironment, and the proportion of Prevotellaceae to Lachnospiraceae flora is kept, so that the normal utilization of in-vivo substances by the intestinal flora and the function regulation effect of flora metabolites are ensured, the normal digestion, absorption and metabolism of nutrients are ensured, and the adverse effect of intestinal microbial disturbance on blood pressure is reduced. Meanwhile, the pea oligopeptide has a regulating effect on kidney and spleen organs, so that the spleen index and the kidney index are kept in a normal range, damage to visceral organs due to hypertension is avoided, and the blood pressure is regulated to be in a normal range.

3. The invention also provides application of the pea oligopeptide in functional foods and supplements, belongs to development of novel pea products, and can be added into the functional foods, medicines or supplements as an additive, so that the added value of agricultural products is improved, side effects caused by traditional medicines are avoided, and the blood pressure is effectively reduced.

Description of the drawings:

FIG. 1: is the chemical structural formula of the pea oligopeptide;

FIG. 2: is a combination graph of pea oligopeptide and ACE protein active sites;

FIG. 3: pea oligopeptide base sequence and structure;

FIG. 4: a method for synthesizing oligopeptide;

FIG. 5: "family" level rat intestinal flora Heatmap.

Detailed Description

The invention will be described in detail and clearly by reference to the accompanying drawings and examples.

The invention provides pea oligopeptide (S) -5-amino-2- ((S) -1- ((S) -2- ((S) -2-amino-3-methylbutanamido) -4-carboxybutyryl) pyrrolidine-2-formamido) -5-oxopentanoic acid with a blood pressure reducing effect, wherein the molecular formula of the pea oligopeptide is as follows: c₂₀H₃₃N₅O₈(ii) a Molecular weight: 471.50.

the pea oligopeptide can be combined with Ala356, Pro407, Val518, Arg522, Tyr523 amino acid residues of ACE and Zn ions, wherein the pea oligopeptide forms hydrogen bonds with Ala356 and Arg522, and the C-terminal carboxyl group of the pea oligopeptide forms metal coordination bonds with the Zn ions. Therefore, the pea oligopeptide is combined with the active site of ACE mainly through the action of hydrogen bonds and the coordination with Zn ions, and has competitive inhibition effect on ACE enzyme, so that the activity of ACE is inhibited. (ACE (angiotensin converting Enzyme).

The pea oligopeptide blocks a renin-angiotensin system by inhibiting the ACE activity, thereby playing a role in remarkably reducing blood pressure.

Therefore, the application mechanism or the application way of the pea oligopeptide in the hypertension field is as follows: by inhibiting ACE, the renin-angiotensin system is inhibited, thereby exerting the effect of lowering blood pressure.

The pea oligopeptide provided by the invention maintains the abundance and diversity of flora within a normal range by regulating the intestinal microenvironment, maintains the proportion of Prevotella ceae and Lachnospiraceae flora, thereby ensuring the normal utilization of in vivo substances by intestinal flora and the function regulation of flora metabolites, and ensuring the normal digestion, absorption and metabolism of nutrients.

Meanwhile, the pea oligopeptide provided by the invention can reduce adverse effects on blood pressure caused by intestinal microbial disturbance by improving the decomposition capability of the lactobacillus flora on carbohydrate. The spleen index and the kidney index are kept in a normal range by regulating the kidney and the spleen organs, so that the damage to visceral organs caused by hypertension is avoided, and the blood pressure is regulated to a normal range.

The preparation method of the pea oligopeptide comprises the following steps:

firstly, using Wang Resin as a carrier to combine Fmoc-Gln (Trt) -OH for reaction to generate Fmoc-Gln (Trt) -Resin, and then introducing Fmoc-Pro-OH, Fmoc-Glu (otBu) -OH and Fmoc-Val-OH in three steps to combine the Fmoc-Gln (Trt) -Resin, the Fmoc-Glu (otBu) -OH and the Fmoc-Val-OH with the reaction product of the previous step respectively;

the products generated in each step in the process are respectively as follows:

Fmoc-Pro-Gln(Trt)-Resin、Fmoc-Glu(otBu)-Pro-Gln(Trt)-Resin、Fmoc-Val-Glu(otBu)-Pro-Gln(Trt)-Resin；

and then cracking the generated Fmoc-Val-Glu (otBu) -Pro-Gln (Trt) -Resin molecules to obtain a mixture of the Val-Glu-Pro-Gln oligopeptide and other short peptide molecular fragments, and finally purifying to obtain a pure Val-Glu-Pro-Gln pea oligopeptide product with the purity of more than 98%.

Wherein Wang resin is Wang resin, Fomc is fluorenylmethoxycarbonyl acyl, and the product has protection effect in the process of amino acid synthesis and can be removed by diethylamine; trt is trityl, which is used to protect the α -carboxyl group of glutamine (Gln), and can be removed with trifluoroacetyl group (TFA). otBu side chain tert-butoxy group for protecting side chain carboxyl group of glutamic acid (Glu). Can be removed with TFA.

The pea oligopeptide can be applied to functional foods and supplements to play a role in reducing blood pressure, and the application dose is 100-150 mg/kg.

Meanwhile, the pea oligopeptide can also be applied to the preparation of the medicine for treating hypertension.