TWI343260B - Novel antihypertensive peptide and use thereof - Google Patents

Description

1343260 IX. Description of the invention: [Technical field to which the invention pertains] The present invention belongs to an antihypertensive polypeptide. [Prior Art] Hypertension, which was present in approximately 26.4% of adults worldwide in 2000, is expected to rise to 29.2% in 2025 (Kearney, α/., 2005, 365: 217-223) 'This symptom is considered to be a major risk factor for cardiovascular diseases such as myocardial infraction, stroke, heart failure, and end stage diabetes. Every 5 mmH of blood pressure is equivalent to a 16% reduction in the incidence of cardiovascular disease (IV) d e/f, 2004, J.M^· 134:980S-988S). The seventh Joint National Committee (JNC7) report: The value of SySt〇nc bl〇〇d pressure/diast〇iic bloodprssure is higher than that of private systolic blood pressure (SySt〇nc bl〇〇d pressure)/diast〇iic bloodprssure At ii5/75mmHg, the age of heart disease and stroke will increase. The Society recommends that some people with a potential group of high blood pressure (systolic blood pressure of 12 (M39 mmHg or diastolic blood pressure of 8〇_89 _Hg), women's life and health changes to the money of the square wire pre-pressure increase and blood officials The disease has occurred. As a result, awareness and demand for ingredients or nutrients that help control blood pressure have increased globally. Many of the biologically active polymorphisms from milk proteins have been found to have healthy nutritional potential. The peptide can directly affect many sputum bacteria, anticoagulation, immune regulation, carrying minerals, opioids or angiotensin converting enzyme (Clare - Swaisg〇〇d, angi〇tensin L purchased 195). In these physiology, vasoconstriction, extensive research is based on its external enzymes, i and in the t-reduction enzyme is a cell located in different tissues in the month is not - vasopressin System (renhi-angk) tennin), blood

The S 6 sulovarin-kinin system (kaiiikrein_kinin) and the immune system appear, Zong Fan. This enzyme is responsible for converting angiotensin-quinone to a contractile angiotensin-2, and also to break down bradykinin and enkephalins that cause vasodilation. Therefore, inhibition of vasoconstriction, invertase is considered to be an effective target for the treatment of hypertension. Until today, serious side effects such as hypotension, elevated potassium levels, decreased eye function, cough, angioedema, skin rash and teratogenicity are thought to be related to drugs that inhibit angiotensin-reversing enzymes. . However, angiotensin-converting enzyme inhibitory polypeptides derived from natural foods are considered to be mild and safe and do not have the side effects of drugs. In the development of new functional foods that control blood pressure, many angiotensin-converting enzyme inhibitory polypeptides derived from milk proteins have been discovered. (Yamamoto et al, 1994, J. Dairy Sci. 77: 917-922; Maeno et al., 1996, J. Dairy Sci. 79: 1316-1321; Yamamoto, 1997, Biopoly. 43: 129-134; Pihlanto- Leppala et al 1998, Int. Dairy J. 8: 325-331; Yamamoto and Takano, 1999, Nahrung 43: 159-164; Clare and Swaisgood, 2000, ut supra; Minervini et al., 2003, Appl. Environ. Microbiol 69: 5297-5305; Li et al, 2004, ut supra-, Robert et al, 2004, J. Agric. Food Chem. 52: 6923-6931; and Quiros et al, 2005, ·/· 88: 3480- 3487). However, these multiple shapes have not yet been verified in vivo. Only a small number of angiotensin-converting enzyme inhibitory polypeptides exhibit anti-hypertensive ability in spontaneously hypertensive rats (SHRs) (FitzGerald® < 2004, W Qing r < 3). There are several products on the market that come from fermented milk or milk protein, such as Calpis sour milk drink (Calpis Co) Japan and C 12 peptide (DMV International, The

Netherlands). Among these products, inhibition of angiotensin converting enzyme polypeptides including: IPP, VPP and FFVAPFEVFGK have been discovered. However, low-dose oral products that inhibit angiotensin-converting enzyme polypeptides still have significant needs. 1343260 f SUMMARY OF THE INVENTION The main object of the invention is to provide a novel polypeptide which is capable of lowering the significant effect of blood pressure on both limbs. The polypeptide can be derived from natural proteins or produced by chemical synthesis and genetic recombination, and can be applied to foods, foods, and medicines to control blood dust or reduce cardiovascular disease. Probability. Thus, the invention features a purified anti-hypertensive polypeptide, and will be. The Hei peptide was named AP-l'' (Ami-hypertension Peptide 1). For example, the API amino acid sequence is:

Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-Ala-Val-Pro-Gln (SEQ ID NO: 1). The term "purified" as used in "purified antihypertensive polymorphism, refers to the property of obtaining a polypeptide", for example, taken from a substance in which the polypeptide is originally present. The polypeptide" refers to a short amino acid polymerization. Further, the present invention is characterized by a pharmaceutical composition comprising the purified peptide and a pharmaceutically acceptable carrier. The pharmaceutical composition is administered orally. The second two is a food containing the antihypertensive polymorphism, for example: API. For example, the food product can be a fermented dairy product. 1343260 "Effective amount" in the present invention means the amount of the antihypertensive polypeptide which achieves a medical effect at the receptor. The effective amount can be varied by a person skilled in the art in accordance with the methods of administration, the use of excipients, or other co-administered agents. [Embodiment] The following abbreviations are used in the present invention: ACE, angiotensin-converting enzyme; CVD, cardiovascular disease; DBP, diastolic blood pressure SBP, systolic blood pressure; SHR, Sp0ntaneously hypertens丨ve rat; WKY, Wistar-Kyoto strained rat; HPLC, high performance liquid chromatography (High Performance Liquid) Chromatography); API, antihypertensive polypeptide (Anti-hypertensi〇npeptide η) The present invention provides an antihypertensive polypeptide comprising an amine of API

Base acid sequence. "Antihypertensive polypeptide" or "API" means a polypeptide having the SEqID NO: 1 amino acid sequence, or a structurally and functionally variant. "Structure in terms of structure and function" means having a similarity to the SEqIDno: 1 amino acid sequence of at least 75% (eg, 80%, 85%, 90% or 95%).

Polymorphous' and the polypeptide has antihypertensive activity. This variant may be derived from a mutation of SEQ ID NO: 1, for example, at a position that does not affect antihypertensive activity. Since SEQ ID NO: I has only 19 amino acids, a person skilled in the art can easily infer the position of the amino acid sequence with or without antihypertensive activity, for example, a Leyla mutation assay. The further step can also be replaced by a similar chemical property of silk acid, which affects the polymorphism. Any of the structural variants of SEQ ID NO: 1 are also pre-retained for their activity against high gold f. This structural variant can be tested for activity by the method of Example. The polypeptide of the invention may be purified from natural sources, such as fermented dairy products,

-S 9 1343260 can also be obtained by recombinant methods or chemical synthesis of nucleotides. The polypeptide of the present invention obtained from the fermented dairy product can be fermented by one or more microorganisms listed below: a lactic acid bacterium, the genus of which is

Lactococcus, Streptococcus, Leuconostoc, and Bifidobactefium, and yeasts, such as Saccharomyces, Candida, and /e/?/a. After low-temperature sterilization, full-fat or skim-free lactating milk, whey or casein can be used as raw materials for milk protein fermentation. The most suitable temperature for fermentation is between _5_45t: and the optimum between the delivery is between 8-180 hours. The polypeptide containing the present invention can be directly used as a raw material for other processed foods or the like. The milk of the fermented dairy product which is normal L to Si can be derived from the β-g of the performance book. The total protein of cattle, sheep, sheep, and pigs can also be modified by p-casein sheep I _ pigs or horses, etc., to make a mosaic or Wei-variable Hydrolyzed milk protein can be synthesized by traditional enzymes, without undue experiment 2 and related variants can be used as an active ingredient, used in the prevention and treatment of hypertension or / and heart disease. - The antihypertensive polypeptide of the present invention is derived from a polypeptide and a functional variant of "dairy product, 7 can be a 妾; ί is a New component of the celite composition towel. In addition to 7 directly for chromatography or other Any polypeptide that can be removed from the tongue can also be obtained from the column. The biochemical method of recording non-transfer and transfer is 7 13432$% 〇9όΠ5346 invention patent 卞 案 • • • • • • • • • • • • • • • • 选η 00 4 4 14 14 ?, — | | 〇〇 〇〇 彳 彳 彳 彳 彳 彳 彳 彳 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。嘁, Friendship, Glucose, the therapeutic composition of the present invention may be in the form of a route of administration. For example, it is suitable for oral administration of any suitable capsules, granules, lozenges and powders, or liquid corrections such as pills, solutions, and suspensions. Other non-baked materials, sugar poly-emulsions and suspensions. VIIIJ includes sterile liquid, in addition to the standard carrier, the oral medicine of the present invention or a plurality of excipients commonly used in oral formulations, for example: two substances can be added - a seed, a stabilizer, an emulsifier, a concentrate, a pigment, Sweet two, :, dissolve and preservatives. These are all those skilled in the art. Additives In the present invention, the t, f drug composition is an effective anti-supplementary blood pressure polypeptide or a functional variant; the σ two blood pressure is 'but Not limited to listed, fermented dairy products, bribery; goods, candy or gum. The gnats and the elderly also provide a method and a limb for lowering the blood pressure of the recipient, and the method U3 gives a stabilizing amount of the antihypertensive polypeptide to lower the blood pressure. In another embodiment, the invention provides a method of preventing or treating a disease, and a composition thereof, comprising: a hypertensive polypeptide or a functional variant to lower blood pressure. The dosage of the polypeptide administered by the government m and the connection = can be determined by the medical expert according to the age and body type according to the condition of the fork. Without further elaboration, the invention has been invented and prepared for antihypertensive work. The skim milk powder was dissolved in 9 g of secondary water to prepare a fermented milk. 2% of the 8 cells of the fermented bacteria were placed in a cooling place for cooling. Zhibiao 囷το ' contains 108 CFU/mL of bacteria from kef丨r grain, including Lactobacillus acidopMlus, Lactobacillm helveticus & ηά and acc7M7Om_yca cerevhk, added to low-temperature sterilized skim milk. The temperature and time of fermentation were 38 t: and 20 hours. Fermented fermented milk (1L) was centrifuged at 12000xg for 0min, and the supernatant was freeze-dried and stored in _2〇ΐΜ#. The supernatant and its cold; Donggan powder-free are rich in antihypertensive peptides, the content of the polypeptide is about 50-500 mg / L in the supernatant, about 1-5-15 mg / in the freeze-dried powder / g, the actual content depends on the fermentation conditions. The ability of a polypeptide to inhibit angiotensin-converting enzyme is not necessarily related to anti-hypertensive C s. 'Because some polypeptides having blood-tubulase-converting enzyme inhibitory activity in vitro lose activity after oral administration. Therefore, as mentioned above, the antihypertensive activity of the supernatant lyophilized powder is obtained from the fermented dairy product, and SHRs and normal blood pressure sputum rats (Wistar-fCyoto rats, WKYs) are used as health foods for evaluating hypertension prevention. potential. The public SHRs and the public WKYs used in the embodiments of the present invention were purchased from the Taiwan National Laboratory Animal Center (Ding A丨pei, Ding 3~Ru), and given a graded feed (Rodent laboratory chow 5001, purina Co., USA). 8-18 SKUs and WKYs SBP and DBP were measured by a rat tail pressure tester (Softron BP16; Softron, Tok; yo, Japan). The antihypertensive ability of the test sample was determined by oral administration of the sample 〇_1〇SHRs in a small time and SBP spear σ DBP in WKYs. Give a single oral dose of 35 mg/kg BW from the fermented milk above the supernatant Lingganzhi (treatment group)' or 疋 give the same dose of the supernatant from the normal raw milk lyophilized powder (control group) '10 hours Between, observe changes in SBP and DBp. The results are presented in Figures 1 and 2. The SHRs from the lyophilized powder of the fermented milk were given an average of i94.3±3.9 mmHg before feeding, and there was a significant decrease in the 3 to 10 hours after oral administration compared with the control group (in the 3rd and 4th , Hiroshi 6, 7, 8, 9 hours, /^<〇.〇1; in the first hour, household <〇〇5). Similarly, the mean DBP administered was 150.6 ± 4.3 mmHg, which was significantly reduced 2 to 1 hour after oral administration (at the 2nd hour, ρ<〇·〇5; at 3, 4, 5, 6 , 7, 8 '9 hours, household <〇.〇]; at 10 hours, p<〇〇5). At 1343260 after oral administration, the maximum decline in SBP and DBP was 42, 2 soils of 2.9 mmHg and 37.3 ± 3.1 mmHg. In WKYS, neither SBP nor DBP had significant changes in the treatment and control groups. This data shows that only in the recipients with high blood pressure, the supernatant lyophilized powder obtained from the fermented milk will show the ability to lower blood pressure. It is advisable to apply [1: Obtain a zone from the supernatant of the fermented milk to break the anti-high blood activity of the "Division A". The above supernatant liquid is redissolved in the secondary water, and the solution is used for Do separation. 1 mL·redissolved liquid (total peptide concentration 2 〇mg/mL) was injected into RP-C18 column () RP-HPLC (reverse-phase HPLC) (UV-VIS detector L-7420; Pump L- 7100, Hitachi Ltd., Japan), solvent A (1% trifluoroacetic acid dissolved in methyl cyanide) and solvent b (secondary water) were used as the mobile phase. The polypeptide was precipitated from the following gradient at a flow rate of ImL/min: from 0% to ι〇〇/ between 0-10 min. The solvent is from H)% to 30% solvent B in 10-120 min, and from 3% to 100% solvent B between i2〇_15〇min. The absorbance is detected at 215 nm. This fraction was depleted in minutes and then used to measure the inhibitory activity of angiotensin converting enzyme. The method for determining angiotensin-converting enzyme inhibitory activity is based on Cushman and

Cheung published in 20:1637-1648 (1971). The activity of this method is based on the ratio of hippurin-L-histidyl-L-leucine (HHL) to hippuric acid based on angiotensin-converting enzyme. Briefly, 30 吣 sample solution to be tested was added to 200 吣 5 mM sodium tetaraborate buffer containing 3 mM potassium hydride (NaCl) and 5 mM equine lysyl leucine (SigmaChemica| C〇, St, Louis, MO). Add angiotensin-converting enzyme (2 mU, E (: 3 4 15 丨, 1 〇〇 mU / mL, Sigma) 'mixed and placed in 37 ° C for 35 min. Add / 25 〇 μί 1 M HCl to stop the reaction In the reaction, the produced hippuric acid was purified from ethyl acetate. After removing the acetaminophen by heating distillation, the absorbance at 228 nm was measured with a spectrophotometer.

-S 1343260 by the following formula s. Inhibition ratio = just absorb the 228 nm absorbance of hippuric acid under the sample reaction, xb is able to suppress blood stasis ====_ 管8 tube age conversion enzymes (more than Diterpenoids of the sputum (the part of the mixture is named as zone A) and between 32-33 mm (this part of the mixture of life) multiple muscles of the purification of the _ zone A and the zone B, _ Example = = =^=SHRS and measured #血嶋 determines its ten-part interval A and B in oral administration of SHRs 1 mg/kg bW, and the changes in SBP and DBP are shown in Figure 3 and 4. The blood pressure of the SHRs fermented milk and the raw material β-milk supernatant lyophilized powder was given as a reference value. Part A exhibited antihypertensive activity, with a maximum of SBp of 42.5 ± 2.4 mmHg and a DBP decrease of 45.9 ± 7. 〇 mmHg after 4 hours of oral administration. Although B showed high angiotensin-converting enzyme inhibitory activity in vitro, the antihypertensive effect in vivo was low. Therefore, the selected area A was subjected to an experiment of lowering blood pressure in the next step. Example 3: Twisting anti-high-gold pressure polymorphism and synthesizing a lot of makeup with a significant anti-hypertensive ability in the living area, after 2 sets of HPLC 31 sets, 'by RP-HPLC Further purification. The precipitation conditions for further purification are different from those described previously. Solvent A (10/〇 trifluoroacetic acid dissolved in sulfhydryl group and > 谷剑 B (primary water) was used as the mobile phase. The multi-form flow rate was 1 mL/min and 1 0 from 0-10 min. 〇. /0 to 20% solvent B and a gradient from 20% to 30% solvent B between 10 and 60 min. θ After further purification there are two main peaks as shown in Figure 5. Each major The peak precipitates were further separated and analyzed by a two-dimensional electrophoresis system (PROTEAN IEF for one-dimensional, Mini-PROTEAN 3 for two-dimensional,

S 1343260

Bio-Rad Laboratories, Inc., USA). The results showed that the material precipitated at the 36th minute showed a distinct point at the positions of pi and 10 and Mw and 2 kD. The other major peaks 'the polypeptide cannot be clearly separated. Therefore, only the sites located in the pi 5 10 and Mw = ? 2 kD fractions were identified by a protein sequencer (perkin Elmer AB [Model 491, USA]). The results of the sequence identification showed that the fraction A contained 19 amino acids:

Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-

Ala-Val-Pro-Gln (SEQ ID NO: 1). This polypeptide was found to be located in a primary structure of beta-casein and may be explained from the casein fraction during fermentation. This amino acid sequence SEQ ID NO: was synthesized by Protech Technology Enterprise Co., Ltd. (Taipei, Taiwan) and subjected to the following test for testing antihypertensive activity on SHRs. Example 4: Determination of the antihypertensive activity of the desired polypeptide The synthetic polypeptide having the same sequence as SEQ ID NO: 1 was tested for its antihypertensive effect in vivo using the method of Example i. As shown in Fig. 6 and Fig. 7, a single oral dose of 1 mg/kg BW administered to SHRs has an antihypertensive effect on both SBP and DBP. In addition, the three peptide VPP was also synthesized and fed SHRs as a control group. Val-Pro-Pro (SEQ ID NO: 2) is derived from β-casein, which has been reported to reduce SBP by 20 mmHg after six hours of oral administration of SHRs 1.6 mg/kg BW ((Nakamura d «/., 1995, J. 5W. 78: 1253-1257; Nakamura ei <3/·, 1995, J· 1) (3/> Less 5W. 78: 777-783) In addition, VPP is currently used on the market. One of the two active ingredients of Calpis fermented milk drink for healthy blood pressure control. As shown in Figure ό and Figure 7, Val_Pro-Pro (SEQ ID NO: 2) can be used up to 4 hours after oral administration of 1 mg/kg BW. Reduce SBP by 26.3±3.2 mmHg and DBP by 18.0 ± 2.1 mmHg. The polypeptide identified in the present invention

Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-Ala_Val-Pro-Gln (SEQ ID NO: 1), SBP most after 5 hours of oral administration It can be reduced by 44.0 ± 2.1 mmHg, and DBP can be reduced by up to 15 1343260 23.0 ± 2.9 mmHg after 6 hours of oral administration. This data shows that the polymorph obtained by the third embodiment has an antihypertensive activity on a living body. In 1996, Μα(9)〇 published two peptides derived from casein hydrolysate: Lys-Val-Leu-Pro-Val, Pro (SEQ ID NO: 3) and Lys-Val-Leu-Pro-Val- Pro-Gln (SEQ ID NO: 4) and confirmed its effect on reducing SBP on SHRs (Maeno β β/·, 1996, wi swpra). According to the experiment, oral administration of 2mg/kg BW for six hours

After that, Lys-Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 4) reduced SBP to 31.5 ± 5.6 mmHg. Because of the identification of the present invention

Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-

Ala-Val-Pro-Gln (SEQ ID NO: 1) contains these two sequences Lys-Val-Leu-Pro-Val-Pro (SEQ ID NO: 3) and

Lys-Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 4), compared to the anti-hypertensive activity of these two polypeptides (synthesis) at the same dose of 1 mg/kg BW. As shown in Figures 8 and 9, the 19 amino acid polypeptides had a maximum decrease of 44.0 ± 2.1 mmHg and 23.0 ± 2.9 mmHg at 5 and 6 hours, respectively, at SBP and DBP. However, Lys-Val-Leu-Pro-Val-Pro (SEQ ID NO: 3) had a maximum decrease of 27.8 ± 3.3 mmHg and 17.5 ± 4.2 mmHg at 2 and 5 hours in SBP and DBP, respectively. Lys-Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 4) had a maximum decrease of 29.2 ± 4.0 mmHg and 14.4 ± 3.2 mmHg at 3 hours and 4 hours, respectively. Figure 1A shows that Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-Ala-Val-Pro-Gln (SEQ ID NO: 1) is relative to Val-Pro-Pro (SEQ ID NO: 2) (P = 0.00001), Lys-Val-Leu-Pro-Val-Pro (SEQ ID NO: 3) (P = 0.00003) and Lys-Val-Leu-Pro- The antihypertensive activity of Val-Pro-Gln (SEQ ID NO: 4) (P = 0.00007) was highly significant. In addition, 'the antihypertensive ability of the polypeptides of the 19 amino acids of the present invention was not derived from hydrolysis to Lys-Val-Leu-Pro-Val-Pro (SEQ ID NO: 3) and Lys-Val- Leu-Pro-Val-Pro-Gln (SEQ ID NO: 4) polypeptide, 16 s

Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-

The synthetic polypeptide of Ala-Val-Pr〇-G丨n (SEQ ID NOM) was hydrolyzed by a method for digesting conditions of the gastrointestinal tract (d///d d in 20: 875-880). Brief Description The polypeptide was first pepsin (pH 2.5) (EC 3, 4.23·1; 1:1 〇〇〇〇; Sigma, USA) at 37. . The hydrolysis was carried out for 1.5 hours, and then hydrolyzed by Corolase PP (pH 7.5) (pancreatic protease; Rohm, Darmstadt, German) at 37 ° C for 2.5 hours. In order to remove the added enzyme, the hydrolyzed polypeptide was filtered through a filter membrane of 3 kDa pore size (Microcon YM-3, 3 kDa NMWL, Millipore Co USA) under centrifugation at 1000 X g for 10 min. The filtered liquid is analyzed by a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) method. As shown in FIG. 11, FIG. 12 and Table 1, the polymorphism obtained from Example 3

Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-

Ala-Val-Pro-Gln (SEQ ID NO: 1) 'The fragment obtained after hydrolysis by gastrointestinal conditions does not contain Lys-Val-Leu-Pro-Val-Pro (SEQ ID NO. and Lys-Val- Leu-Pro-Val-Pro-Gln (SEQ ID NO: 4), and this contains a polybasic acid

Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-

Ala-Val-Pro-Gln (SEQ ID NO: 1) has a specific polypeptide Lys-Val-Leu-Pro-Val-Pro (SEQ ID NO: 3) and

Lys-Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 4) is a significantly higher antihypertensive activity. 1343260 Table 1,

Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-Ala-Val-Pro-Gln (SEQ ID NO: 1) after simulated gastrointestinal digestive test a polypeptide fragment.

No. m/z (mi) m/z (av) Start End Missed Cleavages Sequence 1 414.2347 414.4845 16 19 0 (ΊΟ AVPO M (SEO ID NO: 5) 1 780.4978 780.9917 9 15 0 fK) VLPVPOK (A) (SEQ IDNO: 6) 1 871.5247 872.0586 1 8 0 (-) VLSLSOPK iV) (SEQ ID NO: 7) 1 1175.7147 1176.4534 9 19 1 (K) VLPVPOKAVPO i-) (SEQ ID NO: 8) 1 1633.0047 1634.0276 1 15 1 ( -) VLSLSOPKVLPVPOK (A) (SEQ ID NO: 9) 1 2028.2216 2029.4893 1 19 2 (-) VLSLSOPKVLPVPOKAVPO (SEQ ID NO: 1) Statistical analysis:

All of the above examples use statistical software SAS version 9.1 (SAS)

Institute, Cary, NC) 'Evaluation of Significant Differences by Mann Whitney

Utest. The corpse value <〇.05 was judged to be significantly different. ^, those who are familiar with the art will be able to change and modify according to the actual situation without departing from the spirit of the invention. It is to be noted that the invention is not limited by the scope of the invention disclosed in the second embodiment, but is in the form of all other variations in the scope of the application. [Simple Description of the Drawings] The described and implemented embodiments can achieve better betting by adding the ® formula. In order to enhance the description of the present invention, a diagram of a suitable embodiment will be heard. It is to be noted that the present invention is not limited to the descriptions set forth herein. Fig. 1 is a view showing the centrifugal supernatant of the fermented milk obtained in Example 1 18 § 1343260 Dry powder 'single-dose is administered orally for ten hours, SHRs and WKYs = SB [detected values. The treatment group was SHRs (n=9, ♦) or wKYs (n=6, ◊) to the fermented supernatant liquid green powder (35 mg/kg BW). The control group was given SHRs (η-6' _) or WKYs (_, □) to the granules (3) t^g/kg BW). The data is expressed as the mean soil standard machine difference. The asterisk is expressed as a significant difference from the control group at each T-day point: * indicates p < 〇〇 5 and ** indicates ρ < 〇〇 1 〇 A Figure f is the result obtained by Example 1. The centrifuged supernatant of the fermented milk was lyophilized, and a single dose was administered orally for ten hours, and the DBP detection values of SHRs and WKYs were changed. The treatment group was given SHRs (n=9, ♦) or WKYH ◊) ^5 mg/kg BW from the fermented milk supernatant lyophilized powder. The control group was SJiRs (4), _ or WKYs (4), □) and the raw milk supernatant was given to Kanggan powder (35^ng/kg bW). Data are expressed as mean ± standard machine difference. The asterisk indicates a significant difference from the control group at each time point: * indicates P < 0.05 and ** indicates p < 〇〇 1. Fig. 3 is a graph showing the change in SBp of SHRs between oral administration of the fraction A and the fraction B obtained in the second embodiment for ten hours. The treatment group was given % mg/kg BW from the fermented milk supernatant lyophilized powder obtained in Example 1 (n=9, ♦) 'giving' mg/kg BW compartment A (n=9, ▲), or A portion b (n = 6, Δ) of 1 mg ^ kg BW was administered. The control group was given 35 mg/kg Bw raw milk supernatant lyophilized powder (n=6, ). Data are expressed as mean ± standard machine difference. The asterisk is indicated as having a significant difference from the control group at each time point: * means P < 0.05 and ** means P < ο.ο!. Fig. 4A shows the dbp change of SHRs between the fraction a and the fraction B obtained in the second embodiment by oral administration. The treatment group was given lyophilized powder of n mg/l^gBW fermented milk supernatant (n=9, ♦), 1 mg/kg BW area injury A (n=9, ▲), or 1 mg/kg Bw Part b (n=6 △). For the group, we gave 35 mg/kg B W raw milk supernatant; Donggan powder (n=6). Data are expressed as mean ± standard machine difference. The asterisk is expressed as at each time

Figure 5 is a HPLC chromatogram showing the pattern of the fraction a obtained by the Example II P-HPLC. Two of the main areas were separated and subjected to a = dimensional electrophoresis system. The f-head indicates that the polypeptide contained in the fraction (A) has an isoelectric point (ρι) of about 丨〇 and a molecular weight (Mw) of about 2 kDa. Figure 6 shows oral administration of an anti-hypertensive polypeptide derived from District A

Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys

Ala-Val-Pro-Gln (SEQ ID NO: 1), χ ^ Val-Pro-Pro (SEQ ID NO. 2) SBp change in the 'ten hour' SHRs of the composition. The treatment group was given 1 mg/kg BW

Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-Ala-Val-Pro-Gln (SEQ ID NO: l) (n.9j #) , 1 mg / kg of the abdomen. Jia Che ah 1 〇 2 2 = 6, the control group is given 35 ingredients. Supernatant/Donggan powder (n=6, . The data is expressed as the mean ±' machine difference. The i number is expressed as a point at each point of the day (n=9, #), or Is i mg/kg n==6, x). The control group was given 35

Fig. 8 shows that the oral administration of the synthetic multi-months is not P < 〇.〇1.

Val-Leu-Ser-Leu-Ser-Gln

Vcii-rru-vjin id 1NU: 1)([ BW Val-Pro-Pro (SEQ ID NO: 2) (n

S 20 1343260 -Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-Ala-Val-Pro-Gln (SEQ ID NO: 1) ' Lys-Val-Leu-Pro-Val-Pro (SEQ ID NO: 3) and Lys-Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 4) SBP changes in SHRs for ten hours. The treatment group was Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-Ala-Val-Pro-Gln (SEQ ID NO: 1) given 1 mg/kg BW. ) (n=9,#) ' or Lys-Val-Leu-Pro-Val-Pro (SEQ ID NO: 3) at 1 mg/kg BW (n=6, 〇), or 1 mg/kg BW (n=6, □) of Lys-Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 4). The control group was given 35 mg/kg BW raw milk supernatant lyophilized powder (n=6, ). The data is expressed as the mean i: standard machine difference. The asterisk is expressed as a significant difference with respect to Lys-Val-Leu-Pro-Val-Pro (SEQ ID NO: 3) at each time point _·* indicates P < 0.05 and ** indicates P < 0.01. A positive sign indicates a significant difference with respect to Lys-Val-Leu-Pro-Va丨-Pro-Gin (SEQ ID NO: 4) at each time point: + means < 0.05 and++ means P < 0.01. Figure 9 shows oral administration of a synthetic polypeptide

Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-Ala-Val-Pro-Gln (SEQ ID NO: 1) > Lys-Val-Leu- Pro-Val-Pro (SEQ ID NO: 3) and Lys-Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 4) Ten-times, DBP changes in SHRs. The treatment group was given 1 mg/kg BW

Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-Ala-Val-Pro-Gln (SEQ ID NO: 1) (n=9, Lu)' Or Lys-Val-Leu-Pro-Val-Pro (SEQ ID NO: 3) (n=6, 〇) at 1 mg/kg BW, or Lys-Val-Leu-Pro- at 1 mg/kg BW Val-Pro-Gln (SEQ ID NO: 4) (n=6, mouth). In the control group, 35 mg/kg BW milk supernatant was lyophilized (n=6, _). Data are expressed as mean ± standard machine difference. The asterisk is indicated as having a significant difference with respect to LyS-Val-Leu-Pro-Val-Pro (SEQ ID NO: 3) at each time point: * indicates p < 0.05 and ** indicates P < 0.01.

S 21 1343260 The positive sign is expressed as a significant difference with respect to Lys-Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 4) at each time point: + means < 0.05 and ++ means P < 0_0 Figure 10 shows the polymorphism of a single dose of oral administration.

Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-Ala-Val-Pro-Gln (SEQ ID NO: 1) ^ Val-Pro-Pro (SEQ ID NO: 2) Five hours after Lys-Val-Leu-Pro-Val-Pro (SEQ ID NO: 3) and Lys-Val-Leu-Pro-Val-Pro-Gln (SEQ ID NO: 4) , SHRs SBP decline and its significant differences. All feeding groups were given 1 mg/kg BW. The data is expressed as the mean standard deviation. The p value between the two groups is shown on the graph. Figure 11 shows the synthesized peptide

MALDI-TOF-MS of Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-Ala-Val-Pro-Gln (SEQ ID NO: 1) Mass spectrometry. Figure 12 shows the synthesized peptide

Val-Leu-Ser-Leu-Ser-Gln-Pro-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-Ala-Val-Pro-Gln (SEQ ID NO: 1), after simulated gastrointestinal digestion MALDI-TOF-MS mass spectrum after the test. [Main component symbol description] <»»,

22 S 13.43260 Sincerely, <πο> Dae Ye University and National Chung Hsing University <120> Novel Antihypertensive Peptides and Their Uses

<130> DYU0001-TW <140 096135346 <141> 2007-09-21 <160> 9 <170>Patentln version 3.3 <210> 1 <211> 19 <212> PRT <213> Artificial sequence <220><223> Synthetic amino acid sequence designed according to casein <400>

Val Leu Ser Leu Ser Gin Pro Lys Val Leu Pro Val Pro Gin Lys Ala 1 5 10

Val Pro Gin <210> 2 <211> 3 <212> PRT <213> Artificial sequence 1343260 <220><223> Synthetic amino acid sequence designed according to casein <400> 2 Val Pro Pro <210> 3 <211> 6 <212> PRT <213>Artificial sequence <220><223> Synthetic amino acid sequence designed according to casein <400>

Lys Val Leu Pro Val Pro <210> 4 <211> 7 <212> PRT <213>Artificial sequence <220><223> Synthetic amino acid sequence designed according to casein <400> 4

Lys Val Leu Pro Val Pro Gin <210> 5 1343260 <211> 4 <212> PRT < 213 > artificial sequence <220><223> SEQ ID NO: 1 fragment after hydrolysis via pepsin <400> 5

Ala Val Pro Gin 1 <210> 6 <211> 7 <212> PRT <2]3>Artificial sequence <220><223> SEQ ID NO: 1 fragment after hydrolysis by pepsin <400>; 6

Val Leu Pro Val Pro Gin Lys 1 5 <210> 7 <211> 8 <212> PRT <213>Artificial sequence <220><223> SEQ ID NO: 1 Fragment after hydrolysis via pepsin ;400> 7

Val Leu Ser Leu Ser Gin Pro Lys 1343260 1 5 <210> 8 <211> 11 <212> PRT <213>Artificial sequence <220><223> SEQ ID NO: 1 after hydrolysis by pepsin Fragment <400> 8

Val Leu Pro Val Pro Gin Lys Ala Val Pro Gin 1 5 10 <210> 9 <211> 15 <212> PRT <213>Artificial Sequence<220><223> SEQ ID NO: 1 Via Stomach Fragment after proteolytic hydrolysis <400> 9

Val Leu Ser Leu Ser Gin Pro Lys Val Leu Pro Val Pro Gin Lys 15 10 15 s 4

Claims (1)

f 096135346 invention patent application. . q 矣本doo car 9 q ” mouth V ten, the scope of application patent: 'a purified anti-hypertension peptide AP1 (Anti-hypertension Peptide 1) 'where the antihypertensive peptide Αρι The amino acid sequence is SEQ ID NO: 1. 2. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier. 3. The pharmaceutical composition of claim 2, wherein The composition is administered orally. 4. The food of the invention comprises the antihypertensive polypeptide API and the food ingredient of the first application of the patent scope. 5. The food of the fourth aspect of the patent is a fermented dairy product. A blood pressure composition comprising an effective amount of an antihypertensive polypeptide API, wherein the amino acid sequence of the antihypertensive polypeptide API is SEQ ID NO: 1. 7 - a composition as in claim 6 wherein the combination The composition is a fermented dairy product. A composition for treating cardiovascular disease comprising an effective amount of an antihypertensive polypeptide API, wherein the amino acid sequence of the antihypertensive polypeptide API is SEQ ID NO: 1. 9. 8 The composition of the item wherein the k compound is a fermented dairy product.