KR20150106035A - A pharmaceutical composition for preventing and treating hypertension comprising spirulina sp. gastrointestinal hydrolysate and a peptide derived therefrom - Google Patents

Abstract

The present invention relates to a peptide purified and isolated from a microalgal Spirulina sp. digested hydrolysate and a pharmaceutical composition containing the same for preventing and treating hypertension. Provided is a pharmaceutical composition containing a novel peptide of Spirulina sp. obtained from the following steps of: determining an amino acid sequence of a peptide purified and isolated from a microalgal Spirulina sp. digested hydrolysate; comparing and evaluating effects on an angiotensin converting enzyme (ACE) inhibitory activity, generation of nitric oxide (NO) and reactive oxygen species (ROS) using a peptide obtained from the step as an active ingredient for preventing and treating hypertension.

Description

TECHNICAL FIELD The present invention relates to a composition for prevention and treatment of hypertension, comprising a hydrolyzate of spirulina digestion of marine microalgae and a peptide derived therefrom as an active ingredient. gastrointestinal hydrolysate and a peptide derived therefrom}

The present invention relates to peptides separated and purified from microalgae spirulina digestive hydrolyzate and a pharmaceutical composition for preventing and treating hypertension containing the same.

Hypertension is a major risk factor in the progression of cardiovascular and cerebrovascular disease, occurring in 15-20% of all adults and is rapidly spreading worldwide.

The renin-angiotensin system is an internal modulator of blood pressure, moisture, electrolyte homeostasis, and cell growth. Angiotensinogen released from liver to blood is degraded by renal enzyme lenin to become angiotensin I, and angiotensin converting enzyme (ACE) catalyzes hydrolysis of angiotensin I to produce a strong vasoconstrictor angiotensin II . In this way, angiotensin converting enzyme plays a physiological role to control blood pressure. Angiotensin II is a major physiological effector of the renin-angiotensin system and is a multifunctional hormone and has been known to be systematically linked to blood pressure control and cardiac function. In the endothelial cells, angiotensin II secretes NO and ET-1, which are vascular regulators. NO, an important vasodilator, is a major factor involved in the atherosclerotic character of the vascular wall produced by AT ₂ R stimulated by angiotensin II, and may play an important role in physiological and pathological conditions. In addition, angiotensin II activates transcription of the preproET-1 gene in vascular smooth muscle and endothelial cells, induces peripheral secretion to adjacent cells, and induces vascular smooth muscle cell proliferation with strong vasoconstrictor ability. Thus, high levels of angiotensin II may increase ET-1 synthesis in endothelial cells, and consequently, high concentrations of ET-1 may cause hypertension.

Cyanobacteria Spirulina is a cyanobacterium belonging to the Oscillatoraceae family inhabiting the sea and freshwater. Spirulina, which has a spiral shape, is known as "superfood" because it has the ability to assist the physiological system in response to ROS generation. In addition, spirulina has anti-diabetic effects in reducing blood sugar levels, controlling cholesterol and improving insulin resistance. The effects of spirulina on oxidative stress, hyperglycemia, hypercholesterolemia, and arterial hypertension have been reported.

Since the discovery of ACE inhibitors in snake venom, people have been interested in synthesizing ACE inhibitors such as captopril, enalapril, alacepril, and lisinopril, which are widely used to treat hypertension and cardiovascular disease. However, the synthesis of ACE inhibitors has been associated with side effects such as coughing, taste loss, hyperglycemia, angioedema, and skin rash.

Accordingly, an object of the present invention is to provide an antihypertensive peptide from a marine-derived bioactive substance.

Another object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of hypertension comprising the peptide as an active ingredient.

The above object of the present invention is achieved by a method for determining the amino acid sequence of a peptide isolated and purified from a digestive hydrolyzate of a microalgae spirulina genus; The peptide obtained in the above step was used to compare and evaluate the effects on ACE inhibitory activity, NO and ROS production.

The present invention has an excellent effect of providing a pharmaceutical composition for prevention and treatment of hypertension containing a novel peptide of Spirulina genus as an active ingredient.

FIG. 1 is a graph showing ACE inhibition rates of hydrolysates separated by molecular weight according to the present invention. FIG.
FIG. 2 is a graph showing ACE inhibition rates using fractions obtained after separation and purification of hydrolysates of less than 5 kDa according to the present invention. FIG.
Figure 3 is an amino acid sequence and structural formula of Fraction 5 according to the present invention.
4 is a graph showing an ACE inhibition pattern of peptides isolated and purified according to the present invention.
FIG. 5 is a graph showing cell survival experiment results of peptides isolated and purified according to the present invention.
6 is a graph showing Nitric Oxide inhibition rates of peptides isolated and purified according to the present invention.
FIG. 7 is a fluorescence image and a graph showing the rate of inhibition of reactive oxygen species (ROS) of peptides isolated and purified according to the present invention.
8 is a photograph showing the iNOS and ET-1 inhibition rates of the peptides isolated and purified according to the present invention.
Fig. 9 is a photograph showing that p38 of the mitogen-activated protein kinase (MAPK) of the peptide isolated and purified according to the present invention is regulated.

Hereinafter, the present invention will be described in detail with reference to examples.

Published sample

(ACE) from rabbit lungs, hypersurfactant (ACE), hypersaltreotide (ACE), Greiss reagent, 2- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, protease HiPrep DEAE FF colume (1.6 cm x 10 cm) was cultured in GE healthcare with iNOS, β-actin, phosphorylate (p) p-glycoprotein (Sigma), and angiotensin II and hoechst Specific antibodies against p38, p-JNK, p-ERK and Endothelin-1 were purchased from Santa Cruz and 2 ', 7'-dichlorodihydrofluorescein diacetate from Invitrogen.

Example : In vitro  top Spirulina  Digestion hydrolysis

The method of digestion was done by Kapsokefalou and Miller (1991). 100 mL of 4% (w / v) Spirulina spp. Solution was added to make the pH for digestion environment above 1 M, 10 M HCl and NaOH. Pepsin was added at an enzyme: substrate ratio of 1: 100 and incubated at 37 ° C with agitation. And cultured at pH 2.5 for 2 hours to establish gastrointestinal digestion conditions. Trypsin and a-chymotrypsin were added at an enzyme: substrate ratio of 1: 100 and the solution was further incubated for 2.5 hours at 37 ° C, pH 6.5, the digestion condition of the pancreas. Centrifugation was then carried out for 15 min at 10,000 rpm at 4 ° C. The supernatant was stored frozen at -80 ° C and lyophilized to obtain a dry powder.

Experimental Example  One: Spirulina Fraction ACE  Measure inhibition rate

The method of Cushman and Cheung (1971) was modified to measure ACE inhibition. 50 μL of 2 mg / mL sample solution was added to 50 μL of ACE solution and preincubated at 37 ° C. for 10 minutes. The mixture was added to the substrate (0.5 mM NaCl in 50 mM sodium phosphate buffer, pH 8.3, 4.15 mM HHL ) At the same temperature for 2 hours. The reaction was terminated by the addition of 250 μL of 1.0 M HCl, and then 0.5 mL of ethyl acetate was used to extract the hippuric acid. After centrifugation at 3,000 rpm for 10 minutes, a 0.2 mL top layer was transferred to a test tube and evaporated at 80 for 1 hour. Hippuric acid was re-dissolved in 0.5 mL of distilled water and the absorbance was measured at 228 nm using a microplate reader (PowerWave XS2, BioTek Instrument, Inc., USA). The IC ₅₀ value was defined as the inhibitor concentration needed to inhibit 50% of the ACE activity.

The hydrolysates of spirulina were classified into more than 100 kDa, 10-100 kDa, 5-10 kDa and 5 kDa using an ultrafiltration device (Table 1). As shown in FIG. 1, the ACE inhibitory activity of various molecular weights showed the highest ACE inhibitory activity, with an IC ₅₀ value of less than 5 kDa, of 0.287 mg / mL.

Experimental Example  2: ACE  control Of peptide  refine

The ACE inhibitory peptide was purified from the enzyme hydrolyzate by HiPrep DEAE FF ion exchange column using Rapid Protein Liquid Chromatography (FPLC). Spirulina with the highest activity of spirulina, less than 5 kDa, was loaded on a HiPrep 16/10 DEAE FF ion exchange column, which was kept parallel to the 20 mM sodium phosphate buffer solution, and loaded with NaCl at a flow rate of 2.0 mL / min in the same buffer Were dissolved by dissolving in the preceding gradient. Elution peaks were observed at 280 nm and collected in 4 mL increments.

As a result of the experiment, fraction 5 of the five fractions obtained at less than 5 kDa showed the highest ACE inhibitory activity (72.09%) (FIG. 2).

Experimental Example  3: ACE  control Of peptide  Sympathy

The exact molecular weight and amino acid sequence of the purified peptide was determined using a Q-TOF mass spectrometer (Micromass, UK) equipped with an electrostatic spray ionization (ESI) source. The purified peptide was dissolved in methanol / water (1: 1, v / v) and then injected into the electrostatic injection source and determined by doubly charged condition in the mass spectrum. Following molecular weight determination, the peptide to be fragmented was automatically selected and sequence information was obtained by tandem mass spectrometry.

As a result of the experiment, a peptide (759 Da) represented by SEQ ID NO: 1 (Thr-Met-Glu-Pro-Gly-Lys-Pro) was obtained as shown in FIG.

Experimental Example  4: ACE  Determination of inhibition pattern of inhibitor

Various concentrations of ACE inhibitory peptides were added to each reaction mixture. Enzyme activity was measured with various concentrations of substrate and the ACE inhibition pattern was determined by the Lineweaver-Burk plot.

Experimental Results As shown in FIG. 4, the ACE inhibition pattern of the purified peptide appeared as a mixed inhibition pattern. Thus, ACE inhibitors competitively bind to substrates at the ACE active site. It can also bind to enzyme molecules to generate a dead-end complex, regardless of whether the substrate molecule is bound or not.

Experimental Example  5: Cell culture and cell viability experiments

Human endothelial cell line EA.hy926 represents an immortalized cell line produced by the combination of human umbilical vein endothelial cell and human lung epithelial cell line A549 and maintains the signaling characteristics of human umbilical vein endothelial cells. Cells were placed in DMEM (Invitrogen) supplemented with 10% fetal bovine serum, 100 units penicillin / mL, and 100 μg streptomycin / mL, and cultured in a humidified air environment at 37 ° C and 5% CO ₂ .

Cell viability was determined by 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) reduction experiments. MTT was used as an index of cell viability through reduction of formazan. Cells were treated with various concentrations of peptides (10, 50, 100, 200 μg / mL) for 30 min 24 h prior to treatment with angiotensin II. After the medium was removed, 0.5 mg / mL MTT solution was added to the cells and incubated at 37 ° C for 4 hours. The supernatant was removed and the remaining formazan was dissolved in DMSO. Cell viability was confirmed by observing signals at 550 nm using a microplate reader.

Experimental results did not affect cell viability at various concentrations of peptide (Figure 5).

Experimental Example  6: NO  On the production of Of peptide  effect

EA.hy926 cells were inoculated into phenol red-free DMEM and cultured in the presence or absence of various concentrations of peptides (10, 50, and 100 μg / mL). After the end of the experiment, the medium was collected and the amount of NO released from the cells was determined using the NO assay. After addition of the Griess reagent, the mixture was incubated at room temperature for 10 minutes and absorbance was measured at 540 nm using a microplate reader.

Experimental Results As shown in FIG. 6, at 24 hours after angiotensin II stimulation, the level of NO at the basal level was effectively increased in human endothelial cell, EA.hy926 cell, and was inhibited by the peptide treatment in a concentration-dependent manner.

Experimental Example  7: Intracellular ROS  On the production of Of peptide  effect

EA.hy926 endothelial cells were washed with Hank's Balanced Salt Solution (HBSS). To measure intracellular ROS, 40 μM of 20,70-dichlorofluorescein diacetate (DCF-DA) containing HBSS was added and the cells were cultured at 37 ° C. for 30 minutes. The cells were then observed immediately under a fluorescence microscope.

Experimental Results As shown in FIG. 7, in the group treated with 1 μM angiotensin II, ROS production was significantly increased, whereas peptides-treated cells showed a significant decrease in ROS production in the presence of angiotensin II . Cell shape was restored in a dose dependent manner as ROS decreased.

Experimental Example  8: Western Blot  analysis

Cells were washed 3 times with PBS and resuspended in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.1% Triton X-100, 1 mM EDTA, 1 mM PMSF, 20 mg / mL aprotinin, 20 mg / , 20 mg / mL pepstatin, 1% sodium deoxycholate, 1 mM NaF, 1 mM Na3VO4 in H2O). The same amount of protein was separated by 10% SDS-PAGE gel electrophoresis, transferred to PVDF membrane, and blocked in 5% skim milk. After incubation with the appropriate primary antibody, the membranes were incubated overnight at 4 ° C. The membrane was washed three times in Tris-buffered saline Tween-20 and visualized in the ECL assay kit before the incubation with the secondary antibody for 1 hour at room temperature. Western blotting was visualized using a LAS3200 fluorescence image analyzer and protein expression was quantified using Multi Gauge V3.0 software (Fujifilm Life Science, Japan).

Experimental results confirming the effect of peptides on angiotensin II-induced iNOS and ET-1 production As shown in FIG. 8, 1 μM angiotensin II treatment significantly increased iNOS and ET-1 expression.

Experiments to identify signaling cascades that activate iNOS and ET-1 gene expression in EA.hy926 endothelial cells in response to stimulation with angiotensin II have shown that the peptides according to the present invention significantly inhibit p38 kinase phosphorylation, ERK-1/2 and JNK were not affected by peptide treatment (Figure 9).

<110> Pukyong National University Industry-University Cooperation Foundation <120> Composition for preventing and treating hypertension comprising          spirulina sp. gastrointestinal hydrolysate or a peptide derived          from the same <130> 1 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptide derived from fermented spirulina sp. <400> 1 Thr Met Glu Pro Gly Lys Pro   1 5

Claims (2)

Spirulina sp . ) An inhibitor for angiotensin converting enzyme (ACE) inhibition represented by the amino acid sequence of SEQ ID NO: 1 derived from a hydrolyzate. A pharmaceutical composition for the prevention and treatment of hypertension, which comprises the peptide according to claim 1 as an active ingredient.

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