KR101663550B1 - Pharmaceutical composition and health functional food for prevention or treatment of hypertension comprising tartaric acid as effective component - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating hypertension comprising tartaric acid as an active ingredient, and a health functional food for preventing or improving hypertension. Specifically, the tartaric acid as an active ingredient of the present invention is an angiotensin I converting enzyme ACE) to inhibit vasoconstriction and exhibit little cytotoxicity. Therefore, it can be usefully used as a pharmaceutical composition for preventing or treating hypertension and a health functional food for preventing or improving hypertension.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating hypertension and a health functional food containing tartaric acid as an active ingredient,

The present invention relates to a pharmaceutical composition for preventing or treating hypertension comprising tartaric acid as an active ingredient and a health functional food.

Hypertension is a risk factor for cardiovascular disease worldwide, with 15-20% of adults suffering from it, but the precise cause of hypertension has not been identified. Family history, race, salt intake, insulin resistance, excessive drinking and aging.

Renin-angiotensin system (RAS) plays an important role among various factors of blood pressure rise. It has been known that general physiological activity causes renin-angiotensin system (ROS) by blood flow, sodium reduction and sympathetic activity increase RAS), and renin degrades angiotensin into angiotensin I, which is then converted to angiotensin II, which is a vasoconstrictor of angiotensin I-converting enzyme (ACE) Re-uptake, increased potassium ion release, aldosterone production to increase blood flow, vasoconstriction, and blood pressure.

Tartaric acid, on the other hand, is a dicarboxylic acid with two hydroxyl groups and is an acid naturally present in grapes along with malic acid. It is also called tartaric acid because it is contained in the tin that precipitates when making wine. Syrups, juices, etc., and is used for dyeing industry, confectionary organic synthesis, or coloring of metals. L-Tartaric acid; D-tartaric acid; Racemic mixture (hereinafter referred to as DL-Tartaric acid) which is a mixture of L-Tartaric acid and D-Tartaric acid in the same amount; And optically inseparable isomers such as Meso-Tartaric acid. L-Tartaric acid is mainly present in nature.

Currently known ACE inhibitors include ramipril, D-3-mer-capto-2methylpropanoyl-L-proline, enarapil, risinopril, fosinoril, There is spirapril, which is widely used as a treatment for hypertension. However, since these compounds are easily decomposed in a pharmaceutical dosage form, they are not only inferior in stability but also have other side effects such as vigorous vomiting, coughing, headache, anorexia and taste abnormalities, (Lim SD et al (2008) Korean J. Food Sci., Ani Resour, 28 (5): 587-595). Therefore, development of an ACE inhibitor excellent in ACE inhibitory activity and stability is required.

Korean Patent Publication No. 2012-0092735 and Korean Patent No. 1275766 have reported ACE inhibitors derived from natural substances harmless to humans, but there is no report that tartaric acid is effective for hypertension.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and has completed the present invention by confirming that a mixture of optical isomers of tartaric acid in an equivalent ratio has an ability to inhibit ACE activity and is free from cytotoxicity.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating hypertension comprising tartaric acid as an active ingredient.

The present invention also provides a health functional food for preventing or improving hypertension comprising tartaric acid as an active ingredient.

The composition and the health functional food of the present invention not only have the ability to inhibit ACE activity, but also exhibit little cytotoxicity, so that they are effectively used for blood pressure control, prevention or treatment of hypertension.

Figure 1 is a graph showing the ACE inhibitory effect on tartaric acid isomers and mixtures thereof. Captopril means a positive control of 3 mM of Captopril, DL is 3 mM DL-Tartaric acid, D is 3 mM D-Tartaric acid; L is 3 mM L-tartaric acid; Meso is a mixture of 3mM Meso-Tartaric acid, D: L is 3mM D-Tartaric acid and 3mM L-Tartaric acid, L-Tartaric acid and 3mM of Meso-Tartaric acid, 3mM of L-Tartaric acid, and 3mM of L-Tartaric acid, Meso-Tartaric acid.
Figure 2 is a graph showing MTT assay results for tartaric acid isomers and mixtures thereof. Media means DMEM medium containing 10% fetal bovine serum; Control is dimethyl sulfoxide (DMSO) as a control; DL is DL-Tartaric acid; D is D-tartaric acid; L is L-tartaric acid; Meso is Meso-Tartaric acid; D: L is a mixture of D-Tartaric acid and L-Tartaric acid in an equivalent ratio; D: M is D-Tartaric acid and Meso-Tartaric acid mixed in the same ratio; L: M means L-Tartaric acid and Meso-Tartaric acid mixed at the same ratio.

The present invention provides a pharmaceutical composition for preventing or treating hypertension comprising tartaric acid as an active ingredient. It is preferable that the tarsin acid contained in the composition is a mixture of two or more isomers selected from the group consisting of optical isomers of L-tartaric acid, D-tartaric acid, DL-tartaric acid and meso-tartaric acid, more preferably D- (Meso) - tartaric acid in the same amount ratio; Or a mixture of L-tartaric acid and meso-tartaric acid in an equal ratio. Mixed with the same amount ratio means that each stannic acid isomer is mixed in the same amount.

The optical isomer in the present invention refers to an isomer having a different degree of absorption of polarized light and having an opposite direction of light after passing through the material when the light is shined. When the light is reflected on the mirror like a right hand and a left hand, But it has a structure that never overlaps when covering two. Wherein the D-tartaric acid is a primary tartaric acid, the L-tartaric acid is a left-handed tartaric acid, the DL-tartaric acid is an isomer in which the isomeric mixture of the primary and the left is mixed with the racemic mixture and the meso- do.

The tartaric acid preferably inhibits angiotensin converting enzyme (ACE), but is not limited thereto.

The pharmaceutical composition for preventing or treating hypertension comprising tartaric acid according to the present invention as an active ingredient can be administered by any of oral administration or parenteral administration. Examples of the parenteral administration include intravenous injection and rectal administration. The composition according to the present invention can be formulated as a solid preparation, a semi-solid preparation or a liquid preparation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, suppositories, and the like. Solid form preparations may include, but are not limited to, excipients, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, and the like. Examples of the semi-solid preparation include a cream, a lotion, an emulsifier, a liniment, and the like, but the present invention is not limited thereto, and can be produced by adding a suitable colorant, a flavoring agent, a stabilizer, a tackifier, a surfactant, and the like. Examples of the liquid preparation include water, alcohols, solutions such as solutions of propylene glycol, suspensions, emulsions and the like, but not limited thereto, and they can be prepared by adding appropriate coloring agents, flavoring agents, stabilizers, tackifiers and the like. For example, powders can be prepared by simple mixing of the active ingredient of the present invention with suitable pharmaceutically acceptable excipients such as lactose, starch, microcrystalline cellulose and the like. The granules may contain the active ingredient of the present invention; Suitable excipients which are pharmaceutically acceptable; And a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone and hydroxypropylcellulose, followed by wet granulation using a solvent such as water, ethanol or isopropanol or dry granulation using a compressive force . In addition, the tablet can be prepared by mixing the granule with a suitable pharmaceutically acceptable salt of magnesium stearate or the like and tableting it using a tablet machine.

The present invention also provides a health functional food for preventing or improving hypertension comprising tartaric acid as an active ingredient.

It is preferable that the tartaric acid contained in the health functional food is a mixture of two or more isomers selected from optical isomers of L-tartaric acid, D-tartaric acid, DL-tartaric acid and meso-tartaric acid, more preferably D- And meso-tartaric acid in the same amount ratio or a mixture of L-tartaric acid and meso-tartaric acid in the same ratio.

The tartaric acid contained in the health functional food preferably inhibits angiotensin I converting enzyme (ACE), but is not limited thereto.

The health functional food in the present invention refers to a food prepared or processed by using a raw material or a component having useful functionality and includes, for example, a health supplement, a functional food, a nutrient, and an adjuvant. Therefore, the health functional food according to the present invention is not particularly limited to the kind of the food, and the food functional food according to the present invention is not particularly limited and includes various kinds of foods such as dairy products including dairy products such as meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, , Beverages, tea drinks, alcoholic beverages, and vitamin complexes.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Example  One. Angiotensin  I converting enzyme ( Angiotensin  I- Converting Enzyme ; ACE ) Inhibitory activity assay

In In vitro ACE inhibitory activity assay was performed based on conventional experimental methods (Ronald B. Pegg et al., (2007) Pol. J. Food Nutr. Sci., 57, 447-450; Mariana Fritz et al., 2011) Food Chemistry, 126, 878-884). ACE inhibitory activity was measured by using angiotensin derivative HHL (N-Hippuryl-His-Leu hydrate; Sigma H1635) and angiotensin I converting enzyme (ACE) as Lung acetone powder from rabbit (Sigma L0756 ).

HHL (N-Hippuryl-His-Leu hydrate; Sigma H1635), an angiotensin derivative, was dissolved in a buffer solution (pH 8.3, 150 mM Tris-HCl) to give 3 mM, and 100 mg of Lung acetone powder from rabbit Was dissolved in 1 ml of a buffer solution (containing 150 mM Tris-HCl pH 8.3, 1 μM of ZnCl 2) and then extracted at 4 ° C for 2 hours.

To extract the ACE enzyme, it was separated at 6,600 rpm for 40 minutes at 4 ° C, and only supernatant was used. The unused ACE solution was stored at -20 ° C.

100 μl of HHL (3 mM), 50 μl of sample (15 mM, final concentration of 3 mM) and 100 μl of ACE were used to have a final volume of 250 μl. Captopril (15 mM, Sigma C4042, final concentration: 3 mM) was used as a positive control and the same volume of buffer solution was used as a negative control.

Tartaric acid (Sigma T206, 251380, T400) and meso-tartaric acid (Wako 203-00091) were added to the buffer solution so as to have a concentration of 15 mM in D, L, DL-tartaric acid 1: 1 (by volume) mixture of D-tartaric acid and meso-tartaric acid; 1: 1 (by volume) mixture of L-Tartaric acid and Tartaric acid; A 1: 1 (by volume) mixture of D-tartaric acid and L-tartaric acid was prepared.

The HHL solution (100 μl) and each sample (50 μl) were mixed and allowed to react at 37 ° C. for 60 minutes. An ACE solution (100 μl) was further added thereto and the mixture was incubated at 37 ° C. in a water bath ≪ / RTI > for 30 minutes. Then, 250 μl of HCl (or Glacial acetic acid) was added to stop the reaction. After adding 1 ml of ethyl acetate, the mixture was vigorously shaken at room temperature for 2 minutes, then centrifuged at 3,000 rpm for 5 minutes. The supernatant was transferred to a new tube, and ethyl acetate was evaporated. Then, 500 μl of MeOH was added And analyzed by HPLC at 228 nm.

One) ACE  The reaction by-product, hippuric acid Hippuric acid  analysis

Hipuric acid (hereinafter referred to as 'HA') was prepared by mixing C18 column (2504.6 mm ID, S-6 um, YMC), LC-20AT (liquid chromatograph) module and SPD-20A (UV / VIS dectector) Using high performance liquid chromatography (HPLC). The injection volume (injection volume) of all the samples was the same as 20 μl, and the UV wavelength was measured at 228 nm. The inhibition rate of ACE was measured using HA area and calculated according to the following equation (1).

Equation (1)

ACE inhibition rate = [(HA area of negative control - HA area of sample) / HA area of negative control] × 100

The peptide used as the substrate of ACE was HHL (Sigma), and the amount of peptic acid measured by HPLC was determined by HPLC. The positive control (Captopril) containing Captopril was 85.53 ± 11.80% The activity of DL-tartaric acid (DL) was 85.56 ± 7.57%, the content of D-tartaric acid (D) was 88.28 ± 4.24%, the content of L-tartaric acid (D-tartaric acid) and the isoquinolone (D-tartaric acid) were 91.74 ± 3.23% and 69.27 ± 20.58%, respectively. L-tartaric acid (D: L) was 87.57 ± 9.44%, D-Tartaric acid and Meso-Tartaric acid were the same (D: M) was 93.14 ± 4.25%, and a sample (L: M) containing the same amount of L-tartaric acid and Meso-Tartaric acid showed an activity of 94.35 ± 4.06% (FIG.

Example  2. Confirm cytotoxicity

One) MTT  (3- (4,5- Dimethylthiazole -2- yl ) -2,5- Diphenyltetrazolium Bromide ) Assay

MTT assay was performed to confirm cytotoxicity. First, the medium of 100㎕; SVEC4-10 (blood vessel of mice after (Media Dulbecco's Modified Eagle's Medium containing 10% FBS (Fetal bovine serum), penicillin-streptomycin at 5%) for each division in a 96-well plate, 5x10 ⁴ Endothelial cells, ATCC CRL-2181) cells.

After incubation at 37 ° C and 5% CO ₂ for 20 hours, tartaric acid was dissolved in the medium to a concentration of 400 uM and 200 uM, and 100 μl was added to a 96-well plate to give a final concentration of 100 uM and 200 uM . At this time, the control group maintained the DMSO concentration of 0.1% (v / v) in which tartaric acid was dissolved.

Then, after re-incubated for 20 hours in a 5% CO ₂ and temperature conditions of 37 ℃, MTT reagent (5 mg / ㎖, final concentration 0.5 mg / ㎖) of 20㎕ after handle, CO ₂ After incubation in an incubator for 4 hours, the medium was removed, and 200 μl of each of the dilutions was added to a ratio of DMSO: EtOH of 1: 1. The resulting mixture was slowly mixed and transferred to a new 96-well plate. DMSO: EtOH (1: 1), and the total absorbance was measured at 570 nm using UV spectrophotometer. Cell viability was determined using the following equation (2).

Equation (2)

Cell viability (cell viability) = [OD of the sample ₅₇₀ / of control OD _570] × 100

Toxicities of isomers and mixtures of tartaric acid in SVEC4-10 cells were 100 ± 0.76% at 100 μM at 100 μM and 100 ± 3.55% at 100 μM at 200 μM Media (Control), which contained 0.1% (v / v) of DMSO, was 101.23 ± 1.08% at 200 μM, (L-tartaric acid) was 98.85 ± 0.12% at 100 μM, 103.15 ± 3.10% at 100 μM, 99.91 ± 3.92% at 200 μM for D-tartaric acid, 98.11 ± 1.17% at 100 μM, 98.59 ± 1.63% at 100 μM, 97.77 ± 0.70% at 100 μM, 98.59 ± 1.63% at 200 μM for Meso-Tartaric acid, and 98.59 ± 0.82% at 100 μM for DL-Tartaric acid. 0.11%, the same amount of D-tartaric acid and L-tartaric acid was 101.6 ± 1.68% at 100 μM, 99.49 ± 4.08% at 100 μM, D-tartaric acid Meso-tartaric acid (Meso-Tartari c acid was 99.36 ± 1.33% at 100 μM, 97.45 ± 0.29% at 100 μM, and 101.94 ± 1.22% at 200 μM for the same mixture of L-Tartaric acid and Meso-Tartaric acid. , And cell viability of 100.25 ± 5.78% at 100 μM (FIG. 2). Thus, it was determined that the tartaric acid of the present invention was not cytotoxic.

Claims (10)

A pharmaceutical composition for preventing or treating hypertension comprising tartaric acid as an active ingredient. The pharmaceutical composition according to claim 1, wherein the tartaric acid is a mixture of at least two isomers selected from the group consisting of L-tartaric acid, D-tartaric acid, DL-tartaric acid and Meso-tartaric acid. delete delete The pharmaceutical composition for preventing or treating hypertension according to claim 1, wherein the tartaric acid has an angiotensin I converting enzyme inhibitory activity. A health functional food for preventing or improving hypertension comprising tartaric acid as an active ingredient. 7. The method according to claim 6, wherein the tartaric acid is a mixture of at least two isomers selected from the group consisting of L-tartaric acid, D-tartaric acid, DL-tartaric acid and Meso-tartaric acid. . delete delete The health functional food for preventing or improving hypertension according to claim 6, wherein the tartaric acid has an angiotensin I converting enzyme inhibitory activity.

Images