KR100587179B1 - An Extract of Ligusticum wallichii Having Antihypertensive Function - Google Patents

Abstract

The present invention relates to the extract of Ligusticum wallichii exhibiting antihypertensive function by inhibiting mitogen-activated protein kinase (MAPK) activity. More specifically, the present invention relates to extracts exhibiting antihypertensive function obtained by dissolving the uterus used in folk medicine in an organic solvent.

Cheongung extract having antihypertensive function according to the present invention is useful not only as an antihypertensive agent for treating hypertension, but also as a functional food having a blood pressure lowering effect.

Uterus, hypertension, antihypertensive, MAPK, chloroform

Description

An Extract of Ligusticum wallichii Having Antihypertensive Function

1 shows the effect of Ch1LW on vasoconstrictor-induced contraction in aortic smooth muscle of rats.

Figure 2 shows the activity of MAPK during NE-stimulation in rat aortic smooth muscle. FIG. 2A shows the change over time in ERK1 / 2 induced and phosphorylated by NE, and FIG. 2B shows the change over time in p38 MAPK induced by NE.

Figure 3 shows the difference in ERK1 / 2 and p38 MAPK activity induced by NE in aortic smooth muscle of hypertensive rats and normal blood pressure rats.

Figure 4 shows the effect of Ch1LW on increased activity of ERK1 / 2 induced by NE in rat aortic smooth muscle.

5 shows the effect of Ch1LW on increased activity of p38 MAPK induced by NE in rat aortic smooth muscle.

Figure 6 shows the effect of Ch1LW on blood pressure during cardiac contraction in spontaneous hypertensive rats.

Field of invention

The present invention relates to the extract of Ligusticum wallichii exhibiting antihypertensive function by inhibiting mitogen-activated protein kinase (MAPK) activity. More specifically, the present invention relates to extracts exhibiting antihypertensive function obtained by dissolving the uterus used in folk medicine in an organic solvent.

Background of the Invention

Cheongung is a plant used in folk medicine for the treatment and alleviation of heart disease. Recent experiments have been shown to increase the contractility of heart and coronary blood vessels (Chiou et al., 1991; Hwang, 1993). On the contrary, it has been reported that archery suppresses blood vessel contraction or lowers blood pressure (Hwang, 1993). However, the effects on the blood vessels and purifying of the archery extracts are still unknown.

Hypertension is an increase in pressure on blood vessels, a typical form of vascular aging, and an important phenomenon at the peak of circulatory disease. In humans, hypertension is commonly referred to as systolic blood pressure of 140 millimetre or more and diastolic blood pressure of 90 or more, and is divided into essential hypertension and secondary hypertension, depending on the cause. Essential hypertension is not precisely identified. High blood pressure is equivalent to this, secondary hypertension refers to an increase in blood pressure caused by abnormalities of the kidney, heart, endocrine system caused by various diseases.

This type of hypertension according to adults occurs on the basis of the abnormalities listed here, although the cause is different. In other words, the sympathetic nervous system, dysfunction of the cell membrane, water and Na excretion in the kidney, hyposecretion of hypotensive agents such as PGE ₂ , PGI ₂ , ANP, EDRF, or catecholamine (catecholamine), renin- Angiotensin (renin-angiotensin), such as hypertensive (hypertensive) system, such as abnormal action is caused. Therefore, the nervous system, endocrine system, and abnormalities of the cellular system bring about an increase in pressure of peripheral resistance blood vessels and are caused by hypertension.

For the treatment of hypertension, diuretics, betabroke, Ca ²⁺ channel inhibitors, ACE inhibitors, alpha broccoli, and vasodilators are used. When these drugs are used alone, many side effects due to tissue specificity or resistance are severe. Therefore, the development of new therapeutics or the development of drug use is important for the treatment of hypertension.

On the other hand, many of these drugs for the treatment of hypertension have the main action of reducing the resistance of blood vessels directly or indirectly. In this respect, resistance control of blood vessels is important for blood pressure control and hypertension. Therefore, in terms of solving vascular resistance, solving hypertension is an important and meaningful approach.

The resistance of blood vessels, which have a significant meaning in the development of hypertension, is caused by the reduction of the diameter of blood vessels and the diameter of blood vessels depends on the contraction of the smooth muscle layer that forms the middle layer of blood vessels. Can be.

Smooth muscle contraction is an important parameter for smooth muscle motility and the phosphorylation and contraction of intracellular Ca ²⁺ and MLC is always balanced. However, it is known that phosphorylation and contraction of Ca ²⁺ and MLC do not always occur in equilibrium. After stimulation, intracellular Ca ²⁺ increases rapidly and phosphorylation increases accordingly. However, both decrease slowly later in the stimulus. Shrinkage does not occur in the early stages of stimulation, where Ca ²⁺ and phosphorylation rapidly increases, but slowly increases in later stages when Ca ²⁺ and phosphorylation decreases.

Thus it can be seen that the gap appears to the occurrence of shrinkage for these Ca ²⁺ and phosphorylated, and it is known as Ca ²⁺ sensitized (Ca ²⁺ sensitization). In other words, Ca ²⁺ sensitization is a mechanism that means that even a small Ca ²⁺ can cause a large contraction, and smooth muscle can also cause contractions by methods other than Ca ²⁺ . This Ca ²⁺ sensitization can lead to the ideal contraction of smooth muscle, can cause hypertension in the blood vessels, asthma of the smooth muscle, spasm in the microvascular vessels, premature birth in the uterine muscles.

The modulators causing Ca ²⁺ sensitization are known to be pathways by PKC and pathways by low molecular G protein. Recently, mitogen-activated protein kinase (MAPK) is important. It is known as Ca ²⁺ sensitization modulator. Therefore, it is a meaningful approach to target MAPK in order to examine the effects on blood vessel overreaction and to test the drug effects on hypertension.

MAPK is a representative of the intracellular enzyme system that is activated by extracellular stimulation in humans or animals. There are largely ERK1 / 2, p38 MAPK and JNK. It also has various functions in the cell, ① contributes to the development of cancer by participating in the formation or growth of tumors, ② affects the development of cancer and arteriosclerosis due to the proliferation and differentiation of cells by acting on the cell cycle, ③ also involved in the development of inflammation, immunity and nerves. Therefore, the development of inhibitors of MAPK is a significant field for the development of therapeutic drugs for humans, and recently, global pharmaceutical groups are participating in the development of MAPK inhibitors.

Accordingly, the present inventors have made diligent efforts to find a substance capable of inhibiting MAPK. As a result, the present inventors have confirmed that the uterus extract (Ch1LW) inhibits the activity of MAPK and completed the present invention.

An object of the present invention to suppress the activity of MAPK and to provide a cheongung extract having a high blood-lowering function and a method for producing the same.

Another object of the present invention to provide a pharmaceutical composition and functional food containing the cheongung extract.

In order to achieve the above object, the present invention provides a Ligusticum wallichii extract exhibiting antihypertensive function by inhibiting MAPK activity.

The invention also comprises the steps of: (a) grinding the arch; (b) dissolving the pulverized crossbow in an organic solvent; (c) centrifuging the solution in which the arch is dissolved; And (d) by evaporating the organic solvent to extract a solid component to obtain a cheongung extract provides a method of producing a cheongung extract exhibiting an antihypertensive function by inhibiting MAPK activity.

The present invention also provides a pharmaceutical composition having an antihypertensive function containing the cheongung extract as an active ingredient, and a MAPK activity inhibitor containing the cheongung extract as an active ingredient.

The present invention also provides a functional food having a blood pressure-lowering effect containing the extract as an active ingredient.

The pharmaceutical composition of the present invention may be combined with a conventional carrier in the pharmaceutical field at the time of clinical use, and oral administration of a conventional agent in the pharmaceutical field, for example, tablets, capsules, crokies, solutions, suspensions, and the like. It may be formulated into a variety of preparations, such as injectable preparations in the form of preparations, injectable solutions or suspensions, or ready-to-use injectable dry powders that can be prepared and used as injectable distilled water upon injection.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Preparation of a Archery Sample

The uterus was pulverized and placed in a 10-fold volume (v / v) of chloroform, followed by centrifugation to evaporate the chloroform component and extract the solid component.

Cheongung was purchased, cut into small pieces, and then crushed by a grinder into powder. The powder was mixed in a 10-fold volume of chloroform and then centrifuged at 10000 g for 20 minutes. This was filtered through a filter, the chloroform component was evaporated at 37 ° C, and the solid component was extracted. The extracted Cheongung sample (Ch1LW) was dissolved in 100% ethanol and used for the experiment.

Example 2: Measurement of Vascular Contraction

In describing the experimental procedure, the thoracic aorta of male Sprague Dawley rats weighing 200g to 250g was separated into rings having a width of 2 to 3 mm to prepare a sample with the endothelial removed.

To remove the endothelium, cotton wool soaked with physiological saline (PSS) containing NaCl 136.9 mM, KCl 5.4 mM, CaCl ₂ 1.5 mM, MgCl ₂ 1.0 mM, NaHCO ₃ 23.8 mM, glucose 5.5 mM and EDTA (ethylenediaminetetraacetic acid) 0.01 mM Endothelial was removed by gently rubbing the inner surface of the vessel.

The sample was then attached to the holder under 10mN resting tension and allowed to equilibrate in a 5ml vertical muscle culture bath for 20 minutes and then each piece was repeatedly exposed to 70mM KCl solution until the reaction was stable. And NaCl was changed to a high concentration of K solution by substituting the same molar number of KCl. The solution was saturated with a mixture of 95% oxygen and 5% carbon dioxide at 37 ° C., pH 7.4, and the muscle tension was coupled to a force-displacement transducer (force-coupled to a polygraph system, RPS212, Grass, RI, USA). Displacement transducers (FT03, Grass, RI, USA) were used to record the same size.

Example 3: Measurement of MAPK Phosphorylation

To measure MAPK phosphorylation, the drug was treated in blood vessel specimens for a defined time and then frozen in liquid nitrogen. Frozen samples were milled to obtain cell membranes and cytoplasmic proteins. After the cellular protein components were separated by SDS-PAGE, the degree of activity was measured by binding phosphorylated and non-phosphorylated MAPK antibodies by Western blotting.

Example 4 Effect of Ch1LW on Blood Pressure in Hypertensive Rats

(1) Effect of Ch1LW on vasoconstrictor-induced contraction

The effect of Ch1LW on vasoconstrictor-induced contraction in rat aortic smooth muscle was identified. 1 shows the results. Blood pressure was measured indirectly by attaching a tail-cuff to the tail of the rat.

First, after a response to 10 μM norepinephrine (NE) was established, 1 mg / l and 10 mg / l of Ch1LW were added cumulatively (FIG. 1A). In addition, as shown in Figure 1B are the strip to remove the outside of Ca ²⁺ in the absence of Ca ²⁺ of 1mM EGTA containing medium Pre-incubated for 30 minutes. Then, 1 mM DPB (12-deoxyphorbol 13-isobutyrate) was added to the muscle strip. After DPB-induced sustained contraction was established, 1 mg / l and 10 mg / l Ch1LW were added sequentially.

As described above, when the aortic blood vessels extracted from the rats were contracted by NE administration, when the uterine extract (Ch1LW) was administered, relaxation of the contractions occurred. Shrinkage also occurred when DPB was treated under physiological saline without Ca ²⁺ and EGTA. From this result, it was confirmed that Ch1LW showed a contraction suppression reaction even in Ca ²⁺ independent contraction. Therefore, the uterine extract Ch1LW was found to play a role in relaxing vascular smooth muscle.

(2) Changes in MAPK (mitogen-activated protein kinase) activity during NE-stimulation

The mitogen-activated protein kinase (MAPK) activity during NE-stimulation was examined in rat aortic smooth muscle. 2 shows the results.

Figure 2A shows the change over time in the extracellular signal-regulated kinase (ERK) 1/2, a MAPK isoenzyme induced and phosphorylated by NE, and Figure 2B is p38, another MAPK isoenzyme induced by NE. The change over time in MAPK is shown. Strips were stimulated with 10 mM NE at 0, 5, 15 and 30 minutes, respectively, and Western blotting analysis was performed as usual.

The experimental results showed a relatively phosphorylated rate relative to the resting period. The values are averages (± standard deviations) from the results of three independent experiments. Small boxes in the box show representative results of western blotting with MAPK antibodies.

On the other hand, in DOCA (deoxycorticosterone acetate) hypertensive rats, aldosteone releases Na ⁺ back to the kidney to increase blood volume and eventually cause hypertension. DOCA is a derivative of aldosterone, and when DOCA is planted subcutaneously in rats, symptoms of hypertension occur, which is similar to hypertension caused by an increase in human aldosterone (primarily secondary hypertension), which is widely used in hypertension research. The method of manufacturing DOCA hypertensive rats used in this experiment is as follows. First, the right kidney of the rat (Sprague Dawley, 200 g) was removed under anesthesia. After a week of rest to alleviate the postoperative remission, 200 mg / kg of DOCA was mixed with silicone and a thin film-shaped silicone was implanted subcutaneously. The control group (normal blood pressure rat, sham) was transplanted with silicone without DOCA mixed. DOCA hypertensive rats were fed with brine (0.8% Na ⁺ and 0.2% K ⁺ mixture) and normal tap water was fed to sham rats. In this case, the blood pressure rose rapidly after 4 weeks in the DOCA-administered group (DOCA 185 ± 12 mmHg, sham 112 ± 9.8mmHg).

MAPK was measured using the DOCA hypertensive rats. When 10 μM NE was injected into the blood vessels of DOCA hypertensive rats, the activities of ERK1 / 2 and p38 MAPK increased. Similar results were observed when the same experiment was performed in normal blood pressure rats (Sham). However, MAPK activity by NE was significantly increased in hypertensive rats. The same trend was observed for phosphorylation of p38 MAPK. 3 shows the results.

(3) Effect of Ch1LW on increased activity of ERK1 / 2 induced by NE

The effect of Ch1LW on the increased activity of ERK1 / 2 induced by NE in rat aortic smooth muscle was confirmed. 4 shows the results.

The aortic strips were pretreated with 10 mM NE or saline for 15 minutes and then treated with Ch1LW (10 mg / l) for 15 minutes. Western blotting analysis was performed as usual (FIG. 4A). Figure 4B shows the statistical results of the change in ERK1 / 2 activity, showing the degree of phosphorylation relative to the unstimulated resting period. These values are averages from three independent experiments.

(4) Effect of Ch1LW on increased activity of p38 MAPK induced by NE

The effect of Ch1LW on the increased activity of p38 MAPK induced by NE in rat aortic smooth muscle was confirmed. 5 shows the result.

The aortic strip was pretreated with 10 mM NE or saline for 15 minutes, followed by 10 mg / l Ch1LW for 15 minutes. Western blotting analysis was performed as usual (FIG. 5A). FIG. 5B shows statistical results for the change in p38 MAPK activity, showing the relative percentage of phosphorylation relative to unstimulated resting period. These values are averages from three independent experiments.

As described in (3) and (4), the effect on the increased MAPK activity by NE was examined to determine the mechanism by which the relaxation characteristics of Ch1LW appearing in blood vessels. Ch1LW inhibited the increased activity of MAPK isoenzyme ERK1 / 2 by NE. However, it did not inhibit the activity of another MAPK isoenzyme, p38 MAPK. As a result, Ch1LW inhibits the activity of ERK1 / 2 in MAPK, and it can be seen that vascular relaxation occurs due to this action.

(5) Effect of Ch1LW on blood pressure during cardiac contraction in spontaneous hypertensive rats

The effect of Ch1LW on blood pressure during cardiac contraction in spontaneously hypertensive rats (SHR) was identified. 6 shows the result. As seen in FIG. 6, blood pressure was reduced by treatment with 500 mg / kg of Ch1LW, and inhibition was slowly recovered after 24 hours of treatment.

In the case of administration of the extract, the blood pressure of the spontaneous hypertension rats was markedly reduced in blood pressure as compared to normal Kyoto rats (WKY).

From the results of the above example, the increased activity of MAPK acts as an important factor in the development of hypertension, and it was confirmed that the hormonal extract (Ch1LW) that inhibits this factor has a significant effect on the inhibition of hypertension.

The present invention was able to confirm the potent MAPK inhibitory activity of ERK1 / 2 specificity in the chloroform fraction of the uterus, and MAPK significantly increased its activity in DOCA hypertensive rat blood vessels, thus increasing MAPK activity is important for the development of hypertension. It was found to be a factor. Therefore, it was confirmed that the inhibitory effect of MAPK possessed by the extract of Rhizome is effective in treating hypertension.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention has the effect of providing an extract and a method for producing the extract of Ligusticum wallichii exhibiting antihypertensive function by inhibiting MAPK activity. Cheongung extract having antihypertensive function according to the present invention is also useful as an antihypertensive agent for treating hypertension and a functional food having an antihypertensive effect. In addition, it is expected to contribute to the resolution of diseases related to blood vessels, such as stroke, arteriosclerosis, heart disease, organ narrowing, and premature birth. In addition, it can be developed as an inhibitor of MAPK is expected to be applicable to the development of therapeutics for diseases related to MAPK.

Claims (7)

delete delete Method for producing a cheongung extract exhibiting antihypertensive function by inhibiting MAPK (Mitogen-Activated Protein Kinase) activity, characterized in that it comprises the following steps: (a) milling the arch and then extracting it with about 10-fold volume of chloroform; And (b) centrifuging the solution from which the horoscope was extracted, and then evaporating chloroform and separating solid components to obtain a hormonal extract. delete delete delete delete

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