CN114099494B - Natural pharmaceutical composition for synergistically inhibiting hyperaldosteronism - Google Patents
Natural pharmaceutical composition for synergistically inhibiting hyperaldosteronism Download PDFInfo
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Abstract
The invention discloses a natural medicine composition for synergistically inhibiting aldosteronism, belonging to the technical field of medicines; the natural pharmaceutical composition comprises atractylenolide I and sinapine; the pharmaceutical composition provided by the invention can effectively inhibit the generation of aldosterone at the level of human adrenal cortical adenocarcinoma cell H295R cells, and can obviously inhibit the generation of aldosterone in a mouse aldosterone increase model induced by angiotensin II, and the effect of the pharmaceutical composition is superior to that of the pharmaceutical composition when the pharmaceutical composition is used alone, and the pharmaceutical composition has a synergistic effect; the medicine composition provided by the invention has definite curative effect and can effectively prevent and treat the aldosteronism.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a natural medicine composition for synergistically inhibiting hyperaldosteronism.
Background
Aldosterone (ALD) is a mineralocorticoid synthesized and secreted by cells of the adrenal corticobasal tract. Aldosterone acts on the mineralocorticoid receptors in the kidney and enhances the reabsorption of sodium ions and water by the renal distal tubules and collecting ducts, thereby promoting the excretion of potassium ions, and is the primary regulator of sodium-potassium balance. Hyperaldosteronism can cause a number of metabolic and cardiovascular diseases including primary aldosteronism, secondary aldosteronism, oedema, hypokalemia, hypertension, cardiac fibrosis and heart failure. Therefore, inhibition of aldosterone synthesis and secretion plays an important role in the occurrence and development processes of related diseases caused by abnormal increase of aldosterone, and is receiving wide attention of people.
In the treatment of diseases, natural drugs play an important role. The effective natural pharmaceutical composition can exert synergistic effect to improve the curative effect of the medicine, reduce the occurrence probability of drug resistance of the medicine and reduce the toxic and side effect by reducing the dosage of single medicine in the composition. In recent years, chinese patents also disclose some Chinese medicinal compositions for improving and treating hyperaldosteronism and preparation methods thereof, such as natural medicinal compositions of caulis piperis futokadsurae, flag flower, etc. (publication No. CN 109692260A); a Chinese medicinal composition prepared from selfheal, uncaria and the like (publication No. CN 105902816A). Due to the characteristics of multiple components and multiple targets of the traditional Chinese medicine compound, the precise compatibility proportion and the synergistic index of the traditional Chinese medicine compound are difficult to be determined in the investigation of the synergistic effect. Therefore, the natural medicine composition for treating the aldosteronism with definite active ingredients and definite curative effect is lacked at present. The screening of the natural product pharmacodynamic molecule combination with the synergistic effect of treating the aldosteronism has important significance.
Atractylenolide I (AT-I) with structural formula
Is sesquiterpene compound extracted from rhizome of Atractylodes macrocephala Koidz of Compositae, and is also the main active ingredient of Atractylodes macrocephala Koidz. Modern pharmacological studies show that the bighead atractylodes rhizome has various effects of diuresis, tumor resistance, inflammation resistance, aging resistance and the like. At present, no patent publication of atractylenolide I for regulating aldosterone production exists.
Sinapine (SP) with structural formula
Has antioxidant, blood pressure lowering, and anticancer effects. The application of sinapine in the fields of blood pressure reduction, senile dementia prevention and treatment and the like is disclosed in patent application documents with publication numbers of CN112741832 and CN1615829, and related patents and reports of sinapine for treating aldosteronism do not exist at present. At present, no report that the pharmaceutical composition consisting of atractylenolide I and sinapine can effectively inhibit the production of aldosterone and treat aldosteronism is found.
Disclosure of Invention
In order to overcome the technical problems in the prior art, the invention provides a natural pharmaceutical composition for synergistically inhibiting hyperaldosteronism, so as to effectively inhibit the production of aldosterone and treat hyperaldosteronism.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a natural medicine composition for synergistically inhibiting aldosteronism, which comprises atractylenolide I and sinapine.
Further, based on the cellular level, when the concentration of the atractylenolide I is 1.875 to 7.5 mu M, the molar ratio of sinapine to atractylenolide I is (5-30) to 10; when the concentration of the atractylenolide I is 30 mu M, the molar ratio of sinapine to atractylenolide I is (1.5-15) to 10.
Further, when the pharmaceutical composition is used in combination at an animal level, the mass ratio of sinapine to atractylenolide I is 15: 10.
Further, when the pharmaceutical composition is used in combination at an animal level, the dosage of sinapine is 15mg/Kg, and the dosage of atractylenolide I is 10mg/Kg.
The invention also provides application of the natural medicine composition for synergistically inhibiting the aldosteronism in preparing a medicine for treating diseases or symptoms caused by the excessive exposure of the aldosterone.
Further, the disease or disorder includes primary aldosteronism, secondary aldosteronism, heart failure, chronic renal failure, hypertension, cardiac fibrosis, cardiovascular damage, renal disease, renal fibrosis or sodium water retention.
Still further, the disease or disorder is primary aldosteronism or secondary aldosteronism.
Compared with the prior art, the invention has the following beneficial effects:
the atractylenolide I and sinapine have remarkable diuretic effect on sodium water retention mice loaded by normal saline, and the diuretic effect of the atractylenolide I and sinapine has the characteristics of sodium excretion and potassium retention, and both can provide a natural aldosterone antagonist for clinic; according to the invention, the atractylenolide I and sinapine are used in a combined manner, so that the generation of aldosterone can be effectively inhibited at the cell level of H295R of human adrenocortical adenocarcinoma cells, and the generation of aldosterone can be obviously inhibited in a mouse aldosterone increase model induced by angiotensin II, and the effect is superior to that of the atractylenolide I and sinapine which are used independently, and the synergistic effect is achieved; the pharmaceutical composition provided by the invention has definite curative effect and can effectively prevent and treat the aldosteronism.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a graph showing the results of a study of the diuretic effect of atractylenolide I on water-loaded model mice; wherein a is a urine volume determination result, b is a sodium ion content determination result in urine, c is a potassium ion content determination result in urine, and d is an aldosterone content determination result in serum;
FIG. 2 is a graph showing the results of a study of sinapine diuretic effect on water-loaded model mice; wherein a is a urine volume determination result, b is a sodium ion content determination result in urine, c is a potassium ion content determination result in urine, and d is an aldosterone content determination result in serum;
FIG. 3 is a graph of the effect of atractylenolide I prognosis on aldosterone production in H295R cells;
FIG. 4 is a graph showing the effect of the prognosis for sinapine on aldosterone production in H295R cells;
FIG. 5 shows the results of inhibition of aldosterone production by H295R cells and synergy index (CI) analysis of the combination of atractylenolide I and sinapine;
FIG. 6 shows the effect of combination of atractylenolide I and sinapine on aldosterone-dependent mice, wherein a is the result of measurement of aldosterone content in serum of each group of experimental animals; b is the determination result of the content of sodium ions in the urine of each group of experimental animals; c is the result of measuring the content of potassium ions in the urine of each group of experimental animals.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. It is intended that the specification and examples be considered as exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Atractylodes lactone I adopted in the following examples is purchased from Dorpus jequirolii Biotech limited, cat # PU0047-0025; the sinapine is purchased from Doppel Biotechnology Inc., cat. PS1183-0025; the experimental animal used is a wild type male C57BL/6 mouse, the weight is 18-22 g, and the experimental animal is purchased from the experimental animal center of the national drug and biological product control institute of Beijing, china; the H295R cells used were human adrenocortical carcinoma cells purchased from Procell, wuhan, cat # CL-0399; the content of Aldosterone (ALD) is detected by adopting a mouse aldosterone ELISA kit which is purchased from Shanghai Jianglai Biotechnology Limited company under the product number JL11794; the description will not be repeated below.
Example 1 diuretic Effect of Atractylodes macrocephala lactone I on Water-burdened model mice
Experimental animals were randomized into 3 groups (n = 6), each: (1) a water load model group (physiological saline), (2) a positive control group (spironolactone, spir) (Spir 20 mg/Kg/day), (3) a atractylenolide I group (AT-I) (AT-I20 mg/Kg/day). After continuously gavage the corresponding medicine for 7 days, injecting 100mL/Kg normal saline into the abdominal cavity of each mouse to establish a water-loaded mouse model, collecting mouse serum after the last administration, and carrying out Na-excretion + Urine K + And the analysis of the content of serum aldosterone, and simultaneously collecting urine in 24h of mice by adopting a metabolism cage, the result is shown in figure 1, and as can be seen from figure 1, the atractylenolide I has remarkable diuretic effect compared with a model group (figure 1a, P)<0.01 Increasing urine output and increasing sodium ion excretion in mice (FIG. 1b, P)<0.001 And has a retention effect on potassium ions (FIG. 1c, P)<0.05 While being capable of significantly reducing serum aldosterone levels (figure 1d, p)<0.001 The results show that the diuretic mechanism of atractylenolide I is involved in inhibiting aldosterone production. Compared with the model group<0.05；**，P<0.01；***，P<0.001。
Example 2 diuretic Effect of sinapine on Water-borne model mice
The experimental animals were randomly divided into 3 groups (n = 6) of (1) a water load model group (saline), (2) a positive control group (spironolactone, spir,20 mg/Kg/day), (3) a sinapine group (SP, 40 mg/Kg/day), respectively. After 7 days after continuous gavage of the corresponding drugs, 100mL/Kg of normal saline is injected into the abdominal cavity of each mouse to establish a water load mouse model, and the normal saline is adoptedCollecting urine 24h after the last administration of the mouse by a metabolism cage, collecting mouse serum, and carrying out Na urine + Urine K + And the results of the analysis of serum aldosterone content are shown in FIG. 2. As can be seen from FIG. 2, sinapine has a significant diuretic effect and can significantly increase the urine output of mice as compared with the model group (FIG. 2a, P)<0.01 ); at the same time, the discharge of sodium ions in mice can be increased (FIG. 2b, P)<0.001 Retention of potassium ions (FIG. 2c, P)<0.01 Play a role in discharging sodium and protecting potassium, and simultaneously can effectively reduce the aldosterone level (figure 2d<0.001 Indicating that the diuretic mechanism is involved in inhibiting aldosterone production. Comparing with model group<0.01；***，P<0.001。
Example 3 Effect of Atractylodes macrocephala lactone I on H295R cellular aldosterone production
The experimental method comprises the following steps: taking H295R cells in logarithmic growth phase, inoculating in 96-well plate, 100. Mu.L of medium per well, standing at 37 deg.C, 5% 2 Culturing for 24h in a conditioned incubator. After the cells are attached to the wall, the original culture medium is discarded, atractylenolide I drugs (0, 0.1, 1, 2.5, 5, 10, 20, 50, 100. Mu.M) with different concentrations are diluted in culture medium containing 50nmol/L angiotensin II (Ang II), 100. Mu.L of each well is added into a 96-well plate for incubation for 24h (culture medium without Ang II is added in blank group, no drug is contained, culture medium with Ang II is added in model group, no drug is contained). Collecting supernatant, and testing Aldosterone (ALD) content (n = 4), wherein inhibition rate (%) of atractylenolide I on aldosterone production is defined as = (ALD) Model set -ALD Administration set )/ALD Model set Calculating IC from the inhibition ratio 50 The results are shown in FIG. 3. FIG. 3 shows that atractylenolide I has a significant inhibitory effect on Ang II induced increase in aldosterone secretion levels in H295R cells, IC 50 The concentration was 7.7. Mu.M.
Example 4 Effect of sinapine on aldosterone production in H295R cells
Taking H295R cells in logarithmic growth phase, inoculating in 96-well plate, 100. Mu.L of medium per well, standing at 37 deg.C, 5% 2 Culturing for 24h in an incubator under the condition. Different concentrations of SP (0.01, 0.1, 1, 10, 100. Mu.M) were added, and 50nmol/L of Ang II was added for 24h incubation. Taking the supernatant forAldosterone (ALD) content detection (n = 4), specific detection method refers to example 3, according to inhibition rate (%) = (ALD) Model set -ALD Administration set )/ALD Model set The inhibition of Ang II-induced aldosterone production by sinapine was calculated, and IC was calculated 50 The results are shown in FIG. 4. FIG. 4 shows that sinapine inhibits Ang II-induced increase in aldosterone secretion levels, IC, in H295R cells 50 It was 2.33. Mu.M.
Example 5 synergy index study of the combination of Atractylodes lactone I and sinapine
IC obtained in example 3 and example 4 using combination of atractylenolide I and sinapine 50 The value is taken as the basis, the medicine concentration of the atractylenolide I is 1.875 mu M-30 mu M; the sinapine drug concentration is 2.5-40 μ M.
The inhibition effect of the combination of atractylenolide I and sinapine (the specific mixture ratio is shown in table 1) on the aldosterone production of H295R cells is examined by detecting the content of aldosterone in the H295R cells.
Taking H295R cells in logarithmic growth phase, inoculating in 96-well plates, 100. Mu.L of medium per well, placing at 37 ℃, 5% CO 2 Culturing for 24h in an incubator under the condition. After the cells are attached to the wall, the original culture medium is discarded, the atractylenolide I and sinapine of the combined medicine group are uniformly mixed in the culture medium containing 50nmol/L of Ang II, 100 mu L of each well is added into a 96-well plate for incubation for 24h (the culture medium containing no Ang II is added in a blank group, no medicine is contained, and the culture medium containing Ang II is added in a model group, no medicine is contained). Taking supernatant, detecting the content of aldosterone (n = 4), calculating the inhibiting effect of the combined administration group on the level of aldosterone, wherein the inhibiting rate is expressed by Fa, and the inhibiting rate (%) = (ALD) Model set -ALD Administration set )/ALD Model set ALD stands for aldosterone. The results of using CompuSyn software to analyze the CI value of synergy index after the combination of two drugs, wherein < 1 indicates synergy, =1 indicates additive action, and > 1 indicates antagonistic action are shown in Table 1 and FIG. 5.
TABLE 1 synergy index CI values for atractylenolide I + sinapine combination in H295R cells
As can be seen from table 1 and fig. 5, the combination of atractylenolide I and sinapine at different concentrations showed a synergistic inhibitory effect on aldosterone production by H295R cells at some concentrations, specifically: when the atractylenolide I is 1.875 MuM-7.5 MuM, sinapine is 5 MuM, and the ratio of the sinapine to the atractylenolide I is (5-30) to 10; when the atractylenolide I is 30 MuM, the sinapine is 5 MuM to 40 MuM, and the ratio of the sinapine to the atractylenolide I is (1.5-15) to 10.
Example 6 synergistic Effect examination of angiotensin II-induced aldosterone increment levels in Experimental animals
The effect of this composition on hyperaldosteronism mice was observed with the combined administration of atractylenolide I and sinapine.
Experimental animals were randomly divided into 8 groups (n = 6) in a gastric lavage administration mode, which were (1) a blank group (physiological saline), respectively; (2) model group (Ang II,4 mg/Kg/day); (3) spironolactone group (Spir) (Ang II + Spir 20 mg/Kg/day); (4) atractylenolide I group (AT-I) (Ang II + AT-I10 mg/Kg/day); (5) sinapine group 1 (SP) (Ang II + SP 7.5 mg/Kg/day); (6) sinapine group 2 (SP) (Ang II + SP 15 mg/Kg/day); (7) sinapine group 3 (SP) (Ang II + SP 30 mg/Kg/day); (8) combination 1 (i.p. ang II + AT-I10 mg/Kg/day + SP 7.5 mg/Kg/day) AT sinapine to atractylenolide I = 7.5: 10; (9) combination group 2 (i.p. ang II + AT-I10 mg/Kg/day + SP 15 mg/Kg/day) in sinapine/atractylenolide I = 15: 10; r (R) combined group 3 (i.p. ang II + AT-I10 mg/Kg/day + SP 30 mg/Kg/day), the ratio of sinapine to atractylenolide I = 30: 10. Establishing aldosterone increasing model by intraperitoneal injection of Ang II, continuous intragastric administration for 7 days, collecting urine 24h after the last administration of mouse by using metabolism cage, collecting mouse serum, and performing Na treatment + 、K + And analysis of aldosterone content. The results are shown in FIG. 6 ( ### Represents P in comparison with the blank group<0.001; * Representative of P compared to model group<0.001; compared with sinapine used alone in the combined group, Δ ，P<0.05； ΔΔΔ ，P<0.001; in contrast to the combination group 2, ▲▲ ，P<0.01； ▲▲▲ ，P<0.001 Spironolactone group, atractylenolide I group, sinapine dosage group, combination group 1, combination group 2 and combination group 3 can significantly reduce the content of aldosterone in serum of mice with hyperaldosteronism (fig. 6a, p)<0.001 And shows remarkable sodium discharging (figure 6 b) and potassium protecting (figure 6 c) effects, and the effect of inhibiting the generation of aldosterone of the combined administration group is remarkably superior to that of the atractylenolide I and sinapine single administration group. The combination group 2 can significantly reduce the serum aldosterone level compared with the combination group 1 and the combination group 3, and the sodium discharging and potassium protecting effects are better than those of the combination group 1 and the combination group 3, which indicates that at the animal level, the optimal dose combination of sinapine and atractylenolide I is 15mg/Kg of sinapine and 10mg/Kg of atractylenolide I, and the optimal proportion is 15 mg/Kg: 10mg/Kg, namely 15: 10.
The foregoing is only a preferred embodiment of the present invention, and those skilled in the art can make appropriate modifications based on the teachings herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the method and application of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the method and application described herein, or appropriate variations and combinations thereof, may be made without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (2)
1. Use of a natural pharmaceutical composition for synergistically inhibiting hyperaldosteronism for the manufacture of a medicament for the treatment of a disease or condition caused by hyperaldosteronism, wherein the natural pharmaceutical composition consists of atractylenolide i and sinapine;
based on the cellular level, when the concentration of the atractylenolide I is 1.875 to 7.5 mu M, the molar ratio of sinapine to the atractylenolide I is (5 to 30) to 10; when the concentration of the atractylenolide I is 30 mu M, the molar ratio of sinapine to atractylenolide I is (1.5-15) to 10;
when the pharmaceutical composition is used in combination at an animal level, the mass ratio of sinapine to atractylenolide I is 15: 10.
2. The use according to claim 1, wherein the disease or disorder is primary aldosteronism, secondary aldosteronism.
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