Application of alisol-containing composition in preparation of drugs for treating hypertension

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to application of a composition containing alisol in preparation of a medicine for treating hypertension. According to the present invention, the research results show that after the alisol A, the alisol A-23-acetate and the alisol A-24-acetate are compounded according to the ratio of 1: (0.05-0.3): (0.05-0.5), the renin-angiotensin-aldosterone system can be adjusted, the vascular endothelial function can be significantly improved, and the good treatment effect on hypertension can be provided. Besides, the composition is found to be capable of reducing the expression level of hypertension inflammatory factors and obviously inhibiting inflammatory response, which may be a potential blood pressure reduction mechanism, and the specific mechanism needs to be further researched.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to application of a composition containing alismatol in preparing a medicine for treating hypertension. Background Art

[0002] Hypertension (arterial hypertension) is a clinical syndrome characterized by increased systemic arterial pressure, increased peripheral arteriolar resistance, and varying degrees of increased cardiac output and blood volume. Under the premise of non-drug control, systolic blood pressure ≥140mmHg and / or diastolic blood pressure ≥90mmHg (≥140 / 90mmHg) is generally called hypertension. Patients often show symptoms such as dizziness, headache, blurred vision, tinnitus, insomnia, and fatigue. Long-term hypertension may lead to a variety of cardiovascular diseases and may affect changes in the structure and function of target organs (such as the heart, brain, and kidneys), ultimately leading to serious consequences such as heart failure, renal failure, and stroke.

[0003] Epidemiological data in recent years also show that the incidence of hypertension is on the rise, and it is one of the main causes of death among cardiovascular diseases. Lowering blood pressure is crucial to preventing organ damage and cardiovascular death in patients with hypertension. At present, diuretics, sympathetic nerve inhibitors, angiotensin inhibitors, angiotensin converting enzyme inhibitors, Ca 2+ Although Western medicines such as channel blockers and vasodilators have a definite effect on lowering blood pressure, if the dosage is suddenly reduced or stopped, it will cause blood pressure rebound or fluctuation, and even cause cardiovascular and cerebrovascular diseases. Therefore, how to improve the antihypertensive efficacy while ensuring its safety has become a difficult problem that needs to be solved urgently. In recent years, studies have reported that the combination of traditional Chinese medicine and antihypertensive drugs can increase the antihypertensive effect while reducing adverse reactions. Therefore, finding auxiliary antihypertensive drugs from the treasure house of traditional Chinese medicine is of great significance to the treatment of hypertension and the development of traditional Chinese medicine. Summary of the invention

[0004] In view of this, the present invention provides the use of a composition containing alismatol in the preparation of a drug for treating hypertension.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] Use of the composition in preparing medicine for preventing and treating hypertension;

[0007] The composition comprises alismatol A, alismatol A-23-acetate and alismatol A-24-acetate; the mass ratio of alismatol A, alismatol A-23-acetate and alismatol A-24-acetate is 1:0.05-0.3:0.05-0.5.

[0008] In some specific embodiments, the mass ratio of alisol A, alisol A-23-acetate and alisol A-24-acetate is 1:0.3:0.5, 1:0.1:0.1 or 1:0.05:0.05.

[0009] In the present invention, the composition can be directly ground into powder from commercially available raw materials, or can be an extract or other forms prepared from the medicinal material of Alisma orientalis by conventional means. In some embodiments, alismatol A, alismatol A-23-acetate and alismatol A-24-acetate in the composition are prepared by the following method:

[0010] (1) Extract the medicinal material of Alisma orientalis using method 1 or method 2

[0011] Method 1: Take 1 kg of dried tubers of Alisma orientalis, make them into medicinal slices, and extract them with ethyl acetate reflux twice, each time for 0.5 h; combine the extracts, recover the solvent under reduced pressure, and obtain a dark brown extract for later use.

[0012] Method 2: Take the dried tubers of Alisma orientalis, crush them into coarse particles, percolate with 70% ethanol, combine the percolated liquid, recover the solvent under reduced pressure, and obtain a dark brown extract for later use.

[0013] (2) Preparation of alisol A, alisol A-23-acetate and alisol A-24-acetate

[0014] The preparation of alismatol A includes method a or method b:

[0015] Method a: The extract obtained in method 2 was loaded onto a silica gel column and eluted with petroleum ether-ethyl acetate (3:1), with 500 ml as one fraction. The fraction was detected by HPLC, and the target fraction was collected to obtain alisol A solid.

[0016] Method b: The extract prepared by method 1 was applied to D101 macroporous adsorption resin, and after eluting with 20% ethanol for 10 column volumes, it was eluted with 70% ethanol for 5 column volumes, the 70% ethanol eluate was collected, and the solvent was recovered under reduced pressure to obtain a yellow oil; the yellow oil was dissolved in 65% methanol, and the target compound was separated by preparative HPLC under the following separation conditions: C18 column, 65% methanol as mobile phase, flow rate of 50ml / min, detection wavelength of 208nm, to obtain alisma alcohol A.

[0017] The preparation of alisol A-24-acetate includes method c or method d:

[0018] Method c: Weigh the extract obtained in method 2, apply it to a silica gel column, elute with petroleum ether-ethyl acetate (5:1), 300 ml as one fraction, detect by HPLC, collect the target fraction, and distill under reduced pressure to obtain alisol A-24-acetate solid.

[0019] Method d: Weigh the extract obtained in method 1, apply it to AB-8 macroporous adsorption resin, elute with 30% ethanol for 10 column volumes, then elute with 80% ethanol for 5 column volumes, collect the 80% ethanol eluate, and recover the solvent under reduced pressure to obtain a yellow oily substance; dissolve the yellow oily substance in 68% methanol, and separate the target compound by preparative HPLC. The separation conditions are: C18 column, mobile phase is 68% methanol, flow rate is 50ml / min, and detection wavelength is 208nm. Alismatol A24-acetate solid is obtained.

[0020] Preparation of alismatol A-23-acetate:

[0021] Method e: Weigh the extract obtained in method 1, add sulfuric acid, and reflux in a water bath for 15 minutes. Adjust the pH to 5-6 with 20% sodium hydroxide, add deionized water to adjust the ethanol concentration to 40%, pass through a DM130 macroporous adsorption resin column (diameter-to-height ratio 1:5) at a flow rate of 0.5 BV / h, then elute impurities with 40% ethanol, and then elute the target component with 55% ethanol. Determine the content of the main component (alismulol A23-acetate) in the eluate by HPLC, determine the elution endpoint, combine the eluates, concentrate under reduced pressure, and dry. The macroporous adsorption resin purified product was dissolved twice in 10 times the amount of a mixed solvent of petroleum ether-ethyl acetate (5:2), the residue was dissolved in ethanol and then adsorbed on silica gel, the solvent was evaporated, and the sample was loaded by dry method. The petroleum ether-ethyl acetate mixed solution containing the sample was subjected to silica gel column chromatography (300-400 mesh), and impurities were continuously eluted with petroleum ether-ethyl acetate (5:2), and then the target component was eluted with petroleum ether-ethyl acetate (2:3), and the end point was detected by HPLC, the eluate was collected, concentrated under reduced pressure to near dryness, and dried under reduced pressure at 70°C for 72 hours to obtain a 23-acetate solid of alismatol.

[0022] In the present invention, the prevention and treatment refers to all methods that are beneficial to improving the corresponding symptoms of the patient, including treatment or prevention. In some embodiments, the prevention and treatment includes at least one of the following aspects:

[0023] Regulate the renin-angiotensin-aldosterone system, promote the production of vasodilator active substances, inhibit the release of vasoconstrictor active substances, and reduce the level of inflammatory factors.

[0024] In some embodiments, the vasodilator active substance comprises NO; and the vasoconstrictor active substance comprises ET-1.

[0025] In some embodiments, the inflammatory factors include TNF-α and / or IL-6.

[0026] According to the etiology and pathogenesis, hypertension can be divided into primary hypertension and secondary hypertension. Primary hypertension is a multifactorial disease caused by the interaction of certain congenital genetic genes with many pathogenic pressurizing factors and physiological decompression factors; secondary hypertension is hypertension caused by certain diseases, and common causes mainly include: renal lesions (renal parenchyma, renal vascular hypertension), endocrine abnormalities (primary aldosteronism, pheochromocytoma, Cushing's syndrome, etc.) and other pathogenic factors (sleep apnea syndrome, cardiovascular or cranial lesions, some drugs, etc.). The pathogenesis of hypertension is not yet fully understood, and the current understanding of it is mainly concentrated on the following links: hyperactivity of the sympathetic nervous system; abnormal function of vascular endothelial cells (reduced NO production); renal sodium and water retention; abnormal cell membrane ion transport; insulin resistance, etc. In the present invention, the hypertension treated by the composition is renal vascular hypertension and nitric oxide deficiency hypertension.

[0027] In the present invention, the effective dose of the composition is 1.428 g of crude drug per day.

[0028] The present invention also provides a drug for preventing and treating hypertension, comprising alisol A, alisol A-23-acetate, alisol A-24-acetate and pharmaceutically acceptable excipients;

[0029] The mass ratio of alismatol A, alismatol A-23-acetate and alismatol A-24-acetate is 1:0.05-0.3:0.05-0.5.

[0030] After mixing alisol A, alisol A-23-acetate and alisol A-24-acetate according to the above ratio, suitable excipients are added and prepared according to conventional production methods in the pharmaceutical field to prepare conventional dosage forms acceptable to pharmacy. The pharmaceutically acceptable excipients include the following fillers, disintegrants, lubricants, suspending agents, adhesives, sweeteners, flavoring agents, preservatives, bases, etc. The pharmaceutically acceptable dosage forms include oral dosage forms, injection dosage forms or external dosage forms, and further, specifically include but are not limited to decoctions, granules, capsules, tablets, oral liquids, pills, tinctures, syrups, suppositories, gels, sprays, and injections.

[0031] The Chinese medicine composition of the present invention is prepared from magnolia, perilla leaf, schizonepeta, angelica dahurica, arctium, Xanthium sibiricum, Schisandra chinensis, Pseudostellaria baicalensis and raw liquorice. The present invention finds that the composition can significantly inhibit airway inflammation, significantly improve oxidative stress damage, and reduce bronchoconstriction, and has a good therapeutic effect on bronchial asthma. DETAILED DESCRIPTION

[0032] The present invention provides the application of the composition containing alismatol in the preparation of a medicine for hypertension. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and the relevant personnel can obviously change or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0033] The following experiments, if no specific conditions are specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The APIs or excipients, reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially. Unless otherwise specified, all percentages, ratios, proportions or parts are by weight.

[0034] The present invention will be further described below in conjunction with embodiments:

[0035] Example 1 Extraction of medicinal materials

[0036] Method 1: Take 1 kg of dried tubers of Aliams orientalis (Sam.) Juzep. of the Alismataceae family, prepare them into medicinal slices, and extract them twice with 7000 mL of ethyl acetate under reflux, each time for 0.5 h. Combine the extracts, recover the solvent under reduced pressure, and obtain 35 g of dark brown extract.

[0037] Method 2: Take 3 kg of dried tubers of Aliams orientalis (Sam.) Juzep. of the Alismataceae family, grind them into coarse particles, percolate with 70% ethanol, combine the percolate, recover the solvent under reduced pressure, and obtain 115 g of dark brown extract.

[0038] Example 2 Preparation of Alismatol A

[0039] Method a: Weigh 50 g of the extract of method 2 of Example 1, apply it to a silica gel column (450 g), and elute with petroleum ether-ethyl acetate (3:1), with 500 ml as one fraction. Detect by HPLC, collect the target fraction, and distill under reduced pressure to obtain 0.4 g of a solid. The purity is determined by the area normalization method and is greater than 50% (by weight).

[0040] Method b: Weigh 10g of the extract of Example 1, Method 1, and apply it to D101 macroporous adsorption resin. After eluting with 20% ethanol for 10 column volumes, use 70% ethanol to elute for 5 column volumes, collect the 70% ethanol eluate, and recover the solvent under reduced pressure to obtain 2g of yellow oily substance; dissolve 2g of yellow oily substance in 65% methanol, and separate the target compound by preparative HPLC. The separation conditions are: C18 column, mobile phase is 65% methanol, flow rate is 50ml / min, and detection wavelength is 208nm. 100mg of alismatol A with a purity of 95% (weight) is obtained.

[0041] Example 3 Preparation of alismatol A24-acetate

[0042] Method c: Weigh 50 g of the extract prepared by method 2 of Example 1, apply it to a silica gel column (450 g), elute with petroleum ether-ethyl acetate (5:1), 300 ml as one fraction, detect by HPLC, collect the target fraction, and obtain 0.4 g of solid. The purity is greater than 55% (weight) as determined by the area normalization method.

[0043] Method d: Weigh 20g of the extract prepared by method 1 of Example 1, apply it to AB-8 macroporous adsorption resin, elute with 30% ethanol for 10 column volumes, then elute with 80% ethanol for 5 column volumes, collect the 80% ethanol eluate, and recover the solvent under reduced pressure to obtain 1.5g of yellow oil; dissolve 1.5g of yellow oil in 68% methanol, and separate the target compound by preparative HPLC. Separation conditions are: C18 column, mobile phase is 68% methanol, flow rate is 50ml / min, and detection wavelength is 208nm. 60mg of alismatol A24-acetate with a purity of (95% by weight) is obtained.

[0044] Example 4 Preparation of alismatol A23-acetate

[0045] Method e: Weigh 10 g of the extract obtained by method 1 of Example 1, add 4 mL of sulfuric acid, and reflux in a water bath for 15 min. Adjust the pH to 5-6 with 20% sodium hydroxide, add deionized water to adjust the ethanol concentration to 40%, pass through a DM130 macroporous adsorption resin column (diameter-to-height ratio 1:5) at a flow rate of 0.5 BV / h, then elute impurities with 40% ethanol, and then elute the target component with 55% ethanol, determine the content of the main component (alismulol A23-acetate) in the eluate by HPLC, determine the elution endpoint, combine the eluates, concentrate under reduced pressure, and dry. The macroporous adsorption resin was used to dissolve the purified product twice with 10 times the amount of a mixed solvent of petroleum ether and ethyl acetate (5:2). The residue was dissolved in ethanol and then adsorbed on silica gel. The solvent was evaporated and the sample was loaded by dry method. The petroleum ether-ethyl acetate mixed solution containing the sample was subjected to silica gel column chromatography (300-400 mesh). Impurities were continuously eluted with petroleum ether-ethyl acetate (5:2). The target component was then eluted with petroleum ether-ethyl acetate (2:3). The end point was detected by HPLC, the eluate was collected, concentrated under reduced pressure to near dryness, and dried under reduced pressure at 70°C for 72 hours to obtain the product.

[0046] Example 5 Preparation of the composition of the present invention

[0047] Alismatol A prepared by method b of Example 2, Alismatol A23-acetate prepared by method b of Example 4 and Alismatol A24-acetate prepared by method d of Example 3 were mixed in mass ratios of 1:0.3:0.5, 1:0.1:0.1 and 1:0.05:0.05, respectively, to obtain composition A (mass ratio of 1:0.3:0.5), composition B (mass ratio of 1:0.1:0.1) and composition C (mass ratio of 1:0.05:0.05), respectively.

[0048] Preparation of Comparative Examples 1 to 3 Compositions

[0049] Alismatol A prepared by method b of Example 2, Alismatol A23-acetate prepared by method b of Example 4, and Alismatol A24-acetate prepared by method d of Example 3 were mixed in a mass ratio of 1:0.5:1 and 1:0.01:1, respectively, to obtain composition D (mass ratio of 1:0.5:1), composition E (mass ratio of 1:0.01:1) and composition F (weight ratio of 1:0.01:0.01), respectively.

[0050] Example 6 Effect on the Double Kidney One Clip (2K1C) Hypertensive Rat Model

[0051] 1. Experimental Materials

[0052] Animals: Wistar rats, SPF grade, male, 120 in total, weighing 140-160 g, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0053] Drug: Captopril tablets, Changzhou Pharmaceutical Factory Co., Ltd., batch number: 18110912, specification: 25 mg / tablet.

[0054] Reagents: Nitric oxide (NO) assay kit, endothelin-1 (ET-1) assay kit, angiotensin-Ⅱ (Ang-Ⅱ) assay kit, renin (REN) assay kit, and aldosterone (ALD) assay kit were all purchased from Nanjing Jiancheng Bioengineering Institute with the catalog numbers A012-1-2, H093-1-2, H185, H216, and H188, respectively.

[0055] Instruments: rat non-invasive blood pressure monitor (Beijing Softron Biotechnology Co., Ltd., model: BP-2010A); ultra-low temperature refrigerator (Haier Company, model: DW-86L728); electronic balance (METTLERTOLEOD Company, model: BSA224S-CW); calcium flow workstation (Molecular Devices Company, model: Felxstation3); ultrapure water instrument (Millpore Company, model: Milli-Q); desktop high-speed refrigerated centrifuge (Shanghai Lu Xiangyi Laboratory Instrument Co., Ltd., model: TGL-16M).

[0056] (II) Experimental methods

[0057] Experimental content: 120 Wistar rats, SPF grade, male, weighing 140-160g, were selected. After 6 days of adaptive feeding, 10 rats were selected as the sham operation group, and the remaining rats underwent unilateral renal artery ligation surgery to establish a double kidney one clamp method (2K1C) animal model. The rats were fasted overnight before surgery and had free access to water. They were anesthetized by intraperitoneal injection of 10% chloral hydrate at 0.3mL / 100g, their limbs were fixed, and the skin was disinfected with iodine. The skin and peritoneum were incised 1 cm to the left of the mid-abdomen, the left kidney was exposed, and the left renal artery was bluntly separated. The left renal artery was clamped with a U-shaped silver clamp with an inner diameter of 0.2mm to partially narrow the left renal artery, and then the left kidney was repositioned, the abdominal cavity was sutured layer by layer, and penicillin 2×10 was injected intraperitoneally after disinfection. 5 U 0.5ml to prevent postoperative infection. In the sham operation group, except for the placement of silver clips, the rest of the surgical procedures were the same as above, and the rats were free to eat and drink after the operation. The systolic blood pressure of the rat tail artery was measured 4 weeks after the operation, and those above 140mmHg were considered to be a successful renal hypertension model.

[0058] The rats with successful membrane formation were randomly divided into 8 groups, with 10 rats in each group: model group, captopril group (10 mg / kg), group A (alisol A:alisol A-23-acetate:alisol A-24-acetate, weight ratio = 1:0.3:0.5, 150 mg / kg), group B (alisol A:alisol A-23-acetate:alisol A-24-acetate, weight ratio = 1:0.1:0.1, 150 mg / kg), group C (alisol A:alisol A-23-acetate:alisol A-24-acetate, weight ratio = 1:0.1:0.1, 150 mg / kg), and group D (alisol A:alisol A-23-acetate:alisol A-24-acetate, weight ratio = 1:0.1:0.1, 150 mg / kg). 0.05:0.05, 150 mg / kg), group D (alisol A:alisol A-23-acetate:alisol A-24-acetate, weight ratio = 1:0.5:1, 150 mg / kg), group E (alisol A:alisol A-23-acetate:alisol A-24-acetate, weight ratio = 1:0.01:1, 150 mg / kg), group F (control group, alissol A: alissol A-23-acetate: alissol A-24-acetate, weight ratio = 1:0.01:0.01, 150 mg / kg). Oral administration, continuous administration for 8 weeks. In addition, 10 SD rats were selected as the sham operation group, and the sham operation group and the model group were given an equal volume of normal saline by oral administration every day. The systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the rats were measured before administration and 2 hours after the last administration, and the measurements were performed three times in succession to obtain the average value. After the last administration, the rats in each group were anesthetized by intraperitoneal injection of 10% chloral hydrate at 0.3 mL / 100 g, and blood was collected from the abdominal aorta, and the serum was separated and stored in a -80°C refrigerator.

[0059] Detection indicators: 1. The tail cuff method of non-invasive blood pressure tester was used to measure the SBP and DBP of the rat tail artery.

[0060] 2. ELISA method was used to detect the levels of Ang-Ⅱ, REN, ALD, NO and ET-1 in rat serum.

[0061] (III) Experimental results

[0062] The results showed that before administration, compared with the sham operation group, the SBP and DBP of the rats in the other groups were significantly increased, and the difference was statistically significant (P<0.001), indicating that the model was successfully established; there was no statistically significant difference in the SBP and DBP of the rats in the treatment groups compared with the model group (P>0.05); after 8 weeks of administration, compared with the model group, except for the diastolic blood pressure in group F, the SBP and DBP of the rats in the other groups were significantly decreased, and the differences were statistically significant (P<0.001, P<0.01, P<0.05), among which the SBP of the rats in the captopril group, group A, and group B was more significantly different from that in group C, the SBP of the rats in group C was more significantly different from that in group D, group E, and group F, the DBP of the rats in the captopril group, group A, and group B was more significantly different from that in group C, and the DBP of the rats in group C was more significantly different from that in group D and group E. See Table 1.

[0063] Table 1 Effects of Chinese medicine composition on SBP and DBP of rats ( n=10)

[0064]

[0065] Note: Compared with the sham operation group, ***P<0.001; compared with the model group, ▲▲▲ P < 0.001, ▲▲ P<0.01, ▲ P<0.05.

[0066] Compared with the sham operation group, the serum Ang-Ⅱ content of the rats in the model group was significantly increased, and the difference was statistically significant (P<0.001). Compared with the model group, the serum Ang-Ⅱ content of the rats in the other treatment groups was significantly decreased, and the differences were statistically significant (P<0.001, P<0.01, P<0.05). Among them, the Ang-Ⅱ content in the captopril group, group A, group B, and group C was more significant than that in group D, group E, and group F.

[0067] Compared with the sham operation group, the serum REN content of the rats in the model group was significantly increased, and the difference was statistically significant (P<0.001). Compared with the model group, the serum REN content of the rats in the other treatment groups was significantly decreased, and the differences were statistically significant (P<0.001, P<0.01, P<0.05). Among them, the REN content in the captopril group, group A and group B was more significant than that in group C, group D and group F.

[0068] Compared with the sham operation group, the ALD content in the serum of the rats in the model group was significantly increased, and the difference was statistically significant (P<0.001). Compared with the model group, the ALD content in the serum of the rats in the other treatment groups was significantly decreased, and the differences were statistically significant (P<0.001, P<0.01, P<0.05). Among them, the ALD content in the captopril group, group A, group B, and group C was more significantly different than that in group D, group E, and group F, as shown in Table 2.

[0069] Table 2 Comparison of the expression levels of Ang-Ⅱ, REN and ALD in the serum of rats in each group ( n=10)

[0070]

[0071] Note: Compared with the sham operation group, ***P<0.001; compared with the model group, ▲▲▲ P < 0.001, ▲▲ P<0.01, ▲ P<0.05.

[0072] Compared with the sham operation group, the serum NO content of the rats in the model group was significantly decreased, and the difference was statistically significant (P<0.001). Compared with the model group, except for group F, the serum NO content of the rats in the other treatment groups was significantly increased, and the differences were statistically significant (P<0.001, P<0.01, P<0.05). Among them, the NO content in the captopril group, group A, group B, and group C was more significant than that in group D and group E.

[0073] Compared with the sham operation group, the ET-1 content in the serum of the rats in the model group was significantly increased, and the difference was statistically significant (P<0.001); compared with the model group, the ET-1 content in the serum of the rats in the other treatment groups was significantly decreased, and the differences were statistically significant (P<0.001, P<0.01, P<0.05), among which the ET-1 content in the captopril group, group A, group B, group C, and group D was more significant than that in group E and group F. See Table 3.

[0074] Table 3 Comparison of NO and ET-1 expression levels in serum of rats in each group ( n=10)

[0075]

[0076]

[0077] Note: Compared with the sham operation group, ***P<0.001; compared with the model group, ▲▲▲ P < 0.001, ▲▲ P<0.01, ▲ P<0.05.

[0078] Experimental Example 7 Effects on N-nitro-L-arginine-induced hypertension rat model

[0079] 1. Experimental Materials

[0080] Animals: 90 male SD rats, SPF grade, weighing 160-180 g, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0081] Drug: Captopril tablets, Changzhou Pharmaceutical Factory Co., Ltd., batch number: 18110912, specification: 25 mg / tablet.

[0082] Reagents: L-NNA was purchased from Dalian Meilun Biotechnology Co., Ltd., with the catalog number MB3299-1; nitric oxide (NO) assay kit, endothelin-1 (ET-1) assay kit, tumor necrosis factor-α (TNF-α) test kit, and interleukin-6 (IL-6) test kit were purchased from Nanjing Jiancheng Bioengineering Institute, with the catalog numbers A012-1-2, H093-1-2, H052-1-2, and H007-1-2, respectively.

[0083] Instruments: rat non-invasive blood pressure monitor (Beijing Softron Biotechnology Co., Ltd., model: BP-2010A); ultra-low temperature refrigerator (Haier Company, model: DW-86L728); electronic balance (METTLERTOLEOD Company, model: BSA224S-CW); calcium flow workstation (Molecular Devices Company, model: Felxstation3); ultrapure water instrument (Millpore Company, model: Milli-Q); desktop high-speed refrigerated centrifuge (Shanghai Lu Xiangyi Laboratory Instrument Co., Ltd., model: TGL-16M).

[0084] (II) Experimental methods

[0085] L-NNA is a competitive inhibitor of nitric oxide synthase, which can weaken the vasodilatory effect of nitric oxide and lead to the occurrence of hypertension. In this example, L-NNA was used to establish an animal model of hypertension caused by long-term NO deficiency. The model construction and specific experimental methods are as follows:

[0086] The experiment selected 90 SD rats, SPF grade, male, weighing 160-180g. After 6 days of adaptive feeding, 10 were randomly selected as the blank control group, and the rest were the hypertension modeling group. The modeling group was intraperitoneally injected with L-NNA 15mg / (kg·d), and the blank group was injected with an equal amount of normal saline. After 28 days of administration, the systolic blood pressure of the rats was greater than 140mmHg, indicating that the modeling was successful. The modeled rats were randomly divided into 8 groups, with 10 rats in each group: model group, captopril group (10 mg / kg), group A (alisol A:alisol A-23-acetate:alisol A-24-acetate, weight ratio = 1:0.3:0.5, 150 mg / kg), group B (alisol A:alisol A-23-acetate:alisol A-24-acetate, weight ratio = 1:0.1:0.1, 150 mg / kg), group C (alisol A:alisol A-23-acetate:alisol A-24-acetate, weight ratio = 1:0.1:0.1, 150 mg / kg). 1:0.05:0.05, 150 mg / kg), group D (alisol A:alisol A-23-acetate:alisol A-24-acetate, weight ratio = 1:0.5:1, 150 mg / kg), group E (alisol A:alisol A-23-acetate:alisol A-24-acetate, weight ratio = 1:0.01:1, 150 mg / kg), group F (alisol A:alisol A-23-acetate:alisol A-24-acetate, weight ratio = 1:0.01:0.01, 150 mg / kg). During the administration period, the rats were free to take water and eat. The blank control group and the model group were given an equal volume of normal saline for continuous gavage for 28 days. The systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the rats were measured after the last administration. After the last administration, the rats were fasted but not watered for 12 hours and then anesthetized. 4 mL of blood was collected from the abdominal aorta and placed in a blood collection tube. The serum was separated and stored in a -80°C refrigerator.

[0087] Detection indicators:

[0088] 1. The tail cuff method of non-invasive blood pressure tester was used to measure the SBP and DBP of the rat tail artery.

[0089] 2. ELISA method was used to detect the levels of NO, ET-1, IL-6 and TNF-α in rat serum.

[0090] (III) Experimental results

[0091] The results showed that before administration, compared with the normal group, the SBP and DBP of the rats in the other groups were significantly increased, and the difference was statistically significant (P<0.001), indicating that the model was successfully established; before administration, there was no statistically significant difference in SBP and DBP between the rats in the treatment groups and the model group (P>0.05); after 4 weeks of administration, compared with the model group, except for the diastolic blood pressure (DBP) of group F, the SBP and DBP of the rats in the other groups were significantly decreased, and the differences were statistically significant (P<0.001, P<0.01, P<0.05), among which the SBP of the rats in the captopril group, group A, and group B was more significantly different from that in group C, the SBP of the rats in group C was more significantly different from that in group D, group E, and group F, the DBP of the rats in the captopril group, group A, and group B was more significantly different from that in group C and group D, and the DBP of the rats in group C and group D was more significantly different from that in group E. See Table 4.

[0092] Table 4 Effects of Chinese medicine composition on SBP and DBP in rats ( n=10)

[0093]

[0094] Note: Compared with the normal group, ***P<0.001; compared with the model group, ▲▲▲ P < 0.001, ▲▲ P<0.01, ▲ P<0.05.

[0095] Compared with the normal group, the NO content in the serum of the rats in the model group was significantly decreased, and the difference was statistically significant (P<0.001). Compared with the model group, except for group F, the NO content in the serum of the rats in the other treatment groups was significantly increased, and the differences were statistically significant (P<0.001, P<0.01, P<0.05). Among them, the NO content in the captopril group and group A was more significantly different from that in group B and group C, and the NO content in group B and group C was more significantly different from that in group D and group E.

[0096] Compared with the normal group, the ET-1 content in the serum of the rats in the model group was significantly increased, and the difference was statistically significant (P<0.001); compared with the model group, except for group E and group F, the ET-1 content in the serum of the rats in the other treatment groups was significantly decreased, and the differences were statistically significant (P<0.01, P<0.05), among which the ET-1 content in the captopril group, group A, and group B was more significantly different than that in group C and group D. See Table 5.

[0097] Table 5 Comparison of NO and ET-1 expression levels in the serum of rats in each group ( n=10) Group Dose mg / kg NO (μmol / L) ET-1 (ng / L)

[0098]

[0099] Note: Compared with the normal control group, ***P<0.001; compared with the model group, ▲▲▲ P < 0.001, ▲▲ P<0.01, ▲ P<0.05.

[0100] Compared with the normal group, the TNF-α content in the serum of the rats in the model group was significantly increased, and the difference was statistically significant (P<0.001); compared with the model group, the TNF-α content in the serum of the rats in the other treatment groups was significantly decreased, and the differences were statistically significant (P<0.001, P<0.01, P<0.05), among which the TNF-α content in the captopril group and group A was more significantly different from that in group B and group C, and the TNF-α content in group B and group C was more significantly different from that in group D, group E, and group F.

[0101] Compared with the normal group, the IL-6 content in the serum of the rats in the model group was significantly increased, and the difference was statistically significant (P<0.001); compared with the model group, the IL-6 content in the serum of the rats in the other treatment groups was significantly decreased, and the differences were statistically significant (P<0.01, P<0.05). Among them, the IL-6 content in the captopril group, group A, group B, and group C was more significantly different from that in group E, and the IL-6 content in group E was more significantly different from that in group D and group F. See Table 6.

[0102] Table 6 Comparison of TNF-α and IL-6 expression levels in the serum of rats in each group ( n=10)

[0103]

[0104]

[0105] Note: Compared with the normal control group, ***P<0.001; compared with the model group, ▲▲▲ P < 0.001, ▲▲ P<0.01, ▲ P<0.05.

[0106] The composition proposed by the present invention may play a role in lowering blood pressure by regulating the renin-angiotensin-aldosterone system, improving vascular endothelial function, promoting the increase of vasodilator NO production, and inhibiting the release of vasoconstrictor ET-1. In addition, the composition can reduce the expression levels of inflammatory factors TNF-α and IL-6 and inhibit inflammatory response, which may be its potential mechanism for lowering blood pressure, but the specific mechanism needs further study.

[0107] The above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Use of the composition in the preparation of drugs for preventing and treating hypertension; The composition comprises alisol A, alisol A-23-acetate and alisol A-24-acetate; the mass ratio of alisol A, alisol A-23-acetate and alisol A-24-acetate is 1:(0.05-0.3):(0.05-0.5).

2. The use according to claim 1, characterized in that: The mass ratio of alismatol A, alismatol A-23-acetate and alismatol A-24-acetate is 1:0.3:0.5, 1:0.1:0.1 or 1:0.05:0.

05.

3. The use according to claim 1, characterized in that: The prevention and treatment includes at least one of the following aspects: Regulate the renin-angiotensin-aldosterone system, promote the production of vasodilator active substances, inhibit the release of vasoconstrictor active substances, and reduce the level of inflammatory factors.

4. The use according to claim 3, characterized in that: The active substance that dilates blood vessels includes NO; the active substance that constricts blood vessels includes ET-1.

5. The use according to claim 3, characterized in that: The inflammatory factors include TNF-α and / or IL-6.

6. The use according to claim 1, characterized in that: The hypertension includes renovascular hypertension and / or nitric oxide-deficient hypertension.

7. The use according to claim 1, characterized in that: The effective dosage of the composition is 1.428 g of crude drug per day.

8. A drug for preventing and treating hypertension, characterized in that: It includes alisol A, alisol A-23-acetate, alisol A-24-acetate and pharmaceutically acceptable excipients; The composition comprises: the mass ratio of alisol A, alisol A-23-acetate and alisol A-24-acetate is 1:0.05-0.3:0.05-0.

5.

9. The drug according to claim 8, characterized in that The pharmaceutically acceptable excipients include at least one of a filler, a disintegrant, a lubricant, a suspending agent, a binder, a sweetener, a flavoring agent, a preservative or a matrix.

10. The drug according to claim 8, characterized in that The dosage form of the drug includes decoction, granules, capsules, tablets, oral liquid, pills, tincture, syrup, suppository, gel, spray or injection.

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