LU500100B1 - Antihypertensive pharmaceutical composition, and preparation method and use thereof - Google Patents
Antihypertensive pharmaceutical composition, and preparation method and use thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/438—The ring being spiro-condensed with carbocyclic or heterocyclic ring systems
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/74—Rubiaceae (Madder family)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
Abstract
The present disclosure relates to an antihypertensive pharmaceutical composition, and a preparation method and use thereof, and belongs to the technical field of antihypertensive drugs. The pharmaceutical composition of the present disclosure includes isorhynchophylline (IRN) and sinapine thiocyanate (ST). The pharmaceutical composition of the present disclosure is cheap and easily-available, has definite therapeutic effects and small toxic and side effects, and can prevent and treat hypertension with multiple targets.
Description
BL-5233 LU500100 ANTIHYPERTENSIVE PHARMACEUTICAL COMPOSITION, AND PREPARATION
TECHNICAL FIELD The present disclosure relates to the technical field of antihypertensive drugs, and in particular to an antihypertensive pharmaceutical composition, and a preparation method and use thereof.
BACKGROUND As a common chronic disease, hypertension is most likely to induce cardiovascular disease (CVD) and death. With relatively-high morbidity, disability rate, and fatality rate, hypertension consumes a huge amount of medical and social resources, poses a heavy burden on the entire family and society, and also brings a huge challenge to the public health of all mankind. The use of antihypertensive western medicines can effectively control a blood pressure level in a patient, but shows significant side effects, which is difficult to improve the life quality of the patient. In terms of preventing and treating hypertension, traditional Chinese medicine (TCM) can not only effectively lower a blood pressure, but also significantly improve the clinical symptoms of a patient, which has the advantages of multi-pathway, multi-target, no toxic and side effects, and the like and has become an important means for treating hypertension. In the future, it is of great significance to develop new drugs for preventing or treating hypertension using active ingredients in TCM. However, there is currently a lack of hypotensive products with clear TCM active ingredients and definite therapeutic effects.
SUMMARY The present disclosure is intended to provide an antihypertensive pharmaceutical composition, and a preparation method and use thereof. The pharmaceutical composition of the present disclosure 1s cheap and easily-available, has definite therapeutic effects and small toxic and side effects, and can prevent and treat hypertension with multiple targets.
The present disclosure provides an antihypertensive pharmaceutical composition, including isorhynchophylline (IRN) and sinapine thiocyanate (ST).
Preferably, the pharmaceutical composition may include the following components in parts by weight: 0.3 to 1.2 parts of the IRN and 5 to 20 parts of the ST.
The present disclosure also provides a preparation method of the pharmaceutical composition according to the above technical solution, including the following step: mixing the IRN and the ST to obtain the pharmaceutical composition.
Preferably, a preparation method of the IRN may include: soaking Uncaria rhynchophylla in an ethanol aqueous solution with a volume fraction of 70% at room temperature, and 1
BL-5233 LU500100 filtering a resulting mixture to obtain a filtrate; subjecting the filtrate to vacuum concentration until there is no alcohol smell, and adjusting a pH of a resulting concentrate to 1.0 to 3.0 to obtain a first extraction solution; mixing the first extraction solution with chloroform at a volume ratio of (1-2):(1-2) for extraction to obtain a water phase, and adjusting a pH of the water phase to 10.0 to 12.0 to obtain a second extraction solution; mixing the second extraction solution with chloroform at a volume ratio of (1-2):(1-2) for extraction to obtain an organic phase, and subjecting the organic phase to vacuum recovery to obtain total alkaloids; and subjecting the total alkaloids to silica gel column chromatography to obtain IRN.
Preferably, a preparation method of the ST may include: degreasing radish seeds with petroleum ether, and subjecting a residue obtained from the degreasing to reflux extraction using an ethanol aqueous solution with a volume fraction of 80% to obtain an ST extract; mixing the extract and a potassium thiocyanate (KSCN) aqueous solution with a volume fraction of 20%, and standing a resulting mixture at 0°C to 4°C for 24 h to 48 h to obtain a crude product of ST; and subjecting the crude product to recrystallization to obtain the ST.
The present disclosure also provides use of the pharmaceutical composition according to the above technical solution or a pharmaceutical composition obtained by the preparation method according to the above technical solution in the preparation of a drug for treating hypertension.
Preferably, the hypertension may include spontaneous hypertension and/or high-salt diet (HSD)-induced hypertension.
The present disclosure also provides use of the pharmaceutical composition according to the above technical solution or a pharmaceutical composition obtained by the preparation method according to the above technical solution in the preparation of a drug for reducing an angiotensin (Ang) II content in patients with HSD-induced hypertension.
The present disclosure also provides use of the pharmaceutical composition according to the above technical solution or a pharmaceutical composition obtained by the preparation method according to the above technical solution in the preparation of a drug for inhibiting Ang II-induced vascular endothelial cell injury.
The present disclosure also provides use of the pharmaceutical composition according to the above technical solution or a pharmaceutical composition obtained by the preparation method according to the above technical solution in the preparation of a drug for increasing a NO level and/or reducing an endothelin 1 (ET-1) level in endothelial cells.
The present disclosure provides an antihypertensive pharmaceutical composition. The pharmaceutical composition of the present disclosure 1s cheap and easily-available, has definite 2
BL-5233 LU500100 therapeutic effects and small toxic and side effects, and can prevent and treat hypertension with multiple targets. Compared with TCM extracts, the active ingredients of IRN and ST in the pharmaceutical composition of the present disclosure have clear structures, and are convenient for quality control, cheap and easily-available, and suitable for large-scale industrial production. In addition, the present disclosure provides use of the composition of IRN and ST in the preparation of a drug for preventing and treating hypertension. The HUVCE cell viability experiment confirms that the IRN and ST, when used in combination, show a synergistic effect in reducing hypertensive vascular endothelial injury. In addition, the pharmaceutical composition of the present disclosure can significantly reduce a diastolic blood pressure (DBP) and a systolic blood pressure (SBP) in rat models with spontaneous hypertension and rat models with HSD-induced hypertension, exhibiting a definite effect in reducing and stabilizing a blood pressure.
BRIEF DESCRIPTION OF DRAWINGS FIG 1 shows NO levels in human umbilical vein endothelial cells (HUVECS) after Ang II intervention and drug intervention provided in the present disclosure; and FIG 2 shows ET-1 levels in HUVECs after Ang II intervention and drug intervention provided in the present disclosure.
DETAILED DESCRIPTION The present disclosure provides an antihypertensive pharmaceutical composition, including IRN and ST. The pharmaceutical composition of the present disclosure can synergistically reduce the hypertensive vascular endothelial injury, and can also significantly reduce DBP and SBP in rat models with spontaneous hypertension and rat models with HSD-induced hypertension, exhibiting a definite effect in reducing and stabilizing a blood pressure. IRN is a white crystalline powder with a molecular formula of C»2H>8N204 and a molecular weight of 384.47, which is soluble in chloroform. ST is a white crystalline powder with a molecular formula of CisH24NOs CNS and a molecular weight of 368.45, which is soluble in organic solvents such as methanol, ethanol, and DMSO. IRN has a structural formula shown in formula I, and ST has a structural formula shown in formula II: - / 2 A ere N EX 0 7e 7 HCC PS a NCH SGEN Hao , formula I, and OCH, ; formula II.
In the present disclosure, the pharmaceutical composition may include the following 3
BL-5233 LU500100 components in parts by weight: preferably 0.3 to 1.2 parts of IRN and 5 to 20 parts of ST; and more preferably 0.6 part of IRN and 10 parts of ST.
The present disclosure also provides a preparation method of the pharmaceutical composition according to the above technical solution, including the following step: mixing the IRN and the ST to obtain the pharmaceutical composition.
In the present disclosure, a preparation method of the IRN may preferably include: soaking Uncaria rhynchophylla in an ethanol aqueous solution with a volume fraction of 70% at room temperature, and filtering a resulting mixture to obtain a filtrate; subjecting the filtrate to vacuum concentration until there is no alcohol smell, and adjusting a pH of a resulting concentrate to 1.0 to 3.0 and more preferably of 2.0 to obtain a first extraction solution; mixing the first extraction solution with chloroform at a volume ratio of (1-2):(1-2) and more preferably of 1:1 for extraction to obtain a water phase, and adjusting a pH of the water phase to 10.0 to 12.0 and more preferably 11.0 to obtain a second extraction solution; mixing the second extraction solution with chloroform at a volume ratio of (1-2):(1-2) and more preferably of 1:1 for extraction to obtain an organic phase, and subjecting the organic phase to vacuum recovery to obtain total alkaloids; and subjecting the total alkaloids to silica gel column chromatography to obtain IRN. In the present disclosure, the adjusting a pH to 1.0 to 3.0 may preferably be conducted with HCI, and the adjusting a pH to 10.0 to 12.0 may preferably be conducted with NH3. HO. In the present disclosure, the silica gel column chromatography may be conducted for enrichment and purification. In the present disclosure, the IRN product obtained by the above preparation method may have a purity of more than 95%. The preparation method of the present disclosure is simple, controllable, and easily used in industrialized large-scale production, and an obtained compound has a high purity.
In the present disclosure, a preparation method of the ST may preferably include: degreasing radish seeds with petroleum ether, and subjecting a residue obtained from the degreasing to reflux extraction using an ethanol aqueous solution with a volume fraction of 80% to obtain an ST extract, mixing the extract and a KSCN aqueous solution with a mass fraction of 20%, and standing a resulting mixture at 0°C to 4°C for 24 h to 48 h and more preferably at 4°C for 48 h to obtain a crude product of ST; and subjecting the crude product to recrystallization to obtain the ST. In the present disclosure, before being mixed with the KSCN aqueous solution with a mass fraction of 20%, the extract may preferably be diluted preferably with water. In the present disclosure, the recrystallization may preferably be conducted repeatedly, and the ST product obtained by the preparation method of the present disclosure may have a purity of more than 95%. The preparation method of the present disclosure 4
BL-5233 LU500100 involves easy operations, has strong controllability and low cost, and is easily used in industrialized large-scale production, and an obtained compound has a high purity.
The present disclosure also provides use of the pharmaceutical composition according to the above technical solution or a pharmaceutical composition obtained by the preparation method according to the above technical solution in the preparation of a drug for treating hypertension.
In the present disclosure, the hypertension may include spontaneous hypertension and/or HSD-induced hypertension. Experimental results show that, in the experiment for testing the effects of antihypertensive drugs in rat models with spontaneous hypertension and rat models with HSD-induced hypertension, the IRN and ST have the optimal intragastric dosages of 0.6 mg/kg/d and 10 mg/kg/d, respectively.
The present disclosure also provides use of the pharmaceutical composition according to the above technical solution or a pharmaceutical composition obtained by the preparation method according to the above technical solution in the preparation of a drug for reducing an Ang II content in patients with HSD-induced hypertension.
The present disclosure also provides use of the pharmaceutical composition according to the above technical solution or a pharmaceutical composition obtained by the preparation method according to the above technical solution in the preparation of a drug for inhibiting Ang II-induced vascular endothelial cell injury.
The present disclosure also provides use of the pharmaceutical composition according to the above technical solution or a pharmaceutical composition obtained by the preparation method according to the above technical solution in the preparation of a drug for increasing a NO level and/or reducing an ET-1 level in endothelial cells.
The antihypertensive pharmaceutical composition and the preparation method and use thereof provided by the present disclosure will be further described in detail below with reference to specific examples. The technical solutions of the present disclosure include, but are not limited to, the following examples.
Example 1 Synergistic protection of IRN and ST from HUVEC injury induced by Ang II Experimental materials HUVECs, purchased from ScienCell (USA), where, cells at passages 4 to 7 were used for experiments.
Experimental drugs: IRN, ST, and valsartan (Val), all purchased from Chengdu Chroma-Biotechnology Co., Ltd.; and Ang II, purchased from Beijing Solarbio Science &
BL-5233 LU500100 Technology Co., Ltd.
Experimental reagents: Nitric oxide (NO) (Cat. No.: A013-2-1) purchased from Nanjing Jiancheng Institute of Biological Engineering; ET-1 enzyme-linked immunosorbent assay (ELISA) kit (Cat. No.: JYMO710Hu) purchased from Wuhan ColorfulGene Biological Technology Co., Ltd.; and cell proliferation and toxicity detection (CCK-8) kit (Cat. No.: MAO218-1) purchased from Dalian Meilun Biotechnology Co., Ltd.
Experimental instrument: full-wavelength microplate reader (BioTek Cytation 5), BioTek (USA).
Experimental groups and drug administration: Normal control group (Control): HUVECs were cultivated under normal conditions, without special treatment; model group (Ang II): cells were intervened with Ang II (2 x 10% mol-L™) for 24 h; Val group (Ang II + Val): cells were intervened with Val (5 umol-L") + Ang II (2 x 10° mol-L™) for 24 h; IRN group (Ang II + IRN): cells were intervened with IRN (10 umol:L!) + Ang II (2 x 10“ mol-L”) for 24 h; ST group (Ang II + ST): cells were intervened with ST (200 umol:L*!) + Ang II (2 x 10° mol-L") for 24 h; and combination group (Ang II + IRN + ST): cells were intervened with IRN (10 umol-L*) + ST (200 pmol-L™) + Ang II (2 x 10% mol-L”) for 24 h.
Experimental method
1. The CCK-8 method was used to screen out an appropriate intervention concentration of Val, IRN, and ST for HUVECs: The CCK-8 method was first used to detect the cytotoxicity of the three drug monomers to screen out a drug concentration showing no cytotoxicity. HUVECs were intervened for 24 h with the three monomers at corresponding concentration gradients (Val: 0.1 umol-L*, 1 umol-L*, 5 umol-L*, 10 umol-L*, 15 umol-L*, 20 umol-L*, and 50 umol-L; IRN: 5 umol-L*, 10 umol-L*, 25 umolL*, 50 jumol-L*, 100 umol-L!, 200 umol-L*!, and 300 umol-L*; and ST: 50 umol-L*, 100 umol-L*, 200 umol-L*, 300 umol-L*, 400 umol-L!, 500 umol-L*, and 600 umol-L*!), a CCK-8 solution was added, a resulting mixture was incubated for 2 h, and then an absorbance at 450 nm was detected with a microplate reader to calculate a cell viability. Results showed that, in groups at a Val concentration less than 5 umol-L“, an IRN concentration less than 10 umol-L!, and an ST concentration less than 200 umol-L!, HUVECs had a cell viability showing no significant difference from that in the normal group. The CCK-8 method was then used to detect the 6
BL-5233 LU500100 protective effects of the drug monomers on HUVEC injury induced by Ang II. HUVECs were intervened for 1 h with the three monomers at corresponding concentration gradients (Val: 0.1 umol-L!, 1 umolL*, 2.5 umol-L*, and 5 umol-L*; IRN: 2.5 umol-L*, 5 umol-L*, 7.5 umol-L!, and 10 umol L*; and ST: 25 umol-L*, 50 jumol-L!, 100 umol-L*, and 200 umol-L"!), then Ang II was added at a concentration of 2 x 10% mol-L”!, the cells were further cultivated for 24 h, and an absorbance was determined to calculate a cell viability. According to the effects of monomer drugs on cell viability at different concentrations, the following drug intervention concentrations were screened out: Val: 5 umol-L!, IRN: 10 umol-L*, and ST: 200 umol-L*.
2. Detection of cell NO level: after the drug intervention was completed, a cell culture supernatant was collected, and a NO determination kit was used to detect the cell NO level. After operations according to instructions, an absorbance (OD value) was determined at 550 nm. NO content (umol/L) = (determined OD value - blank OD value)/(standard OD value - blank OD value) x standard concentration x sample dilution factor.
3. Detection of ET-1 level: after the drug intervention was completed, a cell culture supernatant was collected, and the human ET-1 ELISA kit was used to detect a cell ET-1 level. After operations according to instructions, an absorbance (OD value) was determined at 450 nm. With a standard OD value as x-coordinate and a standard concentration as y-coordinate, a standard curve was plotted and a quadratic regression equation of the standard curve was calculated. An OD value of a sample was substituted into the equation to calculate a sample concentration, namely, an actual concentration of the sample.
4. Statistical analysis: The software SPSS statistics 25.0 was used to conduct statistical analysis on data. Measurement data were expressed as mean + standard deviation (X + 5). Measurement data of multiple groups were compared by one-way analysis of variance (ANOVA). P < 0.05 indicates a statistically-significant difference.
Experimental results
1. The effect of Ang II intervention and drug intervention on the NO level in HUVECs Compared with that of the normal control group, the NO level in HUVECs of the model group was significantly reduced, with statistically-significant difference (P < 0.05), indicating that Ang II can significantly inhibit the synthesis and release of NO in endothelial cells. After drug intervention, the NO level in HUVECs of each group was significantly higher than that of the model group (P < 0.05), suggesting that Val, IRN, and ST can significantly increase the NO level in endothelial cells and reduce the damage of Ang II to endothelial cells. The NO levels in the Val group and combination group both were higher than that in the IRN group and ST 7
BL-5233 LU500100 group. Compared with the Val group and combination group, the two monomer groups both showed a statistically-significant difference (P < 0.05). There was no significant difference between the combination group and the Val group (P > 0.05). Each of the composition group and the Val group exhibited no significant difference from the normal control group (P > 0.05). It indicates that, when used in combination, IRN and ST show a better effect than that when used alone, a synergistic effect, and the same effect as Val. Comparison results of NO contents in the groups are shown in FIG 1. Notes: x+sn=3, according to one-way ANOVA, compared with the normal control group, °P < 0.05; compared with the model group, *P < 0.05; compared with the Val group, *P < 0.05; and compared with the combination group, ÀP < 0.05.
2. The effect of Ang II intervention and drug intervention on the ET-1 level in HUVECs After Ang II intervention, the ET-1 level in HUVECs increased significantly (P < 0.05). After drug intervention, the ET-1 level in HUVECs of each group was significantly lower than that of the model group (P < 0.05), suggesting that Val, IRN, and ST all can significantly reduce the ET-1 level in endothelial cells. Moreover, the combination group had a lower ET-1 level than the single component groups, and the two components exhibited a synergistic effect. The IRN group and the ST group showed a statistically-significant difference from the combination group (P < 0.05), indicating that the combination group has the best effect. The combination group exhibited no significant difference from the Val group (P > 0.05), indicating that the IRN and ST, when used in combination, show the same inhibitory effect on ET-1 as val. Comparison results of ET-1 contents in the groups are shown in FIG. 2. Notes: x +s, n = 5, according to one-way ANOVA, compared with the normal control group, *P < 0.05; compared with the model group, *P < 0.05; compared with the Val group, *P < 0.05; and compared with the combination group, “P < 0.05.
Example 2 Antihypertensive pharmacodynamics experiment of the composition of IRN and ST on spontaneous hypertension rats (SHR) Experimental materials Experimental animals: in the model group: SPF 8-week-old male SHRs, with a body weight of 180 g to 200 g; and in the normal group: SPF 8-week-old male Wistar-Kyoto rats (WKYs), with a body weight of 180 g to 200 g, which were all purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK (Beijing): 2016-0005) and raised in an animal laboratory at room temperature of 20°C to 25°C and a humidity of 50% to 65%.
Experimental drugs: IRN and ST, both purchased from Chengdu Chroma-Biotechnology Co., Ltd., with batch No. 20201011, and specification: 2 g/bottle; and Val purchased from 8
BL-5233 LU500100 Beijing Novartis Pharma Co., Ltd., with batch No. x1041 H2020723.
Experimental instruments: BP-98A non-invasive blood pressure meter for rats and mice, produced by Beijing Softron Biotechnology Co., Ltd.; and BioTek Cytation 5 full-wavelength microplate reader produced by BioTek, USA.
Experimental grouping and administration: The rats were randomly divided into: SHR model control group, high, medium, and low-dosage pharmaceutical composition groups, and positive drug control group, and a WKY blank control group was set, with 10 rats in each group.
The following dosages were adopted: high-dosage pharmaceutical composition group: IRN: 1.2 mg/kg/d and ST: 20 mg/kg/d, medium-dosage pharmaceutical composition group: IRN: 0.6 mg/kg/d and ST: 10 mg/kg/d; low-dosage pharmaceutical composition group: IRN: 0.3 mg/kg/d and ST: 5 mg/kg/d; and positive drug control group (Val group): a Val suspension: 14.4 mg/kg/d.
According to the above grouping and corresponding dosage, a dosage volume of 10 mL/kg was adopted, and the WKY blank control group and the SHR model control group were administered with the same volume of distilled water.
Experimental method: The rats in each experimental group were intragastrically administered with the corresponding drug continuously for 30 d (regularly administered once a day in the morning), and the tail-artery blood pressure measurement was used to measure the SBP and DBP in tail arteries before the administration and on day 7, day 15 and day 30 of the administration. The measurement was conducted 3 times continuously and an average was taken as a blood pressure value. Contemporary comparison was conducted in terms of the SBP and DBP to evaluate the antihypertensive effect of a drug.
Experimental results Changes of SBP and DBP in rat tail arteries during 30 days of continuous administration Compared with blood pressure before the administration, the blood pressure in each administration group was reduced on day 7 of the administration (P < 0.05). With the extension of the intervention time, each drug continued to exert its antihypertensive effect. On day 30 of intervention, both SBP and DBP were minimized, with statistical difference from that before the administration (P < 0.01). Compared with the medium-dosage pharmaceutical composition group, the Val group showed no statistical difference in the effect of reducing SBP and DBP (P > 0.05). Results are shown in Table 1 and Table 2.
Table 1 Changes of SBP in rats during 30 days of continuous administration (X + SD, 9
BL-5233 LU500100 mmHg) Group n Before Day 7 of Day 15 of Day 30 of administration administration administration administration WKY blank control 10 132.42 + 2.96 133.41 + 1.89 135.77 + 1.24 138.13 + 1.02 group SHR model control 10 180.19 + 1.61 179.11 + 1.16 180.19 + 1.62 182.21 + 1.39 group Low-dosage group 10 179.96 + 1.85 178.29 £2.54" 172.29 + 2,99""# 164.61 + 1.40™# Medium-dosage group 10 179.67 + 2.26 176.84 + 2,54*# 170.68 + 2.03**# 162.37 + 1.28**# High-dosage group 10 179.434 + 1,87 177.04 + 1,81* 170.93 + 2.48“ # 162.37 + 1.95" # Val group 10 179.31 + 1.91 176.81 + 2.14" 170.25 + 2,17*# 161.83 + 1.73# Note: Compared with SBP before administration, *p < 0.05 and **p < 0.01; and compared with SBP in the model group, *p < 0.05 and *p < 0.01. Table 2 Changes of DBP in rat tail arteries during 30 days of continuous administration (X + SD, mmHg) Group n Before 1 week of 2 weeks of 4 weeks of administration administration administration administration WKY blank control group 10 100.42 + 2.96 101.41 + 1.89 103.77 + 1.24 106.13 + 1.01 SHR model control group 10 137.28 + 1.01 136.64 + 1.06 137.19 + 1.62 139.21 + 1.39 Low-dosage group 10 137.67 + 1.12 135.91 + 1.04” 130.95 + 1.277# 124.84 + 1.217# Medium-dosage group 10 136.77 + 1.04 135.36 + 1.12% 126.68 + 1.12**# 121.87 + 1.28**# High-dosage group 10 136.77 + 1.87 135.20 + 0.99*# 126.91 + 1.05**# 122.07 + 1.95™# Val group 10 136.71 + 1.14 135.41 + 0.99*# 126.25 + 1.19**# 121.50 + 1.03*# Note: Compared with DBP before administration, *p < 0.05 and **p < 0.01; and compared with DBP in the model group, *p < 0.05 and *p < 0.01. Example 3 The antihypertensive effect of a composition of IRN and ST on HSD-induced hypertension rat models Experimental materials Experimental animals: SPF 3-week-old male WKY rats, with a body weight of 70 g to 80 g, which were all purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.
BL-5233 LU500100 (SCXK (Beijing): 2016-0007) and raised in an animal laboratory at room temperature of 20°C to 25°C and a humidity of 50% to 65%.
Experimental drugs: IRN and ST, both purchased from Chengdu Chroma-Biotechnology Co, Ltd, with batch No. 20201011, and specification: 2 g/bottle; and Val purchased from Beijing Novartis Pharma Co., Ltd., with batch No. x1041 H2020723.
Experimental reagent: Ang II (Cat. No. ml058803), purchased from Shanghai Enzyme Linked Biotechnology Co., Ltd.
Experimental instruments: BP-98 A non-invasive blood pressure meter for rats and mice, produced by Beijing Softron Biotechnology Co., Ltd.; and BioTek Cytation 5 full-wavelength microplate reader produced by BioTek, USA.
Experimental modeling (high-salt feed-induced hypertension rat models): WKY rats were randomly divided into: a WKY blank group, with 10 rats; and an HSD-induced model group, with 40 rats; and the WKY blank group was administered with AIN-93 purified feed and the HSD-induced model group was administered with 8% high-salt feed (8% sodium chloride and 92% AIN-93 purified feed). After 3 months of continuous feeding, a blood pressure was measured collectively for the rats. In the model group, qualified rats were screened out based on SBP >130 mm Hg and fed with the AIN-93 purified feed for 1 week, at which time, the rats with the desired SBP were considered as successful models.
Experimental grouping and administration: The rats were randomly divided into: HSD-induced model control group, pharmaceutical composition group, and positive drug control group, and a WKY blank control group was set, with 10 rats in each group. The following dosages were adopted: pharmaceutical composition group: IRN: 0.6 mg/kg/d and ST: 10 mg/kg/d (The dosage group with the optimal effect in Example 2); and positive drug control group (Val group): a Val suspension: 14.4 mg/kg/d.
According to the above grouping and corresponding dosage, a dosage volume of 10 mL/kg was adopted, and the WKY blank control group and the HSD-induced model control group were administered with the same volume of distilled water.
Experimental method:
1. Antihypertensive effect: detection of rat tail artery blood pressure The rats in each experimental group were intragastrically administered with the corresponding drug continuously for 30 d (regularly administered once a day in the morning), and the tail-artery blood pressure measurement was used to measure the SBP and DBP in tail arteries before the administration and on day 7, day 15 and day 30 of the administration. The 11
BL-5233 LU500100 measurement was conducted 3 times continuously and an average was taken as a blood pressure value. Contemporary comparison was conducted in terms of the SBP and DBP to evaluate the antihypertensive effect of a drug.
2. Antihypertensive effect: detection of rat serum Ang II After the blood pressure was determined before the administration and during the 30 d of the administration, blood was collected from the orbital veins of the rats and centrifuged to obtain a supernatant, and 100 uL of serum was used for the detection of Ang II. À sample was prepared according to instructions of an Ang II ELISA kit, and after the reaction was completed, an absorbance (OD450 value) was determined at 450 nm with a microplate reader. With a standard OD450 value as x-coordinate and a standard concentration as y-coordinate, a standard curve was plotted and a regression equation of the standard curve was calculated. An OD450 value of a sample was substituted into the equation to calculate a sample concentration. The Ang II contents in rats of the groups were compared.
Processing of experimental data: the software SPSS statistics 25.0 was used for statistical analysis on the test data, the measurement data was expressed as mean + standard deviation (X + SD), and the / test and one-way ANOVA were used to determine whether there is a significant difference among mean values. P < 0.05 indicates a significant difference and P<0.01 indicates an extremely-significant difference.
Experimental results
1. The effect of the pharmaceutical composition on blood pressure in rats Compared with blood pressure before the administration, the blood pressure in each administration group was reduced on day 7 of the administration (P < 0.05). With the extension of the intervention time, each drug continued to exert its antihypertensive effect. On day 30 of intervention, both SBP and DBP were minimized, with statistical difference from that before the administration (P < 0.01). Compared with the pharmaceutical composition group, the Val group showed no statistical difference in the effect of reducing SBP and DBP (P > 0.05). Results are shown in Table 3 and Table 4.
Table 3 Changes of SBP in rats during 30 days of continuous administration (X + SD, mmHg) Group n Before Day 7 of Day 15 of Day 30 of administration administration administration administration WKY blank control group 10 137.43 £2.40 137.43 + 1.89 136.78 + 1.34 138.32 + 2.30 HSD-induced model 10 184.19 + 1.65 183.31 + 1.24 184.30 + 0.98 185.12 + 2.07 12
BL-5233 LU500100 control group Composition group 10 183.40 + 0.87 181.92 + 1.09 174.81 + 1.34 # 164.58 + 0.96# Val group 10 184.17 + 1.23 182.25 + 0.67% 174.56 + 1.02"*# 163.71 + 1.18"# Note: Compared with SBP before administration, *p < 0.05 and **p < 0.01; and compared with SBP in the model group, *p < 0.05 and **p < 0.01. Table 4 Changes of DBP in rat tail arteries during 30 days of continuous administration (X + SD, mmHg) Group n Before 1 week of 2 weeks of 4 weeks of administration administration administration administration WKY blank control 10 103.167 + 1.74 105.53 + 2.27 105.53 + 1.67 106.32 + 2.44 group HSD-induced model 10 136.20 + 2.61 137.40 + 1.91 137.53 + 1.94 138.27 + 1.63 control group Composition group 10 137.46 + 1.30 135.26 + 1.42 127.14 + 1.04”# 123.19 + 2,91"# Val group 10 136.90 + 1.26 135.08 + 1,12*# 127.25 + 2,33"*# 122.71 + 2,85"*# Note: Compared with DBP before administration, *p < 0.05 and **p < 0.01; and compared with DBP in the model group, *p < 0.05 and *p < 0.01.
2. The effect of the pharmaceutical composition on the serum Ang II level in rats Results showed that, compared with that of the normal group, the serum Ang II content in rats of the model group was significantly increased (P < 0.01); compared with that of the model group, the serum Ang II contents in rats of the drug group and the Val group were decreased, with a significant difference (P < 0.01); and there was no significant difference between the drug group and the Val group (P > 0.05). The comparison of Ang II content among the groups is shown in Table 5 below. Table 5 Changes of serum Ang II content in rats during 30 days of continuous administration Group n Before 1 week of 2 weeks of 4 weeks of administration administration administration administration WKY blank control 10 143.16 + 12.74 145.53 + 12.27 145.53 + 11.67 146.32 + 12.44 group HSD-induced model 10 186.20+1261 187.40 + 19.14 187.53 + 19,39 188.27 + 16,35 13
BL-5233 LU500100 control group Composition group 10 18746+1230 175.26 + 14.22°* 167.16 + 13.39** 163.01 + 12.91°* Val group 10 186.90 + 12.60 174.98 + 11.77°* 165.95 + 13.31°* 162.89 + 15.27°* Note: Compared with serum Ang II content before administration, °p < 0.01; and compared with serum Ang II content in the model group, *p < 0.01.
The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.
14
Claims (10)
1. An antihypertensive pharmaceutical composition, comprising isorhynchophylline (IRN) and sinapine thiocyanate (ST).
2. The pharmaceutical composition according to claim 1, wherein, the pharmaceutical composition comprises the following components in parts by weight: 0.3 to 1.2 parts of the IRN and 5 to 20 parts of the ST.
3. A preparation method of the pharmaceutical composition according to claim 1 or 2, comprising the following step: mixing the IRN and the ST to obtain the pharmaceutical composition.
4. The preparation method according to claim 3, wherein, a preparation method of the IRN comprises: soaking Uncaria rhynchophylla in an ethanol aqueous solution with a volume fraction of 70% at room temperature, and filtering a resulting mixture to obtain a filtrate; subjecting the filtrate to vacuum concentration until there is no alcohol smell, and adjusting a pH of a resulting concentrate to 1.0 to 3.0 to obtain a first extraction solution; mixing the first extraction solution with chloroform at a volume ratio of (1-2):(1-2) for extraction to obtain a water phase, and adjusting a pH of the water phase to 10.0 to 12.0 to obtain a second extraction solution; mixing the second extraction solution with chloroform at a volume ratio of (1-2):(1-2) for extraction to obtain an organic phase, and subjecting the organic phase to vacuum recovery to obtain total alkaloids; and subjecting the total alkaloids to silica gel column chromatography to obtain IRN.
5. The preparation method according to claim 3, wherein, a preparation method of the ST comprises: degreasing radish seeds with petroleum ether, and subjecting a residue obtained from the degreasing to reflux extraction using an ethanol aqueous solution with a volume fraction of 80% to obtain an ST extract; mixing the extract and a potassium thiocyanate (KSCN) aqueous solution with a volume fraction of 20%, and standing a resulting mixture at 0°C to 4°C for 24 h to 48 h to obtain a crude product of ST; and subjecting the crude product to recrystallization to obtain the ST.
6. Use of the pharmaceutical composition according to claim 1 or 2 or a pharmaceutical composition obtained by the preparation method according to any one of claims 3 to 5 in the preparation of a drug for treating hypertension.
7. The use according to claim 6, wherein, the hypertension comprises spontaneous hypertension and/or high-salt diet (HSD)-induced hypertension.
8. Use of the pharmaceutical composition according to claim 1 or 2 or a pharmaceutical composition obtained by the preparation method according to any one of claims 3 to 5 in the
BL-5233 LU500100 preparation of a drug for reducing an angiotensin (Ang) II content in patients with HSD-induced hypertension.
9. Use of the pharmaceutical composition according to claim 1 or 2 or a pharmaceutical composition obtained by the preparation method according to any one of claims 3 to 5 in the preparation of a drug for inhibiting Ang II-induced vascular endothelial cell injury.
10. Use of the pharmaceutical composition according to claim 1 or 2 or a pharmaceutical composition obtained by the preparation method according to any one of claims 3 to 5 in the preparation of a drug for increasing a NO level and/or reducing an endothelin 1 (ET-1) level in endothelial cells.
16
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| CN102266407B (en) * | 2011-08-05 | 2013-01-09 | 李运伦 | Traditional Chinese medicinal composition for treating hypertension and preparation method thereof |
| CN103804207B (en) * | 2012-11-06 | 2015-12-09 | 大连大学 | The preparation method of sinapic acid choline ester chlorate and medical applications thereof |
| CN103432122A (en) * | 2013-08-30 | 2013-12-11 | 苏州天南星生物科技有限公司 | Application of isocorynoxeine in preparation of antihypertensive drugs |
| CN110882247A (en) * | 2019-11-16 | 2020-03-17 | 南方医科大学 | Use of isorhynchophylline in preparing drug-relief medicine |
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