CN112274526A

CN112274526A - Application of dammarane type triterpenoid saponin compound in preparation of medicines for inhibiting PCSK9 from playing role in reducing blood fat

Info

Publication number: CN112274526A
Application number: CN202011277740.1A
Authority: CN
Inventors: 姜新宇; 刘朝晖; 张增勤
Original assignee: Hunan Warburg Pharmaceutical Co ltd
Current assignee: Hunan Warburg Pharmaceutical Co ltd
Priority date: 2020-11-16
Filing date: 2020-11-16
Publication date: 2021-01-29
Anticipated expiration: 2040-11-16
Also published as: CN114748488A; CN114984024A; CN114832008A; CN112274526B; CN114984024B

Abstract

The invention relates to the technical field of natural medicinal chemistry, in particular to dammarane-type triterpenoid saponin compounds separated from gynostemma pentaphylla from Hunan province and Guangxi province, and application of the dammarane-type triterpenoid saponin compounds in preparation of medicines for inhibiting PCSK9 from playing a role in reducing blood fat. Part of monomeric compounds of gynostemma pentaphylla produced in Hunan province and Guangxi province have inhibitory activities to PCSK9 secreted by liver cells to different degrees, so that the aim of individually reducing blood fat or enhancing the blood fat reducing effect of gynostemma pentaphylla by cooperating with statins is fulfilled.

Description

Application of dammarane type triterpenoid saponin compound in preparation of medicines for inhibiting PCSK9 from playing role in reducing blood fat

Technical Field

The invention relates to the technical field of natural medicinal chemistry, in particular to dammarane type triterpenoid saponin compounds separated from gynostemma pentaphylla from Hunan province and Guangxi province and application thereof in preparing medicines for inhibiting PCSK9 from playing a role in reducing blood fat.

Background

Atherosclerosis (as) and cardiovascular disease (cvd) have extremely high morbidity and mortality rates worldwide, and are the main causes of death in our country. Hypercholesterolemia is a major risk factor for atherosclerosis and heart disease, and low density lipoprotein cholesterol (LDL-C) has been identified as an independent and most effective risk factor that can alter the development of cardiovascular disease.

The use of statins has been successful in reducing the risk of cardiovascular events occurring over the last 20 years. The statin is a 3-hydroxy-3-methyl-glutaryl coenzyme (HMGCR) (a rate-limiting enzyme for cholesterol synthesis in a body) inhibitor, can effectively reduce the content of LDL-C in blood, but the statin still has some defects, such as adverse reactions such as rhabdomyolysis and the like of some patients, and when a large amount of statin is used, the cholesterol synthesis is inhibited, and simultaneously the level of proprotein convertase subtilisin/kexin type 9 is increased, so that the lipid-lowering activity of the statin is greatly weakened, and the dosage dependence relationship exists on the enhancement effect of PCSK 9.

PCSK9 is a member of the proprotein convertase family (PC) proteinase K subfamily, named proprotein convertase subtilisin/kexin 9(PCSK9), and is mainly expressed in human liver cells, small intestine, kidney, etc. PCSK9 is a lipid metabolism regulator, and can promote the degradation of LDL-R by binding to low-density lipoprotein receptor (LDL-R) on the surface of liver cell, reduce the clearance ability of LDL-C of liver cell to regulate liver lipid metabolism, and increase plasma LDL-C level. PCSK9 was found to be the causative gene of familial hypercholesterolemia caused by a third gene mutation other than ApoB, LDL-R. At present, only monoclonal antibodies are clinically registered and marketed medicines which play a role in reducing blood fat for PCSK9, but the monoclonal antibodies have the defects of high price, poor patient compliance and the like, and adverse reactions even serious adverse reactions occur in three-phase clinical experiments. Therefore, the safe, effective and acceptable-price PCSK9 inhibitor becomes a hotspot of current lipid-lowering treatment research.

Gynostemma pentaphylla (Gynostemma pentaphylum (Thunb.) Makino) is a perennial climbing herbaceous plant of Gynostemma (Gynostemma) of Cucurbitaceae (Cucurbitaceae), is mainly distributed in China, Japan, Korea, India and southeast Asia, is mainly produced in southern Shaanxi and southern areas of Yangtze river in China, is a rare plant containing ginsenoside except Araliaceae, and is known as 'southern ginseng'. Pharmacological research shows that the gynostemma pentaphylla can effectively improve glycolipid metabolism, has wide pharmacological effects of resisting inflammation, resisting oxidation, resisting tumors, resisting fatigue, protecting liver and the like, and can be used for preventing and treating various metabolic diseases such as diabetes, obesity, dyslipidemia and the like. A large number of compounds with remarkable biological activity, such as triterpenoid saponin, flavone, polysaccharide and other various compounds, have been separated and identified from gynostemma pentaphylla up to now. Earlier studies found that the gypenosides extract can exert lipid-lowering activity by inhibiting PCSK9 (wuliu pines, qian folk chapter. gypenosides have influence on PCSK9 gene expression and simvastatin blood lipid-lowering action, china pathophysiology journal, 2017). However, there are significant differences in chemical composition between gynostemma pentaphylla from different origins, and the specific active ingredients of gynostemma pentaphylla for inhibiting PCSK9 are not clear. By carrying out systematic research on chemical components and pharmacological activities of gynostemma pentaphylla in Hunan province and Guangxi province, the active substances of PCSK9 are reduced, and the medicinal value of the gynostemma pentaphylla is further explored.

Disclosure of Invention

In order to solve the problem that the increase of PCSK9 in a first-line medicament for treating hyperlipidemia, namely a statin medicament, causes the reduction of lipid-lowering activity, the invention provides application of a natural medicament for reducing PCSK9 secretion of liver cells.

In order to achieve the purpose, the invention discloses a new application of gynostemma pentaphylla, and particularly discloses an application of a monomer compound in gynostemma pentaphylla produced in Hunan province and Guangxi province to reduction of activity of PCSK9 secreted by hepatocytes.

Application of compound shown in any one of structural formulas 1-13 in preparation of medicine for reducing PCSK9 lipid secretion of liver cells

Preferably, the dosage concentration is 10 uM.

Compounds of formulae 4, 5, 12-13 are preferred.

The compound is obtained by separating gynostemma pentaphylla from southern Hunan and Guangxi.

In addition, the invention also provides a novel dammarane type triterpenoid saponin compound, the structural formula of which is any one of the compounds shown in formulas 1-6:

the dammarane type triterpenoid saponin compound is obtained by separating gynostemma pentaphylla from Hunan origin and Guangxi origin.

Compared with the prior art, the invention has the following beneficial effects:

1. the novel dammarane type saponin compound is obtained by extracting, separating and purifying gynostemma pentaphylla whole plants in Hunan producing areas and Guangxi producing areas.

2. Part of monomeric compounds of gynostemma pentaphylla produced in Hunan province and Guangxi province have inhibitory activities to PCSK9 secreted by liver cells to different degrees, so that the aim of individually reducing blood fat or enhancing the blood fat reducing effect of gynostemma pentaphylla by cooperating with statins is fulfilled.

3. The invention discloses application of partial monomers of gynostemma pentaphylla in Hunan origin and Guangxi origin in cooperation with statins in treatment of hyperlipidemia.

4. The gynostemma pentaphylla has a long use history in a traditional Chinese medicine treatment system and clinical application of modern Chinese patent medicines, and the activity of reducing blood fat and the safety of medicines of the gynostemma pentaphylla have clinical guarantees.

Drawings

FIG. 1: in example 2, a part of monomers of Gynostemma pentaphyllum Makino produced in Hunan and Guangxi provinces have activity of reducing PCSK9 secretion from hepatocytes under the condition of administration of 20 μ M concentration.

FIG. 2: in example 3, a part of monomers of Gynostemma pentaphyllum Makino produced in Hunan and Guangxi provinces have activity of reducing PCSK9 secretion from hepatocytes under the condition of 10 μ M concentration.

FIG. 3: example 3 Total gypenosides secreted PCSK9 level activity in HepG2 cells at a concentration of 50. mu.g/mL.

Detailed Description

The essential matters of the present invention will be described in detail with reference to the drawings and examples. However, the present invention is not limited to the following embodiments. The experimental methods in the following examples are all conventional methods unless otherwise specified; materials, reagents and the like used in the following examples are commercially available from public unless otherwise specified.

Part of monomeric compounds of gynostemma pentaphylla produced in Hunan province and Guangxi province can reduce the activity of high PCSK9 expression induced by combination of lipoprotein serum and simvastatin on the level of HepG2, and achieve better lipid-lowering effect.

In order to explore the activity of the monomer compounds in gynostemma pentaphylla from Hunan province and Guangxi province on a high-expression PCSK9 cell model, develop in-vitro cell experiments, detect the expression of PCSK9 protein, and verify the effect of the monomer compounds in gynostemma pentaphylla from Hunan province and Guangxi province on the PCSK9 high-expression model by adopting an ELISA kit experiment.

In a preferred embodiment of the invention, the pathological feature is high expression of PCSK9, and the cell is human liver cancer cell HepG 2. The experimental results of the specific embodiment show that part of monomers in the gynostemma pentaphylla from Hunan province and Guangxi province have obviously reduced activity on PCSK9 on the cellular level, which indicates that the components in the gynostemma pentaphylla from Hunan province and Guangxi province have better activity of reducing lipid in cooperation with statins.

Example 1 preparation of monomer Compound from Gynostemma pentaphyllum Makino in Hunan and Guangxi provinces

Respectively taking two local gynostemma pentaphyllum medicinal materials, extracting for two times by a decoction method, extracting for 2 hours by 8 times of water each time, combining extracting solutions, filtering and concentrating to obtain the gynostemma pentaphyllum total saponin extract. Extracting herba Gynostemmatis total saponin extract with 8 times of methanol under ultrasound for 5 times, each for 20min, mixing filtrates, and concentrating under reduced pressure to obtain methanol soluble fraction. And (3) performing appropriate amount of the methanol soluble part extract by deactivated silica gel column chromatography, performing gradient elution with chloroform-methanol-water (100:0:0 → 0:100:0), performing TLC inspection, and mixing to obtain 5 fractions (Fr.a-Fr.i). And respectively carrying out repeated silica gel, ODS, Sephadex LH-20 column chromatography, Flash rapid preparation and semi-preparation of high performance liquid phase to obtain compounds 1-24, wherein the structural formula of the compounds is as follows:

the physicochemical properties and spectral data of the novel compounds 1 to 6 (structural formulae 1 to 6) were as follows:

compound 1: a white amorphous powder of a crystalline substance,

(c 0.1,MeOH)，IR(KBr)cm^-1:3370,2915,1594,1077，UVλ_max(CH₃OH):201nm，HR-ESI-MS m/z 1117.5756[M+Na]⁺(C₅₃H₉₀NaO₂₃,calcd.1117.5771)，¹H NMR(C₅D₅n,500MHz) and¹³C NMR(C₅D₅n,125MHz) is shown in tables 1 and 2, is not reported in the literature and is a novel compound;

compound 2: a white amorphous powder of a crystalline substance,

(c 0.51,MeOH)，IR(KBr)cm^-1:3395,2918,1599,1077，UVλ_max(CH₃OH):202nm，HR-ESI-MS m/z 1085.5858[M+Na]⁺(C₅₃H₉₀NaO₂₁,calcd.1085.5872)，¹H NMR(C₅D₅n,500MHz) and¹³C NMR(C₅D₅n,125MHz) is shown in tables 1 and 2, is not reported in the literature and is a novel compound;

compound 3: a white amorphous powder of a crystalline substance,

(c 0.11,MeOH)，IR(KBr)cm^-1:3363,2937,1597,1041，UVλ_max(CH₃OH):202nm，HR-ESI-MS m/z 1231.6431[M+Na]⁺(C₅₉H₁₀₀NaO₂₅,calcd.1231.6451)，¹H NMR(C₅D₅n,500MHz) and¹³C NMR(C₅D₅n,125MHz) is shown in tables 1 and 2, is not reported in the literature and is a novel compound;

compound 4: a white amorphous powder of a crystalline substance,

(c 0.11,MeOH)，IR(KBr)cm^-1:3397,2920,1659,1079，UVλ_max(CH₃OH):200nm，HR-ESI-MS m/z 1221.6276[M-H]^-(C₅₉H₉₇O₂₆,calcd.1221.6268)，¹H NMR(C₅D₅n,500MHz) and¹³C NMR(C₅D₅n,125MHz) is shown in tables 1 and 2, is not reported in the literature and is a novel compound;

compound 5: a white amorphous powder of a crystalline substance,

(c 0.11,MeOH)，IR(KBr)cm^-1:3397,2921,1659,1078，UVλ_max(CH₃OH):201nm，HR-ESI-MS m/z 1239.6373[M-H]^-(C₅₉H₉₉O₂₇,calcd.1239.6374)，¹H NMR(C₅D₅n,500MHz) and¹³C NMR(C₅D₅n,125MHz) is shown in tables 3 and 4, is not reported in the literature and is a novel compound;

compound 6: a white amorphous powder of a crystalline substance,

(c 0.12,MeOH)，IR(KBr)cm^-1:3397,2920,1659,1078，UVλ_max(CH₃OH):200nm，HR-ESI-MS m/z 915.5326[M-H]^-(C₄₇H₇₉O₁₇,calcd.915.5317)，¹H NMR(C₅D₅n,500MHz) and¹³C NMR(C₅D₅n,125MHz) is shown in tables 3 and 4, is not reported in the literature and is a novel compound;

TABLE 1 NMR data (C) of 1-4 aglycones of the compounds₅D₅N,J in Hz)

^a)Measured at 500MHz；^b)Measured at 125MHz；^c)Overlapped signals.

TABLE 2 NMR data (C) for sugar groups of Compounds 1 to 4₅D₅N,J in Hz)

^a)Measured at 500MHz；^b)Measured at 125MHz；^c)Overlapped signals；Glc:glucopyranosyl；Ara:arabinopyranosyl；Rha:rhamnopyranosyl；Xyl:xylopyranosyl.

TABLE 3 NMR data (C) for Compounds 5 and 6 aglycones₅D₅N,J in Hz)

^a)Measured at 500MHz；^b)Measured at 125MHz；^c)Overlapped signals.

TABLE 4 NMR data (C) for sugar radicals of Compounds 5 and 6₅D₅N,J in Hz)

^a)Measured at 500MHz；^b)Measured at 125MHz；^c)Overlapped signals；Glc:glucopyranosyl；Rha:rhamnopyranosyl；Xyl:xylopyranosyl.

Example 2 part of monomeric compounds of Gynostemma pentaphyllum Makino in Hunan and Guangxi provinces have significant PCSK9 reduction activity on HepG2 cells under the condition of drug concentration of 20 mu M

Human hepatoma cells (HepG2) were divided into groups of three parallel wells, and 1 x 10 cells were seeded per well⁵The individual cells were plated in 12-well plates, cultured in DMEM low-sugar medium containing 10% fetal bovine serum, and cultured in 5% CO₂The culture is carried out in an incubator at 37 ℃; after inoculation of the cells, the groups of cells were treated as follows: group 1 was blank group, cultured with DMEM low-sugar medium containing 5% Fetal Bovine Serum (FBS), group 2 was model group, cultured with DMEM low-sugar medium containing 5% lipoprotein-deficient serum (LPDS), several groups thereafter were administration groups (20. mu.M) of different monomeric compounds, cultured with DMEM low-sugar medium containing 5% LPDS, blank group treated for 48 hours, model group and administration group were cultured with DMEM low-sugar medium containing 5% LPDS for 23 hours, then simvastatin (1. mu.M) was added, model group was cultured for 25 hours, administration group was added with different monomeric compounds (20. mu.M) after 1 hour of simvastatin action for 24 hours. After the above groups of cells were treated as described above, the culture medium was collected and subjected to treatment and measurement according to the ELISA kit protocol. The results are shown in FIG. 1.

FIG. 1 shows that a part of monomeric compounds of Gynostemma pentaphyllum Makino produced in Hunan and Guangxi provinces have the activity of reducing the level of PCSK9 secreted by HepG2 cells due to combination of LPDS-poor and simvastatin (1 μ M) at an administration concentration of 20 μ M.

Example 3 part of monomeric compounds of Gynostemma pentaphyllum Makino in Hunan and Guangxi provinces at a drug concentration of 10. mu.M, and the total glycosides of Gynostemma pentaphyllum Makino have significant PCSK 9-reducing activity on HepG2 cells

Dividing human liver cancer cells (HepG2) into several groups, each group having three parallel wells, inoculating 1 × 105 cells per well into 12-well plates, culturing with DMEM low-sugar medium containing 10% fetal bovine serum, and culturing in a 37 deg.C incubator containing 5% CO 2; after inoculation of the cells, the groups of cells were treated as follows: group 1 was blank group, cultured in DMEM low-sugar medium containing 5% FBS, group 2 was model group, cultured in DMEM low-sugar medium containing 5% LPDS, several groups thereafter were separately administration group of different monomeric compounds (10. mu.M) or total glycosides (50. mu.g/mL) cultured in DMEM low-sugar medium containing 5% LPDS, blank group was treated for 48 hours, model group and administration group were cultured in DMEM low-sugar medium containing 5% LPDS for 23 hours and then simvastatin (1. mu.M) was added, model group was cultured for 25 hours, administration group was added with different monomeric compounds (10. mu.M) after 1 hour of simvastatin action for 24 hours. After the above groups of cells were treated as described above, the culture medium was collected and subjected to treatment and measurement according to the ELISA kit protocol. The results are shown in FIGS. 2 and 3.

FIG. 2 shows that a part of monomeric compounds of Gynostemma pentaphyllum Makino produced in Hunan and Guangxi provinces have activity of reducing the level of PCSK9 secreted by HepG2 cells due to combination of LPDS and simvastatin (1 μ M) at an administration concentration of 10 μ M.

FIG. 3 shows that gypenosides have activity to reduce the levels of PCSK9 secreted by HepG2 cells in combination with simvastatin (1. mu.M) at a concentration of 50. mu.g/mL. The research shows that the statins can increase the expression of PCSK9 protein and mRNA while reducing blood fat on the animal level, and the application of gypenosides can obviously reduce the protein expression of PCSK 9. However, the gynostemma pentaphylla has a lot of production places and disordered basic sources, so that the gynostemma pentaphylla total glycoside extracts from different sources cannot be confirmed to have the effect. The patent indicates that the gypenoside used in the experiment can reduce the PCSK9 level rise caused by statins in a cell experiment, and is consistent with the report of the results of previous animal experiments. Based on systematic research analysis, the patent firstly discovers the active monomeric compound which remarkably reduces the level of PCSK9 at the cellular level, in particular the monomeric compound which shows excellent activity at low concentration (10 mu M). This result suggests that the active monomeric compound described in this patent is an active substance of gynostemma pentaphylla for inhibiting PCSK9 expression, and the activity is superior to total glycosides.

It is to be understood that the above examples are for the purpose of clearly and specifically describing the present invention, and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that variations may be made in other forms based on the teachings of the invention. The present invention is neither necessary nor exhaustive of all embodiments. Obvious variations or modifications of this invention are possible within the scope of the invention.

Claims

1. Application of compound shown in any one of structural formulas 1-13 in preparation of medicine for reducing PCSK9 lipid secretion of liver cells

2. The use according to claim 1, wherein the compound is administered at a concentration of 10 uM.

3. The use according to claim 1, wherein the compound is a compound of formula 4, 5, 12-13.

4. Use according to claim 1, characterized in that: the compound is obtained by separating gynostemma pentaphylla from southern Hunan and Guangxi.

5. Use of a compound of any one of formulae 1-13 and a statin in the preparation of a lipid lowering medicament.

6. The use according to claim 5, wherein the statin is simvastatin.

7. A dammarane type triterpene saponin compound has a structural formula shown in any one of formulas 1-6:

8. the dammarane-type triterpene saponin compound according to claim 7, which is characterized in that: the dammarane type triterpenoid saponin compound is obtained by separating gynostemma pentaphylla from Hunan origin and Guangxi origin.