CN110038041B

CN110038041B - SIRT1 agonist and application thereof

Info

Publication number: CN110038041B
Application number: CN201910410704.9A
Authority: CN
Inventors: 詹姆斯周; 谢百波; 臧新钰; 张纲
Original assignee: Guangxi Hebabiz Pharmaceutical Co ltd; Beijing Hebabiz Management Co ltd
Current assignee: Guangxi Hebabiz Pharmaceutical Co ltd; Beijing Hebabiz Management Co ltd
Priority date: 2019-05-16
Filing date: 2019-05-16
Publication date: 2021-06-08
Anticipated expiration: 2039-05-16
Also published as: CN110038041A

Abstract

The invention provides a SIRT1 agonist which is a crystalline product of Z018A, comprises 3 components, and has three characteristic peaks in an HPLC detection pattern. The agonist has stable quality, can obviously improve the in-vivo expression level of SIRT1, and can be used for resisting aging. In addition, the invention also provides a pharmaceutical preparation based on the agonist, application and the like.

Description

SIRT1 agonist and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an agonist of SIRT1, which can improve the expression level of SIRT1 and can be used for resisting aging.

Background

The Z018A developed by the inventor in an early stage is disclosed in Chinese patent ZL201610169121.8 (the whole content of which is incorporated by reference herein), has stable quality and three characteristic peaks on an HPLC detection map, and can be used for preventing or treating fatty liver injury and obesity, reducing blood lipid level and reducing inflammatory factor level.

After the patent publication, the inventors did not find reports of new indications or other improvements for Z018A. For example, intracellular activated oxygen is thought to be the most prominent cause of cellular damage, and accumulation of cellular damage ultimately leads to cellular senescence, whereas SIRT 1(SIRT1) slows cellular senescence by alleviating the intracellular activated oxygen load by inhibiting the NF- κ B pathway, and thus activated SIRT1 contributes to anti-senescence (Hubbard B P et al Trends Pharmacol Sci 2014,35(3): 146-. However, the prior art has no report of its correlation with anti-aging or SIRT 1.

Nevertheless, the inventors were unaffected by the prior art's research and development of Z018A due to silence, and, after long-term research, surprisingly found that its crystalline product (ZAG019) is an agonist of SIRT1 and is useful for anti-aging.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a novel SIRT1 agonist, which can improve the expression level of SIRT1 in vivo and is used for resisting aging. In addition, the invention also provides a medicinal preparation of the agonist, a preparation method, application in preparing medicaments and the like.

In particular, in a first aspect, the invention provides a SIRT1 agonist which is a crystalline product of Z018A. As used herein, the term "SIRT 1 agonist" refers to a substance that increases the level of expression of SIRT1 in vivo. The substance may be a compound or a composition. The SIRT1 agonist of the first aspect of the invention is named as an HPLC detection map, comprises three components and is a composition.

Herein, the term "Z018A" refers to the refined extract of licorice described in Chinese patent ZL201610169121.8, which can be prepared by the following method:

(1) soaking Glycyrrhrizae radix in water, and retaining solid part;

(2) leaching the solid fraction obtained in step (1) with an alcohol having a concentration greater than 85% (V/V), retaining the liquid fraction and drying;

(3) dissolving the dried product obtained in the step (2) by using alcohol with the concentration of 50-57% (V/V), loading the dissolved product to a macroporous adsorption resin chromatographic column, collecting eluent eluted by using alcohol with the concentration of 59-70% (V/V), and drying the eluent; and the combination of (a) and (b),

(4) and (4) dissolving the dried product obtained in the step (3) by using alcohol with the concentration of 30-60% (V/V), loading the dissolved product to a polyamide resin chromatographic column, eluting the polyamide resin chromatographic column by using alcohol with the concentration of 30-60% (V/V), and collecting the eluent.

Preferably in the process for the preparation of Z018A, the alcohol is methanol and/or ethanol, preferably ethanol.

It is also preferred that in the process for the preparation of Z018A, in step (2), the concentration is greater than 90% (V/V), preferably greater than 93% (V/V), such as 95% (V/V).

It is also preferable that in the preparation method of Z018A, in step (3), the concentration of the dissolved alcohol is 53-56% (V/V), such as 55% (V/V); and/or the concentration of the eluted alcohol is 60-62% (V/V), such as 60% (V/V).

It is also preferable that in the process for preparing Z018A, the concentration in step (4) is 40 to 50% (V/V), preferably 43 to 48% (V/V), such as 45% (V/V).

Preferably the agonist of the first aspect of the invention is the ethanol crystalline product of Z018A. That is, the agonist of the first aspect of the present invention is a product obtained by crystallizing Z018A in a solvent containing ethanol and then filtering. In a specific embodiment of the invention, the content of ethanol is 50-55% (V/V).

Preferably, the agonist of the first aspect of the invention may be prepared by a method comprising the steps of:

(5) after the preparation step (4) of Z018A, concentrating the eluate obtained in the step (4), preferably to 5-20 times, preferably to 10 times, the volume of the eluate; and the combination of (a) and (b),

(6) and (3) adding ethanol into the concentrated solution obtained in the step (5) to enable the concentration of the ethanol to be 50-55% (V/V), standing for crystallization, filtering to collect a crystallization product, optionally washing with the ethanol with the concentration of 15-25% (V/V) and drying.

The agonist of the first aspect of the invention is named "ZAG 019" and comprises three components. From the HPLC detection profile, the agonist of the first aspect of the present invention has three characteristic peaks, which have the same retention time as the three characteristic peaks in the HPLC detection profile of Z018A, respectively, but different peak areas.

Preferably, the agonist of the first aspect of the invention has an HPLC profile with three characteristic peaks as shown in figure 1. More preferably, the ratio of the three characteristic peaks is also stable, for example, as shown in fig. 1, the ratio of the peak areas of the three characteristic peaks from left to right is 0.9 to 1.1: 20-24: 900-1100, in the ratio of 1: 22: 977 or so.

In a second aspect, the present invention provides a process for the preparation of an agonist of the first aspect of the invention comprising the steps of concentrating, crystallizing and optionally washing a solution comprising Z018A.

Preferably the method of the second aspect of the invention comprises:

More preferably, in the production method of the second aspect of the present invention, in the step (6), the concentration of ethanol used for washing is 20% (V/V).

In a third aspect, the invention provides a pharmaceutical formulation comprising an agonist of the first aspect of the invention and a pharmaceutically acceptable excipient. Herein, the term "pharmaceutically acceptable excipient" includes pharmaceutically acceptable carriers, excipients, diluents and the like, which are compatible with the pharmaceutically active ingredient. The use of pharmaceutically acceptable excipients for the preparation of pharmaceutical preparations is well known to those skilled in the art. The pharmaceutical preparation of the present invention comprises the composition of the first aspect of the present invention as an active ingredient, and the composition is combined with pharmaceutically acceptable adjuvants (such as carriers, excipients, diluents, flavoring agents, etc. well known to those of ordinary skill in the art) to prepare various preparations, preferably solid preparations and liquid preparations, such as tablets, pills, capsules (including sustained release or delayed release forms), powders (such as lyophilized powders), suspensions, granules, tinctures, syrups, emulsions, suspensions, injections, etc., and various sustained release forms, so as to be suitable for various administration forms, such as oral administration, parenteral injection, mucosal administration, intramuscular administration, intravenous administration, subcutaneous administration, intraocular administration, intradermal administration, or transdermal administration, etc., and most preferably oral administration. In a particular embodiment of the invention, the pharmaceutical formulation of the third aspect of the invention is a liquid oral formulation.

In a fourth aspect, the invention provides the use of a SIRT1 agonist of the first aspect of the invention in the manufacture of a medicament for increasing the level of SIRT1 expression in vivo. The subject whose expression level is elevated is preferably an aging subject. In this context, the subject may be a mammal, such as a mouse, preferably a human.

In a fifth aspect, the invention provides the use of a SIRT1 agonist of the first aspect of the invention in the manufacture of a medicament for anti-aging.

The invention has the beneficial effects that: the invention provides a novel SIRT1 agonist, which can obviously improve the expression quantity of SIRT1 in vivo and is used for resisting aging; in addition, the SIRT1 agonist has stable quality, and the quality characteristics are hardly influenced by the liquorice source.

The present invention incorporates prior publications which are intended to describe the invention more clearly and are incorporated herein in their entirety as if they were repeated herein.

For the purpose of facilitating understanding, the present invention will be described below with reference to specific drawings and examples. It is to be expressly understood that the description is illustrative only and is not intended as a definition of the limits of the invention. Other technical schemes of the invention can also be obtained by using the method of the embodiment of the invention. Many variations and modifications of the present invention will be apparent to those skilled in the art in light of the teachings of this specification.

Drawings

Fig. 1 shows the HPLC detection results of ZAG019 of the invention.

FIG. 2 shows a set of western blot photographs of the effect of each drug on D-galactose senescence model rat SIRT1, grouped as: normal Control group (Control), Model group (Model), Silibinin Control group (Silibinin (100mg/kg)), ZAG019 low dose group (ZAG019(5mg/kg)) and ZAG019 high dose group (ZAG019(15 mg/kg)).

FIG. 3 shows the statistical results of the effect of each drug on D-galactose senescence model rat SIRT1, grouped as: normal Control group (Control), Model group (Model), Silibinin Control group (Silibinin (100mg/kg)), ZAG019 low dose group (ZAG019(5mg/kg)) and ZAG019 high dose group (ZAG019(15 mg/kg)); p<0.01, relative to a normal control group;^#p<0.05 and^##p<0.01, relative to the model set.

Detailed Description

The plant materials and chemical agents in the specific examples are conventional materials purchased from commercial sources, wherein pharmacological experiments follow the relevant animal experimental guidelines of CFDA.

Example 1 preparation of ZAG019

Basically, the preparation method is carried out by referring to the method for preparing Z018A in the Chinese patent ZL201610169121.8, except that polyamide resin chromatographic column eluent is collected, decompressed and concentrated at 60 ℃ until the volume of the collected eluent is one tenth of the volume of the eluent, ethanol is added until the final concentration of the ethanol is 50-55% (V/V), the mixture is kept stand for crystallization, crystals are collected by filtration, washed by 20% (V/V) ethanol, and dried to obtain ZAG 019.

A small amount of ZAG019 was dissolved in methanol and subjected to HPLC detection, using a Diamonsil C18 column (4.6 mm. times.250 mm, 5 μm) at 25 ℃ and a methanol-0.2% phosphoric acid aqueous solution as a mobile phase at a flow rate of 0.8mL/min and a detection wavelength of 379nm, and gradient elution was carried out under the conditions described in Table 1.

The different liquorice sources and batches of liquorice are refined, the yield of the ZAG019 obtained by the method is quite different, but the quality is stable, HPLC detection results are basically shown in figure 1, three characteristic peaks still appear, and the ratio of the peak areas of the three peaks from left to right is about 1: 22: 977.

table 1: gradient elution chart

Example 2 ZAG019 anti-aging experiments

ZAG019 prepared according to the preparation method of example 1 was administered to aging model rats, and the effect on SIRT1 was studied.

1, Experimental drugs

High and low dose ZAG 019: ZAG019 was formulated in 0.5% CMC-Na solution to give 1.5mg/ml and 0.5mg/ml solutions, respectively.

Silibinin (Silibinin): silybin is prepared into a solution with the concentration of 10mg/ml by 0.5 percent of CMC-Na.

2, experimental animals

Wistar rats, male, weighing 180-. A breeding environment: SPF grade animal house, free feeding, 12h light/12 h dark.

3, procedure of experiment

After 50 wistar rats were acclimatized for one week, they were randomly divided into a normal Control group (Control), a Model group (Model), a Silibinin Control group (Silibinin (100mg/kg)), a ZAG019 low dose group (ZAG019(5mg/kg)) and a ZAG019 high dose group (ZAG019(15mg/kg)) by weight, and 10 rats were administered to each group. Except for the normal control group, the neck and the back of the rats of the other 4 groups are injected with 120mg/kg of D-galactose subcutaneously; normal control group injected equal volume of normal saline subcutaneously; the rats of the normal control group and the model group are gavaged with about 0.2ml of 0.5% CMC-Na solution; the rats of the silibinin control group, ZAG019 low dose group and ZAG019 high dose group were administered with 100mg/kg silibinin, 5mg/kg ZAG019 and 15mg/kg ZAG019 by gavage respectively, and the injections and gavage were administered 1 time per day for 56 days. Rats were fasted without water deprivation after the last dose, sacrificed 16 hours later, and rat liver tissues were taken for western blot detection of SIRT1 and the SIRT1 expression level (GAPDH as a reference) of each group was calculated.

4, results of the experiment

The results are shown in fig. 2 and 3, and D-galactose administration significantly reduced SIRT1 expression levels; compared with a model group, each administration group has an agonistic effect on SIRT1, promotes the increase of SIRT1 expression, and has significant difference (p <0.01 or p <0.05) compared with the model group, wherein, the agonistic effect of ZAG019 on SIRT is in positive correlation with the dosage, but the effect of ZAG019 with low dosage exceeds the effect of the positive medicament silybin.

Claims

Use of a SIRT1 agonist for the preparation of a medicament for increasing the expression level of SIRT1 in wistar rats with significantly reduced SIRT1 expression level due to D-galactose administration, wherein the HPLC detection profile of the agonist has three characteristic peaks as shown in fig. 1, and the ratio of the peak areas of the three characteristic peaks from left to right is 0.9-1.1: 20-24: 900-1100, and the agonist is prepared by a method comprising the following steps:

(1) soaking Glycyrrhrizae radix in water, and retaining solid part;

(2) leaching the solid fraction obtained in step (1) with ethanol at a concentration greater than 85% (V/V), and retaining the liquid fraction and drying;

(3) dissolving the dried product obtained in the step (2) by using 53-56% (V/V) ethanol, loading the dissolved product to a macroporous adsorption resin chromatographic column, collecting eluent eluted by using 60-62% (V/V) ethanol, and drying the eluent;

(4) dissolving the dried product obtained in the step (3) by using ethanol with the concentration of 43-48% (V/V), loading the dissolved product to a polyamide resin chromatographic column, eluting the polyamide resin chromatographic column by using ethanol with the concentration of 43-48% (V/V), and collecting eluent;

(5) concentrating the eluent obtained in the step (4) at 60 ℃ under reduced pressure to 5-20 times of the volume of the eluent; and the combination of (a) and (b),

(6) adding ethanol into the concentrated solution obtained in the step (5) to enable the concentration of the ethanol to be 50-55% (V/V), standing for crystallization, filtering and collecting a crystallization product, washing with the ethanol with the concentration of 15-25% (V/V), and drying;

the SIRT1 agonist is prepared into a solution with the concentration of 0.5mg/ml, and the administration dosage is 5 mg/kg; or the SIRT1 agonist is prepared into a solution with the concentration of 1.5mg/ml, and the administration dosage is 15 mg/kg.
2. The use of claim 1, wherein the ratio of the peak areas of the three characteristic peaks from left to right is 1: 22: 977.
3. the use according to claim 1, wherein, in step (5), the eluate obtained in step (4) is concentrated at 60 ℃ under reduced pressure to 10 times the volume of the eluate.
4. The use of claim 1, wherein the medicament comprises the agonist and a pharmaceutically acceptable excipient.
5. The use of claim 4, wherein the medicament is an oral formulation.