Use of a chinensis-type triterpenoid compound or its glycoside in the preparation of a hepatoprotective drug

Abstract

The application discloses application of a triperpenoid compound of Ailanthus-type or a glycoside thereof in preparation of a liver-protecting medicine. The application also performs glycosylation modification on the triperpenoid compound of Ailanthus-type to obtain a liver-protecting active triperpenoid glycoside of Ailanthus-type. The compound has potential application value in preparation of a medicine for preventing or treating liver diseases.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of ailanthus triterpenoids or their glycosides in the preparation of hepatoprotective drugs. Background Technology

[0002] Triterpenes are a class of compounds with a wide range of biological and pharmacological activities. The most widely distributed triterpenes in plants are tetracyclic and pentacyclic triterpenes, while ailanthus-type 6-6-5-cyclic triterpenes are relatively rare. To date, more than 50 species of *Ailanthus altissima* and its ailanthus glycosides have been reported in plants (Domingo, V.; Arteaga, JF; Quílez Del Moral, JF; Barrero, A.F. *Unusually Cyclized Triterpenes: Occurrence, Biosynthesis and Chemical Synthesis*. *Nat. Prod. Rep.* 2009, 26, 115–134. *Stonik, VA; Kolesnikova, S. *A. A. *A ...

[0003] In summary, current research on ailanthus-type 6-6-5 cyclic triterpenes and their analogues faces the following limitations and shortcomings: On the one hand, natural resources of ailanthus-type 6-6-5 cyclic triterpenes are scarce, and their chemical synthesis is difficult; on the other hand, research on the cyclases and their genes responsible for the biosynthesis of the ailanthus-type 6-6-5 cyclic triterpenes skeleton is very limited, especially in the application research of enzymatic synthesis of active ailanthus-type 6-6-5 cyclic triterpenes or their analogues. Summary of the Invention

[0004] This invention provides the use of ailanthus triterpenoid compounds or their glycosides in the preparation of drugs for the prevention or treatment of liver diseases. The ailanthus triterpenoids have a structure as shown in Formula 1 or 2.

[0005]

[0006] In some embodiments of the present invention, the glycoside is a monosaccharide or disaccharide of the ailanthus-type triterpenoid compound.

[0007] In some embodiments of the present invention, the glycoside is a glucosinolate of the ailanthus-type triterpenoid compound.

[0008] In some embodiments of the present invention, the glycosides of the ailanthus-type triterpenoid compound are obtained by an enzymatic reaction using glycosyltransferase with the ailanthus-type triterpenoid compound as a glycosyl acceptor. Preferably, the enzymatic reaction uses UDP-glucose as the glycosyl donor.

[0009] In some embodiments of the present invention, the glycosyltransferase used in the enzymatic reaction has an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0010] In some embodiments of the present invention, the glycosides of the ailanthus-type triterpenoid compounds have structures as shown in formula 1a or 1b:

[0011]

[0012] In some embodiments of the present invention, the liver disease is drug-induced liver injury, such as liver injury caused by drugs like acetaminophen.

[0013] Supplementary Explanation

[0014] Unless otherwise stated, the technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. This invention may also be practiced using any methods and materials similar to or equivalent to those described herein. While specific embodiments and preferred methods and materials are described herein, they do not impose any limitation on the invention.

[0015] Beneficial technical effects

[0016] This invention discovers that specific ailanthus triterpenoid compounds have potential application value in the development of drugs for treating liver diseases. Furthermore, this invention employs glycosyltransferases to glycosylate ailanthus triterpenoid compounds, obtaining ailanthus triterpenoid glycosides with hepatoprotective activity, providing new chemical entities for drug development. Attached Figure Description

[0017] Figure 1 HR-SEI-MS spectrum of compound 1a;

[0018] Figure 2 IR spectrum of compound 1a;

[0019] Figure 3 HR-SEI-MS spectrum of compound 1b;

[0020] Figure 4 IR spectrum of compound 1b. Detailed Implementation

[0021] The following specific embodiments are provided to further illustrate various aspects of the present invention. These embodiments are non-limiting and should not be construed as limiting any aspect of the invention. The scope of protection of the present invention is limited only by the claims. Various modifications and improvements can be made to various aspects of the present invention without departing from the scope of the claims, and these modifications and improvements also fall within the scope of protection of the present invention. For example, replacing the promoters and expression vectors used in the embodiments with other promoters and expression vectors commonly used in the art is something that those skilled in the art can understand and implement.

[0022] Additionally, it should be noted that, unless otherwise specified, all materials and reagents used in the following embodiments are commonly used in the art and can be obtained through conventional commercial means; all methods used are conventional methods known to those skilled in the art.

[0023] Example 1: Preparation and structural analysis of glycosylated products

[0024] Using 0.4 mM compound 1 (Arabidiol) as the glycosyl acceptor and 0.8 mM UDP-glucose (UDP-Glc) as the glycosyl donor, a 2 mg / mL purified glycosyltransferase was added, and an in vitro enzymatic reaction was carried out in a 20 mL system in 50 mM Tris-HCl buffer. The reaction was carried out in a 35 °C water bath for 16 h, and the reaction was terminated when the substrate no longer underwent transformation as detected by HPLC. The reaction solution was extracted three times with water-saturated n-butanol, the organic layer was evaporated to dryness under reduced pressure, redissolved in methanol, filtered, and purified by reverse semi-preparative HPLC. Elution conditions: isocratic elution with 100% acetonitrile; flow rate: 3 mL / min; column temperature: 30 °C. The obtained glycosylated product was dissolved in deuterated pyridine and analyzed by 1D NMR and 2D NMR, and IR and ORD were also measured.

[0025] A novel compound 1a was obtained by enzymatic reaction using the glycosyltransferase AmUGTa (with the amino acid sequence shown in SEQ ID NO. 1). The compound was then analyzed by HR-ESI-MS (…). Figure 1 The molecular formula of 1a is determined to be C. 36 H 62 O7. The NMR data were assigned using a combination of 1D and 2D NMR spectra (Table 1), and the structure of 1a was identified as arabidiol3-O-β-D-glucoside (infrared spectrum shown in Table 1). Figure 2 (As shown).

[0026] A novel compound 1b was obtained by enzymatic reaction using the glycosyltransferase AmUGTb (with the amino acid sequence shown in SEQ ID NO.2). The compound was then analyzed by HR-ESI-MS (…). Figure 3The molecular formula of 1b is determined to be C. 42 H 72 O 12 The NMR data were assigned by combining 1D NMR and 2D NMR spectra (Table 1), and the structure of 1b was identified as 3-(2′-O-β-D-glucopyranosy)-O-β-D-glucopyranosyl arabidiol (infrared spectrum shown in Table 1). Figure 4 (As shown).

[0027] Table 1: Compounds 1a and 1b 1 H and 13 C NMR (δinppm, Jin Hz)

[0028]

[0029]

[0030] Example 2: Protective effect against acetaminophen-induced hepatocyte damage in vitro

[0031] The protective effects of ailanthus triterpenoid compounds 1–7 and ailanthus triterpenoid glycosides 1a and 1b against acetaminophen-induced in vitro hepatocyte damage were detected using the MTT assay.

[0032] The structures of compounds 3-7 are shown below:

[0033]

[0034] HepG2 cells were seeded in 96-well cell culture plates and cultured for 24 h. Then, a non-toxic concentration of the test compound and paracetamol (APAP, final concentration 8 mM) were added. A positive control group (glutathione GSH), a solvent blank control group, and a model group were also included. Cells were cultured for another 24 h. The culture medium was discarded, and 100 μl of MTT (0.5 mg / ml) solution was added to each well. Cells were cultured for another 4 h, and the MTT solution was discarded. 150 μl of DMSO was added to each well, and the cells were shaken. The absorbance was measured at 570 nm using a microplate reader. Cell viability (%) = (average OD of the drug-treated group / average OD of the solvent control group) × 100%.

[0035] As shown in Table 2, compounds 2 and 1a have significant hepatoprotective activity and can be used as lead compounds for the development of hepatoprotective drugs.

[0036] Table 2: Results of the hepatoprotective activity of the compounds

[0037]

[0038] Note: *** P < 0.001, ##P<0.01,and # P<0.05。

Claims

1. Use of a Meliaceous triterpene compound or a Meliaceous triterpene compound glycoside in the preparation of a medicament for preventing or treating drug-induced liver injury, characterized in that, The ailanthus triterpenoid compound has the structure shown in Formula 2, and the ailanthus triterpenoid compound glycoside has the structure shown in Formula 1a. 。

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