Flavonolignan glycoside for preparing PTR1 inhibitor as well as preparation method and application of flavonolignan glycoside

Abstract

The invention discloses flavonolignan glycoside and pharmaceutically acceptable salts thereof. The flavonolignan glycoside is shown as a structural formula I, the invention also discloses a preparation method of the flavonolignan glycoside and an application of the flavonolignan glycoside in preparation of a pteridine reductase-1 inhibitor. The flavonolignan glycoside provided by the invention can effectively inhibit the activity of pteridine reductase-1, and can be used for preparing medicines for relieving leishmaniasis.

Description

Technical Field

[0001] The present invention relates to the field of pteridine reductase-1 inhibitors, and more specifically, to a flavonolignan glycoside for preparing a PTR1 inhibitor, a preparation method and an application thereof. Background Art

[0002] Leishmania is the causative agent of various forms of leishmaniasis infection. The life cycle of Leishmania has two stages, the first stage consisting of a motile, flagellated promastigote stage that is found in the digestive tract of sand fly species, and the second stage being a non-motile amastigote stage that occurs in its mammalian host. Leishmania species are pterin auxotrophs; the parasites take up pterin from the host and reduce it to its dihydro and tetrahydro forms. To this end, the parasite utilizes pteridine reductase-1 (PTR1), an enzyme of the short-chain dehydrogenase family, which can reduce biopterin to its dihydro (H2B) and tetrahydro (H4B) forms, respectively, achieving inhibition of dihydrofolate reductase-thymidylate synthase (DHFR-TS). PTR1 also provides a bypass mechanism for the reduction of folate (conjugated pterin). Therefore, the parasite is resistant to antifolates while ensuring the continued synthesis of thymidine (a component of DNA) required for its survival, which makes PTR1 an important target for the discovery of anti-leishmaniasis drugs. Drug compositions that can inhibit the activity of pteridine reductase-1 have a certain alleviating effect on leishmaniasis.

[0003] Pachycarpus plants are deciduous trees, and their bark can be used to extract tannin, the stem bark fiber can be used to weave coarse cloth, and the wood can be used to make furniture. The medicinal value of Pachycarpus plants is that the bark of Pachycarpus plants has the effects of scavenging free radicals, anti-tumor and inhibiting cancer cells. So far, there has been no report that Pachycarpus plants and their extracts can be used to prepare pteridine reductase-1 inhibitors and alleviate leishmaniasis. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] Another object of the present invention is to provide a flavonolignan glycoside for preparing a pteridine reductase-1 inhibitor, which can effectively inhibit pteridine reductase-1 and is expected to be used in the preparation of a drug for alleviating leishmaniasis.

[0006] Another object of the present invention is to provide a method for preparing the flavonolignan glycosides.

[0007] Another object of the present invention is to provide the use of the flavonolignan glycosides in the preparation of pteridine reductase-1 inhibitors.

[0008] Still another object of the present invention is to provide a pharmaceutical composition comprising the flavonolignan glycosides.

[0009] In order to achieve these purposes and other advantages according to the present invention, a flavonolignan glycoside and a pharmaceutically acceptable salt thereof are provided, wherein the flavonolignan glycoside is as shown in structural formula I;

[0010]

[0011] The present invention further claims a method for preparing flavonolignan glycosides for alleviating leishmaniasis, comprising:

[0012] Step 1, extracting the dried stem bark of Lannea acida A.Rich with an ethanol solution with a volume fraction of 70-90%, filtering the supernatant, and concentrating under reduced pressure to obtain a paste extract, suspending the obtained paste extract with water, and extracting with n-hexane and ethyl acetate once, taking the ethyl acetate extract and separating it by silica gel column chromatography, and eluting with n-hexane-ethyl acetate-methanol gradient to obtain fractions E1-E11;

[0013] Step 2: Separate the obtained fraction E4 by silica gel column chromatography, and use n-hexane-acetone-methanol gradient elution to obtain fractions E4-1 to E4-8;

[0014] Step 3: Use a mixed solution of acetonitrile and water in a mass ratio of 7:3 to purify E4-4 by RP-HPLC to obtain the flavonolignan glycoside.

[0015] Preferably, the extraction time in step 1 is 5 to 9 days, the number of extractions is 2 to 4 times, and the supernatants filtered from multiple extractions are combined and concentrated under reduced pressure.

[0016] The present invention further claims to protect the use of the flavonolignan glycosides and pharmaceutically acceptable salts thereof in the preparation of pteridine reductase-1 inhibitors.

[0017] The present invention further claims protection for a pharmaceutical composition, which comprises the flavonolignan glycoside or a pharmaceutically acceptable salt thereof as claimed in claim 1 and excipients.

[0018] The present invention at least includes the following beneficial effects: The present invention provides flavonolignan glycosides and pharmaceutically acceptable salts thereof, which can effectively inhibit pteridine reductase-1 and can be used to prepare drugs for alleviating leishmaniasis.

[0019] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The flavonolignan glycosides of the present invention 1 H NMR spectrum;

[0021] Figure 2 The flavonolignan glycosides of the present invention 13 C NMR spectrum;

[0022] Figure 3 is the DEPT spectrum of the flavonolignan glycosides described in the present invention;

[0023] Figure 4 The flavonolignan glycosides of the present invention 1 H– 1 H COSY spectrum;

[0024] Figure 5 is the HSQC spectrum of the flavonolignan glycosides described in the present invention;

[0025] Figure 6 is the HMBC spectrum of the flavonolignan glycosides described in the present invention;

[0026] Figure 7 is the NOESY spectrum of the flavonolignan glycosides described in the present invention;

[0027] Figure 8 The flavonolignan glycosides of the present invention 1 Diagram of key related signals of HSQC, HMBC and NOESY spectra. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can implement the invention with reference to the description.

[0029] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0030] The present invention separates a new flavonolignan glycoside from the stem bark of Lannea acida A.Rich plant, which has a structure shown in Formula I:

[0031]

[0032] The following is a specific embodiment of the present application:

[0033] Plant raw materials:

[0034] The stem bark of Lannea acida A.Rich was collected from Agbaragun River in Egbe forest, Yagba West, Kogi State, Nigeria, by the University of Ilorin in January 2020. The material was identified and collected at the Herbarium Unit of the Department of Plant Biology, Forestry Research Institute of Nigeria (FRIN) under the specimen number (UILH / 001 / 1414 / 2019).

[0035] Extraction and separation

[0036] Step 1: Soak the stem bark (10.0 kg) of Lannea acida A. Rich in ethanol: water (90:10) for 7 days, then filter and concentrate under reduced pressure, and repeat the process three times. Suspend the extract (1.2 kg) in water and extract with n-hexane and ethyl acetate in turn. The ethyl acetate portion (300 g) is subjected to silica gel column chromatography and eluted with a gradient solvent system of n-hexane: ethyl acetate: methanol (100:0:0-0:100:0:-0:0:100) to obtain 11 fractions E1-E11. The elution solvent solubility corresponding to each fraction of E1 to E11 is shown in Table 1.

[0037] Table 1 Elution solvent concentration and fractions

[0038]

[0039] Step 2: The E4 fraction was subjected to silica gel column chromatography again, and eluted with a gradient solvent system of n-hexane: acetone: methanol (100:0:0-0:100:0:-0:0:100) to obtain fractions E4-1-E4-8. The elution solvent solubility corresponding to each fraction E4-1-E4-8 is shown in Table 2.

[0040] Table 2 Elution solvent concentration and fractions

[0041]

[0042] Step 3: Use ACN:H2O (7:3) to RP-HPLC purification of E4-4, and the obtained compound is the flavonolignan glycoside (50 mg).

[0043] Characterization of flavonolignan glycosides:

[0044] H NMR spectrum (500MHz, DMSO-d6), C NMR spectrum (125MHz, DMSO-d6), DEPT NMR spectrum, and 2D NMR spectrum of flavonolignan glycosides 1 H– 1 The H COSY spectrum, 2D NMR HSQC spectrum, 2D NMR HMBC spectrum and 2D NMR NOESY spectrum are shown in Figure 1 to Figure 7 As shown, the flavonolignan glycosides 1 H– 1 Key related signals of H COSY, HMBC and NOESY spectra are as follows: Figure 8 As shown, the flavonolignan glycosides 1 H(500MHz) and 13 The C (125 MHz) NMR data are shown in Table 3.

[0045] Table 3 1 H(500MHz) and 13 C (125MHz) NMR data (deuterated methanol)

[0046]

[0047]

[0048] The flavonolignan glycoside prepared by the present invention is a light yellow powder; the melting point is 270-272°C; Figures 1 to 7 As shown, the spectrum information is as follows: Vλ max (logε)229(3.92);

[0049] IR(KBr disk)υ max (cm -1 ):3392(OH),1689(C=O); FAB-MS m / z:593.1[MH] - ; HRFAB-MS m / z: 593.1266[MH] - (calcd.for 593.1295,C 30 H 25 O 13 ).

[0050] like Figure 1 As shown in Table 1, 1 H-NMR (CD3OD, 500MHz) spectrum information is as follows: δ H5.10(d,J=6.0Hz,H-2),4.17(q,J=5.5Hz,H-3),2.91(dd,J=16.5,6.0Hz,H-4a);2.76(dd,J=16.5,5.0Hz,H-4b),6.70(s,H-8),6.72 (d,J=2.0Hz,H-2′),6.70(d,J=8.5Hz,H-5′),6.61(dd,J=8.5,2.0Hz,H-6′),6.75(s,H-3″),7.33(s,H-6″),6.10(s,H-8″),4.51(br s, H-1″′), 3.38 (overlapped, H-2″′), 3.30 (overlapped, H-3″′), 3.11 (t, J=9.5Hz, H-4″′), 3.33 (overlapped, H-5″′), 1.07 (d, J=6.5Hz, H-6″′).

[0051] like Figure 2 As shown in Table 1, 13 C-NMR (CD3OD, 125MHz) spectrum information is as follows: δ C 79.2(C-2),71.0(C-3),23.8(C-4),151.6(C-5),99.7(C-6),148.7(C-7),97.2(C-8),157.5(C-9),102 .9(C-10),128.8(C-1′),113.6(C-2′),145.2(C-3′),145.2(C-4′),115.5(C-5′),117.5(C-6′),106.0( C-1″),152.5(C-2″),103.2(C-3″),146.6(C-4″),143.7(C-5″),109.1(C-6″),141.5(C-7″),92.0(C-8″ ), 161.0(C-9″), 100.1(C-1″′), 70.4(C-2″′), 70.5(C-3″′), 71.8(C-4″′), 69.1(C-5″′), 17.7(C-6″′).

[0052] like Figure 2 As shown in Table 1, the molecular formula was deduced by HRFAB-MS analysis to be C 30 H 26 O 13 ([MH] + m / z 593.1266, calculated value 593.1295C 30 H 25 O 13 ), showing an unsaturation of 18,13 The C NMR spectrum (Table 1) shows 30 carbon atoms, which can be distinguished into 14 quaternary carbons, 14 methines, 1 methylene and 1 methyl by DEPT spectrum. 1 In the H NMR spectrum, seven aromatic hydrogen signals are visible, including one ABX coupling system δ H 6.72 (d, J = 2.0 Hz), 6.70 (d, J = 8.5 Hz), 6.61 (dd, J = 8.5, 2.0 Hz), and 4 single peak signals δ H 7.33,6.75,6.70,6.10. From 1 H NMR spectrum H 5.10 (d, J = 6.0 Hz), 4.17 (q, J = 5.5 Hz), 2.91 (dd, J = 16.5, 6.0 Hz), and 2.76 (dd, J = 16.5, 5.0 Hz) can be inferred that there is a flavan-3-ol ring in compound I. The four hydrogen signals correspond to H-2, H-3, H-4a and H-4b on the C ring of flavan-3-ol, respectively. The COSY correlation signals between them can also confirm this inference. The ABX coupling system should come from the B ring. Combining carbon spectrum and two-dimensional can confirm the existence of catechin fragments (Dubeler et al, Phytochemistry, 1997; Chao et al, J. Agric. Food Chem., 2010). According to the HMBC spectrum with C-9 (δ C 157.5), C-10(δ C 102.9), δ H The single peak at 6.70 can be located at H-8. Based on the H-8 related signal in the HMBC spectrum, δ C 99.7 and Delta C 148.7 were identified as C-6 and C-7, respectively. In addition to the data for the C6-C3-C6 skeleton, 13 More aromatic carbons can be observed in the C NMR spectrum, including tetrasubstituted benzene rings (δ C 106.0, 152.5, 103.2, 146.6, 143.7 and 109.1) and α,β-unsaturated ketones (δC 161.0, 93.5, 142.4), indicating the presence of C6-C3 units similar to the caffeic acid skeleton (Rong et al, Magn. Reson. Chem., 2005). According to the HSQC and HMBC related information, 1 The three remaining aromatic hydrogen singlet signals in the H NMR spectrum are H 7.33, 6.75 and 6.10 are located within the caffeic acid skeleton. H 6.10 and δC The HMBC correlation signal between 99.7 (C-6) confirmed that it was located at H-8″, H-3″ (δ H 6.75) and H-6″(δ H 7.33) is also located by the HMBC long-range correlation signal of the nearby quaternary carbon. The C-2 / C-3 trans relationship in the C ring is determined by H-2 (δ H 5.10) and H-3(δ H 4.17), and the coupling constant was very small in the case of C-2 / C-3 cis relationship. The above data are close to those of muurin A isolated from Brosimum acutifolium (Takashima et al., Planta Med., 2002).

[0053] also, 13 A group of carbon signals (δ C 100.1, 70.4, 70.5, 71.8, 69.1 and 17.7), 1 H NMR spectrum observed δ H The sugar terminal proton signal at 4.51 (br s), δ H The four oxygen-linked methine proton signals at 3.38, 3.33, 3.30, and 3.11 and δ H The methyl signal at 1.07 (d, J = 6.5 Hz) was determined to be L-rhamnose after acid hydrolysis (Dai et al, Molecules, 2023). H 4.51) and Rha-H-2″′)(δ H 3.38), the NOESY correlation indicates that the terminal group configuration is α-configuration (Wang et al, Planta Med., 2014). The HMBC spectrum shows that H-3 (δ H 4.17) and the terminal carbon signal Rha-C-1″′(δ C 100.1), indicating that rhamnose is attached to C-3 (δ C 71.0). Flavonolignan glycoside is a new type of flavonolignan rhamnopyranoside with a highly fused ring. Therefore, the structure of flavonolignan glycoside is inferred to be muurin A-3-O-α-L-rhamnopyranoside.

[0054] According to the mass spectrum and the above-mentioned chromatographic information, the molecular formula was deduced by HRFAB-MS analysis to be C 30 H 26 O 13 ([MH]- The theoretical mass number of C is 593.1295. 30 H 25 O 13 ), the spectrum shows m / z 593.1266[MH] - The molecular formula of the flavonolignan glycoside prepared by the present invention is C 30 H 26 O 13 , which is consistent with the theoretical value.

[0055] Method for determination of pteridine reductase-1 inhibitory activity:

[0056] The resulting flavonolignan glycosides were analyzed for in vitro PTR1 inhibitory activity using a cytochrome-c coupled assay in a buffer containing 20 mM sodium acetate and 20 mM NaCl at pH 4.5 (obtained by the addition of glacial acetic acid). Each test compound was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 500 μM, serially diluted to obtain a range of concentrations (i.e., 3.906, 7.812, 15.625, 31.25, 62.5, and 125 μM), and the diluted test compounds were dispensed into a 96-well plate with a final DMSO level of 5% in all samples (including controls). The plates were then pipetted using a multichannel pipette (Eppendorf plus) The final reaction mixture containing LmPTR1 (5 μM), biopterin (0.35 μM) and cytochrome C (81 μM) was added to each well. Each reaction was performed in triplicate. After addition, the 96-well plate was incubated at 30°C for 10 minutes using an ELISA spectrophotometer, and a pre-reading was obtained at 550 nm. In the second stage, NADPH (final concentration: 500 μM) was added to start the reaction, followed by incubation at 30°C for 40 minutes, with moderate shaking for 1 minute between each reading. The results were converted into inhibition percentage (%), and the results of the determination of pteridine reductase-1 inhibitory activity are shown in Table 4.

[0057] Table 4 Inhibition rate of flavonoid lignans with different solubility on PTR1

[0058]

[0059] The blank control group refers to the sample that is not treated with any inhibitory drugs. The trimethoprim group is used as a positive control. The trimethoprim molecule has a 2,4-diaminopyrimidine skeleton. Molecules with this type of pharmacophore are potent inhibitors of enzymes such as dihydrofolate reductase-thymidylate synthase (DHFR-TS) and pteridine reductase-1 (PTR1). As can be seen from Table 4, flavonolignan glycosides showed a significant inhibitory effect on PTR1. With the increase of flavonolignan glycoside concentration, the inhibition rate of flavonolignan glycosides on pteridine reductase-1 also increased. When the concentration reached 62.5 μM, the inhibition rate of flavonolignan glycosides on pteridine reductase-1 reached 81.45%, even exceeding the inhibition rate of the positive control trimethoprim on pteridine reductase-1 (76.8%). Further, Prism-Graphpad software was used for interpretation to calculate IC 50 Value, flavonolignan glycoside IC 50 The value is 56.43±2.14 μM. Therefore, the flavonolignan glycosides with a concentration greater than 56.43 μM have a significant inhibition rate on pteridine reductase-1.

[0060] Cytotoxicity assay (MTT method):

[0061] Furthermore, we conducted toxicity experiments on flavonolignan glycosides with a concentration greater than 56.43 μM. Considering the rationality of the medical concentration, 100 μM flavonolignan glycosides were selected for toxicity experiments. Normal human BJ cells were obtained from the American Type Culture Collection (ATCC). They were cultured in DMEM medium supplemented with 5% FBS, 100 IU / mL penicillin and 100 μg / mL streptomycin and stored in a 5% CO2 incubator at 37°C. 100 μL / well cell solution (5×10 4 Cells / mL BJ cells) were added to a 96-well plate and incubated at 37°C for 24 hours, and then treated with 100 μM flavonolignan glycosides or negative control DMSO for 48 hours. 20 μL MTT (5 mg / mL) dye was then added to each well, and the plate was incubated at 37°C for 4 hours. After incubation of 100 μL, DMSO was added to each well to dissolve the purple formazan crystals, and then the absorbance was measured at 570 nm using an ELISA plate reader to calculate the growth inhibition rate of BJ cells. Five parallel experiments were designed, and the results of the five parallel experiments are shown in Table 5.

[0062] Table 5 Growth inhibition rate of BJ cells

[0063]

[0064] Generally speaking, if the growth inhibition rate of a drug on normal human BJ cells is less than 50%, it means that the drug is non-toxic. The results of the cytotoxicity experiment showed that flavonolignan glycosides did not show cytotoxicity to normal human BJ cells at the level of 100 μM (the growth inhibition rate of BJ cells was less than 5%). 50 Within the safe concentration above the concentration of 500 mg / kg, flavonolignan glycosides show a significant inhibitory effect on pteridine reductase-1, which is even superior to the inhibitory effect of trimethoprim on pteridine reductase-1. The clinical use of trimethoprim is accompanied by many complications, such as susceptibility to certain infections, slow healing and gingival bleeding. The flavonolignan glycosides provided in the present application are derived from natural herbs, have no side effects, and can be used as a potential candidate for the preparation of pteridine reductase-1 inhibitors.

[0065] In summary, flavonolignan glycosides can be used as a potential candidate for inhibiting pteridine reductase-1, and can be used to prepare pteridine reductase-1 inhibitors and drugs for alleviating leishmaniasis.

[0066] The present invention further claims protection for a pharmaceutical composition, which comprises the flavonolignan glycoside or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition may be in the form of pills, granules, tablets, capsules or injections.

[0067] As described above, according to the present invention, a flavonolignan glycoside for alleviating leishmaniasis is provided, which can effectively inhibit pteridine reductase-1 and can be used to prepare a drug for alleviating leishmaniasis.

[0068] The number of equipment and processing scales described here are used to simplify the description of the present invention. Modifications and variations of the flavonolignan glycosides, preparation methods and uses of the present invention for preparing pteridine reductase-1 inhibitors will be obvious to those skilled in the art.

[0069] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the specific embodiments shown and described herein.

Claims

1. Flavonolignan glycosides and pharmaceutically acceptable salts thereof, characterized in that: The flavonolignan glycosides are shown in structural formula Ⅰ; 2. A method for preparing flavonolignan glycosides and pharmaceutically acceptable salts thereof, characterized in that: include: Step 1, extracting the dried stem bark of Lannea acida A.Rich with an ethanol solution with a volume fraction of 70-90%, filtering and concentrating the supernatant under reduced pressure to obtain a paste extract, suspending the obtained paste extract with water, and extracting with n-hexane and ethyl acetate in sequence, separating the ethyl acetate extract with silica gel column chromatography, and eluting with n-hexane-ethyl acetate-methanol gradient to obtain fractions E1-E11; Step 2: Separate the obtained fraction E4 by silica gel column chromatography, and use n-hexane-acetone-methanol gradient elution to obtain fractions E4-1 to E4-8; Step 3: Use a mixed solution of acetonitrile and water in a mass ratio of 7:3 to purify E4-4 by RP-HPLC to obtain the flavonolignan glycoside.

3. The method for preparing the flavonolignan glycosides and pharmaceutically acceptable salts thereof according to claim 2, characterized in that: In step 1, the extraction time is 5 to 9 days, the number of extractions is 2 to 4 times, and the supernatants filtered from multiple extractions are combined and concentrated under reduced pressure.

4. Use of the flavonolignan glycosides and pharmaceutically acceptable salts thereof as claimed in claim 1 in the preparation of pteridine reductase-1 inhibitors.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the flavonolignan glycoside or a pharmaceutically acceptable salt thereof according to claim 1 and excipients.

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