Arbutin, its polyhydroxy derivatives, their compositions and uses

Abstract

This invention belongs to the field of pharmaceutical technology and discloses arbutin, its polyhydroxy derivatives, their compositions, and uses. Using a microbial transformation method, this invention successfully modified the structure of arbutin, obtaining three novel arbutin derivatives with polyhydroxy substitutions. In vitro neuronal inflammation assays and neuronal oxidative damage protection assays confirmed that these compounds possess good anti-neuroinflammatory and neuronal damage protective activities. Simultaneously, in vivo Alzheimer's disease mouse model experiments demonstrated that these compounds can significantly improve the learning and memory abilities of AD mice, and can serve as active ingredients in drugs for treating neurological diseases. In vitro antitumor cell assays confirmed that compounds I-III possess significant antitumor activity and can serve as active ingredients in antitumor drugs, with broad applications.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to arbutin, its polyhydroxy derivatives, their compositions and uses. Background Technology

[0002] Arbutin, also known as ursolicol, is a natural ursane-type pentacyclic triterpenoid compound widely distributed in olives, including olive leaves, bark, fruit, and extra virgin olive oil. The arbutin content is one of the important indicators for evaluating the quality of olive oil. It can also be isolated from different parts of plants such as bayberry, camellia, oleander, lavender, and apple. Preliminary studies have shown that arbutin possesses various pharmacological activities, including anti-inflammatory, cardiotonic, antiarrhythmic, and antitumor effects, as well as the ability to promote skin wound healing. Currently, there are no reports on the application of arbutin in the treatment of nervous system diseases.

[0003] Furthermore, arbutin's poor solubility, low bioavailability, and limited number of hydrogen bond donors in its parent nucleus and rotatable bonds in its rigid framework restrict its bioactivity in anti-tumor and other disease-related applications. To improve these properties, functional groups need to be introduced onto saturated CH bonds, which is difficult to achieve with conventional chemical modifications. Microbial transformation, however, is a simple and effective technique for modifying the structure of natural compounds, allowing for regioselectivity and stereoselectivity in introducing functional groups, offering advantages such as high efficiency, economy, and environmental friendliness. The transformation products obtained through microbial transformation can yield derivatives with enhanced bioactivity for direct use in drug development. On the other hand, the newly introduced chemically active groups on the parent nucleus after microbial transformation increase the sites for chemical modification and alteration, thus solving the problem of limited reaction sites in the organic chemical preparation of triterpenoid derivatives. However, compared to chemical synthesis, microbial transformation is highly random; the results cannot be predicted as accurately as chemical synthesis, nor can they be designed purposefully. Moreover, even similar substrates transformed by the same or different strains can yield significantly different products. Furthermore, the same substrate transformed by different strains may also yield significantly different transformation products. Therefore, selecting appropriate strains is crucial when using microbial transformation methods to modify the structure of compounds. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide arbutin, its polyhydroxy derivatives, compositions thereof, and uses.

[0005] In a first aspect, the present invention provides an arbutin polyhydroxy derivative represented by any of the following structural formulas:

[0006]

[0007] A second aspect of the present invention provides a method for preparing the above-mentioned arbutin polyhydroxy derivative, comprising the following steps:

[0008] 1) Fermentation culture of microorganisms, after adding arbutin, transformation culture, and removal of mycelium to obtain fermentation broth, wherein the microorganism is Actinomucor elegans CGMCC 3.3393;

[0009] 2) After extracting the fermentation broth with an organic solvent, the extract is evaporated to dryness to obtain the crude extract;

[0010] 3) The crude extract was purified by reversed-phase silica gel column chromatography using a methanol-water two-phase system with gradient elution, and the fractions were collected and combined.

[0011] 4) The components obtained in step 3) are purified by reversed-phase high-performance liquid chromatography to obtain the arbutin polyhydroxy derivative.

[0012] A third aspect of the present invention provides the use of arbutin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating nervous system diseases, wherein the arbutin has the following structural formula:

[0013]

[0014] In a fourth aspect, the present invention provides the use of the above-described arbutin polyhydroxy derivatives or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating nervous system diseases.

[0015] Furthermore, the drug for treating nervous system diseases is a drug that repairs neurological oxidative stress damage.

[0016] Furthermore, the drug used to treat nervous system diseases is an anti-neuroinflammatory drug.

[0017] Furthermore, the drug for treating neurological diseases is a drug for treating Alzheimer's disease.

[0018] In a fifth aspect, the present invention provides the use of the above-mentioned arbutin polyhydroxy derivative in the preparation of an antitumor drug, wherein the tumor includes one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer, and colon cancer.

[0019] Furthermore, the present invention provides the use of the above-mentioned arbutin polyhydroxy derivative in the preparation of a drug for killing drug-resistant leukemia cells.

[0020] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the above-described arbutin polyhydroxy derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0021] Furthermore, the pharmaceutically acceptable excipient is one or more of the following: diluent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, surfactant, adsorbent carrier, and lubricant.

[0022] Compared with existing technologies, this invention utilizes microbial transformation technology to successfully modify the structure of arbutin, obtaining a novel class of polyhydroxy-substituted arbutin derivatives. In vitro neuronal oxidative stress injury protection experiments and microglial cell inflammation experiments confirmed that these compounds possess good neuronal damage repair and anti-neuroinflammatory activities. In vivo AD mouse model experiments demonstrated that the compounds can significantly improve the learning and memory abilities of mice, and can serve as active ingredients in drugs for treating nervous system diseases. In vitro anti-tumor cell experiments confirmed that compounds of formulas I-III possess significant anti-tumor activity and can serve as active ingredients in anti-tumor drugs, with broad applications. Attached Figure Description

[0023] Figure 1 The time the mouse spent in the platform quadrant in Example 4 is represented by the time spent in the platform quadrant. Detailed Implementation

[0024] To further illustrate the present invention, the preparation method of the polyhydroxy arbutin derivative provided by the present invention and its application in the preparation of antitumor drugs are described in detail below with reference to embodiments.

[0025] Example 1: Preparation of compounds with structural formulas I-III

[0026] This invention employs a microbial transformation method, using arbutin as a raw material, to prepare the compound of this invention through fermentation, extraction, and separation. *Actinomucor elegans* CGMCC 3.3393 was purchased from the Chinese Academy of Sciences Microbial Culture Collection Center (CGMCC), and stored on potato slant agar at 4°C.

[0027] Taking *Actinomucor elegans* CGMCC 3.3393 as an example, the process for preparing compounds with structural formulas I-III is as follows:

[0028] 1) Fermentation, conversion and extraction

[0029] *Actinomucor elegans* CGMCC 3.3393 was inoculated into a 250 mL Erlenmeyer flask (containing 100 mL of potato broth) as a seed culture. After shaking and incubating for 12 hours at 160 rpm and 26 °C, when the mycelial growth was vigorous, 1 mL of the seed culture was added to 20 1000 mL shake flasks (containing 400 mL of potato broth). After shaking and incubating for 24 hours, 25 mg of arbutin (0.25 mL, 100 mg / mL ethanol solution) was added to each shake flask, for a total of 500 mg of substrate. Transformation was continued for 7 days under the same conditions. The fermentation broth was filtered to remove the mycelium, and the filtrate was extracted three times with an equal volume of ethyl acetate. The extract was concentrated to dryness under reduced pressure to obtain approximately 1.05 g of crude transformant.

[0030] 2) ODS-C18 column chromatography separation

[0031] The crude extract was separated by ODS-C18 column chromatography. Methanol:water gradient elution (20:80, 30:70, 60:40, 80:20, 100:0). Fractions were collected, analyzed by HPLC, and combined to obtain combined fractions Fr.1-Fr.5.

[0032] 3) Reversed-phase high-performance liquid chromatography purification

[0033] Component Fr.2 was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The preparative conditions were: a YMC ODS A-5 μm column (12.0 × 250 mm), acetonitrile-water (40:60, V / V), flow rate 3.0 mL / min, and detection wavelength 203 nm. Transformation products with structures of formulas I, II, and III were obtained. The mass spectrometry and wavelength spectrometry data for compounds of formulas I–III are shown below.

[0034] Compound I: 7β-hydroxy-21-carbonylarbutin; optical rotation High-resolution mass spectrometry m / z 495.3442 [M+Na] + (ca l cd.C 30 H 48 O4Na, 495.3450); the proton and carbon NMR spectra are shown in Table 1.

[0035] Compound II: 7β,21β,23-Trihydroxyarbutin; Optical rotation High-resolution mass spectrometry m / z 513.3560 [M+Na] + (ca l cd.C 30 H 50 O5Na, 513.3556); the proton and carbon NMR spectra are shown in Table 1.

[0036] Compound III: 7β,21β-dihydroxy-28-acetylarbutin; optical rotation High-resolution mass spectrometry m / z 539.3719, [M+Na] + (ca l cd.C 32 H 52 O5Na, 539.3712); the proton and carbon NMR spectra are shown in Table 1.

[0037] Table 1. 1H and 1C NMR spectra of compounds I through III

[0038]

[0039]

[0040]

[0041] The above results indicate that the structures of the obtained compounds, formulas I through III, are correct.

[0042] Example 2: The repair and protective activities of compounds I-III on nerve cells damaged by hydrogen peroxide.

[0043] 1) Experimental materials

[0044] CO2 incubator (Jouan IGO150); ELISA reader (Bio-TEK ELx800); inverted fluorescence microscope (Olympus IX51); MTT cell proliferation and cytotoxicity assay kit (Beyotime Biotechnology Research Institute); DMEM high glucose medium (Gibco l BRL); fetal bovine serum; dimethyl sulfoxide (DMSO); trypsin (Shanghai Bioengineering Co., Ltd.); 30% hydrogen peroxide (H2O2) (Tianjin Ruijinte Chemical Co., Ltd.); PC-12 cells (Cancer Institute, Chinese Academy of Medical Sciences).

[0045] 2) Experimental methods

[0046] The effect of each test compound on the viability of H2O2-damaged PC-12 cells was determined using the MTT assay: cells were digested with trypsin and counted, and the cell density of the cell suspension was adjusted to 5 × 10⁻⁶. 4Cells were cultured at a concentration of 10 cells / mL, with 200 μL added to each well of a 96-well plate. The plates were then incubated in a 5% CO2 incubator at 37°C for 12 h. After cell attachment, the cells were divided into three groups: control group, model group (damaged with 700 μmol / L H2O2 for 4 h), and model + test compound (1, 5, 10 μM) groups. Each well had a final volume of 200 μL, and each concentration was tested in triplicate. After 24 h of drug treatment, 10 μL of MTT solution (5 mg / mL, i.e., 0.5% MTT) was added to each well, and the plates were incubated for another 4 h. The absorbance of each well was measured at 490 nm using a microplate reader, and cell viability was calculated as: Cell viability = OD value of drug-treated group / OD value of control group.

[0047] 3) Experimental Results

[0048] Based on the MTT assay results, the effects of arbutin and compounds I–III of this invention on the survival rate of PC-12 cells damaged by H2O2 were calculated and are shown in Table 2.

[0049] Table 2. Effects of test samples on the survival rate of PC-12 cells damaged by H2O2.

[0050]

[0051] (Compared with the model group, * P < 0.05, ** P<0.01, *** P<0.001)

[0052] Compared with the control group, the cell survival rate of the H2O2 treatment group was significantly reduced, indicating that the cell model was successful. Compared with the H2O2 treatment group, donepezil hydrochloride (positive control drug) and compounds I-III can significantly improve cell survival rate, indicating that compounds I-III of the present invention have significant nerve cell repair effects and show a good dose-dependent relationship within a certain dose range, and can be used as active ingredients for treating neurooxidative damage.

[0053] Example 3: Anti-neuroinflammatory activity of compounds of formulas I-III of the present invention

[0054] 1) Experimental materials

[0055] Instruments and reagents: CO2 incubator (Jouan I GO150); microplate reader (Bi o-TEK ELx800); fluorescence inverted microscope (Olympus IX51); MTT cell proliferation and cytotoxicity assay kit (Beyotime Biotechnology Research Institute); RPM I 1640 medium (Gibcol BRL); microglia BV-2, RNase A, fetal bovine serum, dimethyl sulfoxide (DMSO), trypsin (Shanghai Bioengineering Co., Ltd.).

[0056] Test samples: arbutin and compounds I–III synthesized in Example 1, with a purity of over 95%; L-monomethylarginine (L-NMMA) was selected as a positive control drug, and all compounds were dissolved in DMSO and then diluted.

[0057] 2) Experimental methods

[0058] The effect of each tested compound on the viability of BV-2 microglia was determined using the MTT assay: BV-2 cells in logarithmic growth phase were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution, and the cell concentration was adjusted to 5 × 10⁶ cells / year. 4 Cells were seeded at a density of 100 μL / mL in 96-well culture plates. For the drug treatment group and the cell control group, 100 μL of cell suspension was added to each well, with three replicates per group. The blank control group received only 100 μL of DMEM complete culture medium per well, with three replicates. The 96-well culture plates were incubated at 37°C in a 5% CO2 incubator for 24 h. Then, different concentrations of the test sample were added to achieve a final concentration of 0.1-100 μM, and the plates were incubated for another 72 h. The absorbance (A) at 490 nm was measured using the MTT assay, and the inhibition rate was calculated [Inhibition rate = (1 - A value of experimental group / A value of control group) × 100%].

[0059] The effect of each test compound on LPS-induced NO release in BV-2 cells was determined using the Griess method: the cell concentration was adjusted to 2 × 10⁻⁶. 5 Cells were seeded at a density of 1 mL / mL in 96-well plates, with 3 replicates per well. The blank control group received only DMEM culture medium and also had 3 replicates. After incubating the 96-well plates at 37°C and 5% CO2 for 24 hours, different concentrations of the test sample were added to achieve a final concentration of 0.1-100 μM. After further incubation for 12 hours, the supernatant was collected, and the NO level in the culture medium was measured according to the kit instructions. Data were analyzed using SPSS Statistical 25 software, and the half-maximal inhibitory concentration (IC50) for each test sample was calculated. 50 ).

[0060] 3) Experimental Results

[0061] Based on the results of the MTT and Griess assays, the effects of arbutin and compounds I–III of this invention on LPS-induced NO release in BV-2 cells were calculated, and the results are shown in Table 3.

[0062] Table 3. Results of test samples inhibiting LPS-induced NO release in BV-2 cells

[0063] compound <![CDATA[IC 50 (μM)]]> Cell viability (%) L-NMMA 25.15±1.32 101.33±2.61 Arbutin 15.11±1.10 92.90±1.03 Compound I 16.23±1.43 89.49±1.02 Compound II 8.84±1.03 97.45±1.10 Compound III 4.94±1.92 104.22±2.07

[0064] The results showed that compounds I–III of the present invention did not have a significant inhibitory effect on BV-2 cells, but could significantly reduce the release level of the inflammatory factor NO induced by LPS in BV-2 cells, and had good anti-neuroinflammatory activity. Their anti-neuroinflammatory activity was also significantly better than that of the positive control L-NMMA, and they can be used as active ingredients of anti-neuroinflammatory drugs.

[0065] Example 4: Effects of the present invention's compounds arbutin and compounds I–III on the learning and memory abilities of an AD mouse model.

[0066] 1) Establishment of AD mouse model

[0067] SPF-grade male C57BL / 6 mice, weighing 22–25 g, were randomly divided into 6 groups: a control group, a model group (D-gal (150 mg / kg) + NaNO2 (70 mg / kg) + AlCl3 (40 mg / kg)), an arbutin group (10 mg / kg), compound I group (2 mg / kg), compound II group (2 mg / kg), and compound III group (2 mg / kg). All drug powders were dispersed in 0.05% sodium carboxymethyl cellulose (CMC-Na). Except for the control group, mice in the other groups were subcutaneously injected with D-gal and NaNO2 and orally administered AlCl3 by gavage for 8 consecutive weeks to simulate the aging process (the normal group received an equal volume of 0.05% CMC-Na). Four weeks after modeling, all mice were administered arbutin and compounds I–III by gavage at a dose of 10 or 2 mg / kg daily for another 4 weeks.

[0068] 2) Experimental methods

[0069] The water maze laboratory experiment forces experimental animals to swim and find platforms hidden in the water. The animals need to memorize spatial location and orientation to locate the platforms. The learning and memory abilities of the animals are assessed by testing the time it takes to find the platforms. This invention uses a circular pool with a diameter of 1 meter and a water temperature of 20°C. A platform with a diameter of 8 cm is placed in the pool, 0.9 cm below the water surface. Model mice are trained in the pool for 4 days before the experiment. The mice are placed in the water with their heads facing the pool wall, randomly in one of the four cardinal directions (north, south, east, or west). The time it takes for the animal to find the platform is recorded within 60 seconds. If it takes longer than 60 seconds, the animal is guided to the platform and stays there for 10 seconds. Each animal is trained 5 times a day, with 30-minute intervals between training sessions. After the last training session, the platform is removed, and the animal is placed back in the pool. The number of times and the time taken to enter and stay in the quadrant where the platform was originally placed are recorded within 120 seconds. Each test group has 4 replicates.

[0070] (3) Experimental Results

[0071] Compared with the control group, the model group mice spent significantly less time in the plateau quadrant, while the arbutin group and the compound I–III groups spent significantly more time there. The results are as follows: Figure 1 As shown. The results indicate that arbutin and compounds I–III of the present invention can significantly improve the learning and memory abilities of mice and can be used as active ingredients in drugs for treating nervous system diseases.

[0072] Example 5: Effects of the present invention's compounds arbutin and compounds I–III on the learning and memory abilities of an AD mouse model.

[0073] 1) Experimental materials

[0074] Instruments and reagents: CO2 incubator (Jouan I GO150); fluorescence inverted microscope (Olympus IX51); MTT cell proliferation and cytotoxicity assay kit (Beyotime Biotechnology Research Institute), RPM I 1640 medium (Gibco lBRL), RNase A, fetal bovine serum, dimethyl sulfoxide (DMSO), and trypsin (Shanghai Bioengineering Co., Ltd.).

[0075] The tumor cell lines used for testing were: HeLa cells (human cervical cancer cells), K562 cells (human leukemia cells), K562 / ADR cells (human drug-resistant leukemia cells), SH-SY5Y cells (human neuroblastoma cells), Du-145 cells (human prostate cancer cells), HePG2 cells (human liver cancer cells), MCF-7 cells (human breast cancer cells), and CT26 cells (colon cancer cells), purchased from the Cancer Institute of the Chinese Academy of Medical Sciences.

[0076] Test samples: arbutin and compounds of formulas I to VI synthesized in Examples 1 and 2, with a purity of over 95%; cisplatin was selected as a positive control drug, and all compounds were dissolved in DMSO and then diluted.

[0077] 2) Experimental methods

[0078] The half-maximal inhibitory rate (IC50) of each tested compound against tumor cell lines was determined using the MTT assay. 50 Value: Tumor cells in the logarithmic growth phase were collected and their concentration adjusted to 5 × 10⁻⁶ cells using RPM I 1640 culture medium containing 10% fetal bovine serum. 5Cells were seeded in 96-well plates at a concentration of 100 μL / well. The drug treatment group and cell control group received 100 μL of cell suspension per well, with three replicates per group. The blank control group received only 100 μL of RPM I 1640 complete culture medium per well, also with three replicates. After incubating the 96-well plates at 37°C and 5% CO2 for 24 h, different concentrations of the test sample were added to achieve a final concentration of 0.1-100 μM, and incubation continued for another 72 h. The absorbance (A) at 570 nm was measured using the MTT assay, and the inhibition rate was calculated [Inhibition rate = (1 - A value of experimental group / A value of control group) × 100%]. The experiment was repeated three times. SPSS 11.5 software was used to perform regression equations to calculate the half-maximal inhibitory concentration (IC50) of each test sample against tumor cells after 72 h. 50 ).

[0079] 3) Experimental Results

[0080] Based on the MTT assay results, the IC50 of the test sample for the above-mentioned cells was calculated. 50 The values ​​are shown in Table 2.

[0081] Table 2. Screening results of in vitro cytotoxic activity of test samples

[0082]

[0083] The results show that the compounds represented by formulas I to III of this invention have good antitumor activity and can be used as active ingredients in antitumor drugs. Simultaneously, the compounds represented by formulas I to III of this invention can effectively kill drug-resistant human leukemia cells.

Claims

1. A bear fruit alcohol polyhydroxy derivative represented by any one of the following structural formulae:

2. A method for preparing the urocanic acid polyhydroxy derivative of claim 1, characterized by, The preparation method comprises the following steps: 1) fermenting and culturing microorganisms, adding bear fruit alcohol for conversion culture, removing mycelium to obtain a fermentation liquor, wherein the microorganism is Actinomucor elegans CGMCC 3.3393; 2) after the fermentation liquor is extracted by an organic solvent, the extract is evaporated to dryness to obtain a conversion crude extract; 3) the conversion crude extract is purified by reverse-phase silica gel column chromatography, the reverse-phase silica gel column chromatography adopts a methanol-water two-phase system gradient elution, and the combined components are collected; 4) the combined components obtained in step 3) are purified by reverse-phase high-performance liquid chromatography to obtain the bear fruit alcohol polyhydroxy derivative. 3.Use of the bear fruit alcohol polyhydroxy derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for treating nervous system diseases.

4. Use according to claim 3, characterized in that, The drug for treating nervous system diseases is a drug for repairing oxidative stress damage of nerves.

5. Use according to claim 3, characterized in that, The drug for treating nervous system diseases is an anti-neuroinflammatory drug.

6. Use according to claim 3, characterized in that, The drug for treating nervous system diseases is a drug for treating Alzheimer's disease. 7.Use of the bear fruit alcohol polyhydroxy derivative according to claim 1 in the preparation of an anti-tumor drug, wherein the tumor is selected from one of cervical cancer, leukemia, neuroblastoma, prostate cancer, liver cancer, breast cancer and colon cancer. 8.Use of the bear fruit alcohol polyhydroxy derivative according to claim 1 in the preparation of a drug for killing leukemia drug-resistant cells. 9.A pharmaceutical composition comprising the bear fruit alcohol polyhydroxy derivative or a pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutically acceptable excipient.

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