A cucurbitane-type triterpenoid compound and its preparation and application

Abstract

The invention discloses a novel tannic acid-lowering hyacinthane-type triterpenoid compound and its preparation and application. The compound is made of medicinal watermelon as raw material, and a cold soaking extraction method is adopted to concentrate the medicinal watermelon extract, and a silica gel and ODS-C 18 The target compound was prepared by HPLC. 1 H-NMR, 13 The compound was structurally identified by spectral analysis methods such as C-NMR, 2D NMR, and high-resolution mass spectrometry, and an octacorucane-type triterpenoid compound represented by formula (I) was obtained. The compound has a novel structure and an IC50 value of 5.7 μM for acetylcholinesterase. The extraction and separation method is simple, which facilitates further pharmacological research on the compound and creates conditions for the development of a new type of anti-Alzheimer's disease drug with high efficiency and low toxicity.

Description

(I) Technical field

[0001] The present invention relates to the field of extraction, separation and preparation of effective components of natural products, and relates to a method for extracting and separating new cucurbitane-type triterpenoid compounds from medicinal watermelon materials and applications thereof. (II) Background technology

[0002] Citrullus colocynthis is the fruit of the plant of the genus Citrullus in the family Cucurbitaceae. It is a hairy climbing vine plant that lives in desert areas. It is also called bitter watermelon and bitter apple. This medicinal plant is traditionally used to treat diabetes, jaundice, asthma, cancer, gastrointestinal diseases, etc. The main reported components in Citrullus colocynthis are cucurbitane-type triterpenoids. Cucurbitane-type triterpenoids have a 19-losing carbon-9β-methyl-10α-lanosterene-5 skeleton and are highly oxidized tetracyclic triterpenoids. At present, there are only more than 20 types of cucurbitane-type triterpenoids isolated from Citrullus colocynthis. Cucurbitane-type triterpenoids are the main active ingredients that exert pharmacological activity. It is reported that cucurbitacin B and E have inhibitory activity against various tumor cells such as human leukemia cells. Zheng et al. found that cucurbitacin B inhibited SH-SY5Y human neuroblastoma tumor cells; Marzouk et al. reported that a cucurbitacin E glucoside had significant in vivo anti-inflammatory and analgesic activity.

[0003] Chen et al. reported that two cucurbitacins A and B were separated from Hemsleya endecaphylla, but no other reports on cucurbitacin-type triterpenoid compounds with C-17 side chain missing were found. The present invention separates a new cucurbitacin-type triterpenoid compound from medicinal watermelon. The compound has a novel structure and is a rare cucurbitacin-type triterpenoid compound with C-17 side chain missing. (III) Summary of the invention

[0004] The present invention aims to provide a triterpenoid compound of the cucurbitane type and its preparation method and its application in the preparation of anti-Alzheimer's disease drugs. The compound is made from medicinal watermelon fruit, extracted by solvent, silica gel and ODS-C 18 Column chromatography and preparative high performance liquid chromatography are used for separation to obtain a new norcarbazine triterpenoid compound represented by formula (Ⅰ).

[0005] The technical solution adopted by the present invention is:

[0006] In the first aspect, the present invention provides a cucurbitane-type triterpenoid compound represented by formula (I):

[0007]

[0008] In a second aspect, the present invention provides a method for preparing a cucurbitane-type triterpenoid compound represented by formula (I), the method comprising the following steps:

[0009] (1) Extraction: The dried fruits of the medicinal watermelon are crushed to obtain medicinal watermelon particles, and the particles are immersed in an ethanol aqueous solution for extraction at room temperature. The extract is filtered, and the filtrate is concentrated under reduced pressure until no liquid flows out, and then dried to obtain a crude extract; the volume of the ethanol aqueous solution is 3-5 mL / g based on the mass of the medicinal watermelon particles; the volume concentration of the ethanol aqueous solution is 80-95%, preferably 95%; the extraction is preferably performed 3 times; and the drying is performed at 60° C. for 5 h;

[0010] (2) Separation:

[0011] a. The crude extract obtained in step (1) is dry-loaded on a silica gel chromatography column (preferably a column height of 70 cm, an inner diameter of 8-12 cm, and a silica gel of 100-200 mesh), and eluted with petroleum ether, a volume ratio of 1:1 petroleum ether: ethyl acetate, and a volume ratio of 5:1 dichloromethane-methanol as eluents, the elution rate is 50-200 mL / min (preferably 150 mL / min), and each eluent is eluted for 3-4 column volumes; each 2.5 L of the effluent is collected as a fraction to obtain 8 fractions, which are respectively recorded as fractions A to H;

[0012] b. Load fraction E onto a silica gel column (preferably 50 cm high, 3-6 cm inner diameter, 200-300 mesh silica gel), and use dichloromethane:methanol as eluents in a volume ratio of 60:1 and 20:1, respectively, for gradient elution. Each eluent is eluted for 3-5 column volumes, and each 700 mL of the effluent is collected as one fraction to obtain 6 fractions, which are recorded as fractions Ea to Ef.

[0013] c. The fraction Ee collected in step b was then subjected to ODS-C 18 Column chromatography (ODS-C 18 The filler particle size is 50 μm, the inner diameter of the chromatography column is 2 to 4 cm), and methanol-water with a volume ratio of 30:70, 50:50, 70:30, and 90:10 is used for elution. Each gradient elution is 1 L, and each 500 mL of the effluent is collected as one fraction, obtaining 8 fractions, which are recorded as fractions Ee-1 to Ee-8.

[0014] d. The fraction Ee-5 was separated by preparative high performance liquid chromatography, eluted isocratically with methanol-water in a volume ratio of 55:45 at a flow rate of 10 mL / min, and the fraction at 26 min was collected and concentrated under reduced pressure and evaporated to dryness to obtain the cucurbitane-type triterpenoid compound represented by formula (I); the chromatographic column model of the preparative high performance liquid chromatography was Shimadzu Shim-Pack GIST, with a particle size of 5 μm, a specification of 20×250 mm, and a detection wavelength of 210 nm.

[0015] In a third aspect, the present invention provides the use of a cucurbitacane-type triterpenoid compound represented by formula (I) in the preparation of an acetylcholinesterase activity inhibitor.

[0016] The present invention provides the use of a cucurbitane-type triterpenoid compound represented by formula (I) in the preparation of an anti-Alzheimer's disease drug.

[0017] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: the compound (I) separated by the present invention has a novel structure, has good anti-acetylcholinesterase inhibitory activity, and its IC 50 The value reaches 5.7μM, and the extraction and separation method is simple, which facilitates further pharmacological research on this compound and creates conditions for the development of new anti-Alzheimer's drugs with high efficiency and low toxicity. (IV) Description of the drawings

[0018] Figure 1 : High resolution mass spectrum of the compound of formula (I).

[0019] Figure 2 : The compound of formula (I) 1 H NMR spectrum.

[0020] Figure 3 : The compound of formula (I) 13 C NMR spectrum.

[0021] Figure 4 : HSQC spectrum of the compound of formula (I).

[0022] Figure 5 : HMBC spectrum of the compound of formula (I).

[0023] Figure 6 : NOSEY spectrum of the compound of formula (I). (V) Specific implementation methods

[0024] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0025] The room temperature in the present invention refers to 25-30° C. The medicinal watermelon (Citrullus colocynthis) used in the present embodiment is the fruit of the plant of the genus Citrullus in the family Cucurbitaceae, a hairy climbing vine plant living in desert areas, also known as bitter watermelon and bitter apple.

[0026] Example 1: Preparation of the compound of formula (I)

[0027] (1) Extraction: 5 kg of dried medicinal watermelon fruits were crushed into coarse powder particles of about 0.5 cm, and extracted three times with 95% ethanol aqueous solution at room temperature, with 25 L of ethanol aqueous solution used each time. The extracts were combined and filtered, and the filtrate was concentrated under reduced pressure until no liquid flowed out, and then dried at 60°C for 5 h to obtain 420 g of crude extract.

[0028] (2) Separation:

[0029] a. The crude extract (420 g) obtained in step (1) was loaded onto a silica gel chromatography column (column height 70 cm, inner diameter 10 cm, silica gel 100-200 mesh, loading amount 1000 g) by dry method, and eluted with petroleum ether, petroleum ether-ethyl acetate with a volume ratio of 1:1, and dichloromethane-methanol with a volume ratio of 5:1, respectively, at a flow rate of 150 mL / min, and three gradient eluents were eluted for 3 column volumes respectively. Each 2.5 L of effluent was collected as a fraction to obtain 8 fractions, recorded as fractions A to H. After reduced pressure concentration and drying at 60° C. for 5 h, fractions A (9.1 g), B (42.9 g), C (25.6 g), D (36.2 g), E (21.5 g), F (27.2 g), G (52.7 g), and H (43.3 g) were obtained respectively.

[0030] b. Fraction E (21.5 g) was then loaded onto a silica gel column (column height 50 cm, inner diameter 5 cm, silica gel 200-300 mesh, filling amount 210 g) for separation, and gradient elution was performed using dichloromethane:methanol with a volume ratio of 60:1 and 20:1 as eluents, respectively. The flow rate was 50 mL / min, and the elution volume of each gradient eluent was 4 column volumes. Every 700 mL of effluent was collected as one fraction, and 6 fractions were obtained, recorded as fractions Ea to Ef. After concentration under reduced pressure and drying at 60°C for 5 h, fractions Ea (1.3 g), Eb (2.6 g), Ec (2.4 g), Ed (4.9 g), Ee (3.2 g) Ef (2.4 g), and Eg (2.9 g) were obtained, respectively.

[0031] c. The fraction Ee (3.2 g) collected in step b was loaded onto ODS-C 18 Chromatographic column (column height 30cm, inner diameter 3cm, filled with 120g ODS-C 18, filler particle size is 50μm), and eluted with methanol-water with volume ratios of 30:70, 50:50, 70:30, and 90:10, respectively. Each gradient elution was 1L, and each 500mL of effluent was collected as one fraction, and 8 fractions were obtained, recorded as fractions Ee-1 to Ee-8. After concentration under reduced pressure, it was dried at 60°C for 3h to obtain 51mg of Ee-1, 32mg of Ee-2, 43mg of Ee-3, 57mgg of Ee-4, 53mg of Ee-5, 26mg of Ee-6, 21mg of Ee-7, and 19mg of Ee-8, respectively.

[0032] d. Fraction Ee-5 was separated using a preparative HPLC column (chromatographic column model: Shimadzu Shim-Pack GIST, 5 μm, 20×250 mm) with methanol-water in a volume ratio of 55:45 as the mobile phase, isocratic elution at a flow rate of 10 mL / min, and the detection wavelength was 210 nm. The fraction at 26 min was collected, concentrated under reduced pressure and evaporated to dryness to obtain 13.8 mg of a cucurbitacin-type triterpenoid compound represented by formula (I).

[0033] Example 2: Structural Identification of Compounds of Formula (I)

[0034] The obtained compound (I) was characterized by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy ( 1 H NMR, 13 C NMR, 2D NMR) were used for structural identification, and the results are shown in Figure 2 to Figure 6 shown.

[0035] The compound is a white amorphous powder, easily soluble in chloroform. HRESIMS gives a molecular ion signal of m / z 359.2224 [MH] - .exist 1 In the H NMR spectrum, there is one olefinic proton in the low-field region [δ H 5.71 (br s)], two oxygen protons [δ H 3.47 (br d), 3.94 (m)], three groups of double peak signals [δ H 3.10,2.33(d,J=14.4Hz),δ H 2.02, 2.20 (d, J = 17.4 Hz); δ H 2.51,1.94 (d,J=18.0Hz)]. There are 5 methyl singlets in the high field region (δ H 0.98,1.02,1.13,1.19,1.23). 13 There are 22 carbon signals in the C NMR spectrum, among which δ C 138.1 and 120.4 are a pair of olefin signals, and also include two hydroxyl carbon signals (δ C68.4,78.6), 5 methyl signals (δ C 19.3, 20.2, 24.4, 25.4, 26.6), 2 keto carbonyl carbon signals (δ C 211.2, 216.5). The HMBC spectrum shows that H-3 (δ H 3.47) and C-28 (δ C 26.6),C-29(δ C 25.4), C-1(δ C 29.5), C-5(δ C 138.1) related, H-6 (δ H 5.71) and C-7(δ C 24.4), C-8(δ C 43.4), C-10(δ C 34.0), it can be seen that C-5 / C-6 is a double bond, and from H-12 (δ H 3.10,2.33) and H-19(δ H 1.19) and C-11(δ C 211.2) related, H-15 (δ H 2.02,2.20) and H-17(δ H 2.51,1.94) and C-16(δ C 216.5), it can be known that there are two keto carbonyl signals at the 11th and 16th positions, respectively. Based on the above information, it can be inferred that the compound is a tetracyclic triterpenoid compound of the nor-carboxycucurbitane type. In the NOESY spectrum, the correlations of H-2 / H-10, H-2 / CH3-29, and H-10 / CH3-29 indicate that H-10 is in α configuration, indicating that 2-OH is in β configuration. According to the coupling constant J=1.8HZ of the protons of H-2 and H-3, it is determined to be a cis configuration. It can be known that H-2 and H-3 are on the same side, both in β configuration, and CH3-29 is in α configuration, so the structural formula of this compound can be determined to be the structure shown in (I). 1 H and 13 C NMR signals were assigned (Table 1).

[0036]

[0037] Table 1. Compounds of formula (I) 1 H and 13 C NMR chemical shift value (solvent is CDCl3)

[0038]

[0039]

[0040] Example 3: Evaluation of the acetylcholinesterase (AChE) inhibitory activity of the compound of formula (I)

[0041] The AChE inhibition activity test method is the Ellman method, and the specific steps are as follows:

[0042] Sample solution: The compound of formula (I) was dissolved in DMSO and diluted to the required concentration with Tris-HCl buffer (50 mM, pH=8.0), and the DMSO content in the prepared solution was controlled to be less than 1%.

[0043] 160 μL of 1.5 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) aqueous solution, 50 μL of AChE solution (0.22 U mL -1 , solvent is Tris-HCl buffer) and 10μL sample solution. Incubate at 37℃ for 10min, then quickly add 30μL iodinated acetylcholine solution (15mM, solvent is Tris-HCl buffer). Measure the absorbance values ​​at 0, 5, 15 and 20min at a wavelength of 405nM. Under the same conditions, replace the AChE solution with an equal volume of Tris-HCl buffer as a blank control and calculate the inhibition rate.

[0044] Seven concentrations of the sample (0.008, 0.04, 0.2, 1, 5, 20, 100 μM) were selected to determine the enzyme inhibition rate, and the negative logarithm of the molar concentration was linearly regressed with the enzyme inhibition rate to obtain the molar concentration at 50% inhibition, which is the IC of the sample. 50 Each experiment was repeated three times.

[0045] The inhibition rate was calculated as: [1-(absorbance change of experimental group / absorbance change of blank group)]×100%.

[0046] The compound of formula (I) is used as a sample, and its inhibitory activity on acetylcholinesterase is: IC 50 =5.7μM.

Claims

1. A cucurbitane-type triterpenoid compound represented by formula (I):

2. A method for preparing the cucurbitane-type triterpenoid compound represented by formula (I) according to claim 1, characterized in that: The method comprises the following steps: (1) Extraction: The dried fruits of the medicinal watermelon are crushed to obtain medicinal watermelon particles, and the particles are immersed in an ethanol aqueous solution for extraction at room temperature. The extract is filtered, and the filtrate is concentrated under reduced pressure until no liquid flows out, and then dried to obtain a crude extract; (2) Separation: a. The crude extract obtained in step (1) was dry-applied to a silica gel chromatography column and eluted with petroleum ether, petroleum ether: ethyl acetate in a volume ratio of 1:1 and dichloromethane-methanol in a volume ratio of 5:1 as eluents, the elution rate was 50-200 mL / min, and each eluent was eluted for 3-4 column volumes; each 2.5 L of the effluent was collected as a fraction to obtain 8 fractions, which were recorded as fractions A to H; b. Load fraction E onto a silica gel column and perform gradient elution using dichloromethane:methanol in a volume ratio of 60:1 and 20:1, respectively, as eluents. Each eluent is used for 3-5 column volumes. Each 700 mL of the effluent is collected as one fraction to obtain 6 fractions, which are recorded as fractions Ea to Ef. c. The fraction Ee collected in step b was then subjected to ODS-C 18 Column chromatography was performed using methanol-water with a volume ratio of 30:70, 50:50, 70:30, and 90:

10. Each gradient elution was 1 L, and each 500 mL of the effluent was collected as one fraction, to obtain 8 fractions, which were recorded as fractions Ee-1 to Ee-8. d. Fraction Ee-5 was separated by preparative high performance liquid chromatography with isocratic elution using methanol-water in a volume ratio of 55:45 at a flow rate of 10 mL / min. The fraction at 26 min was collected and concentrated under reduced pressure to dryness to obtain the cucurbitane-type triterpenoid compound represented by formula (I).

3. The method according to claim 2, characterized in that The volume dosage of the ethanol aqueous solution in step (1) is 3-5 mL / g based on the mass of the medicinal watermelon particles; the volume concentration of the ethanol aqueous solution is 80-95%.

4. The method according to claim 2, characterized in that Step a: The silica gel chromatography column has a column height of 70 cm, an inner diameter of 8-12 cm, and a silica gel mesh of 100-200.

5. The method according to claim 2, characterized in that Step b: The silica gel chromatographic column has a height of 50 cm, an inner diameter of 3-6 cm, and a silica gel of 200-300 mesh.

6. The method according to claim 2, characterized in that ODS-C in step c 18 The filler particle size is 50 μm and the inner diameter of the chromatography column is 2 to 4 cm.

7. The method according to claim 2, characterized in that The chromatographic column model of the preparative high performance liquid chromatography in step d is Shimadzu Shim-Pack GIST, with a particle size of 5 μm, a specification of 20×250 mm, and a detection wavelength of 210 nm.

8. Use of the cucurbitane triterpenoid compound of formula (I) according to claim 1 in the preparation of an acetylcholinesterase activity inhibitor.

9. Use of the cucurbitane triterpenoid compound of formula (I) according to claim 1 in the preparation of anti-Alzheimer's disease drugs.

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