New pentacyclic triterpenoid compounds and their extraction methods and uses

Abstract

The present invention has isolated a new class of pentacyclic triterpenoid compounds with novel structures from jasmine roots, which have strong inhibitory activity against tumors. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and specifically relates to a method for extracting new pentacyclic triterpenoid compounds from jasmine roots and the use thereof in treating tumors. Background Art

[0002] Cancer has become the leading cause of death worldwide. Cancer can occur in various organs and tissues at any age.

[0003] Most cancer patients are diagnosed in the middle to late stages of the disease, resulting in poor clinical treatment outcomes. The increasing emergence of multidrug resistance, in particular, complicates cancer treatment. While some small-molecule anticancer chemotherapy drugs and antibody-based drugs have entered clinical trials, the intellectual property rights for most clinically used new cancer drugs are controlled by international pharmaceutical companies. my country has developed relatively few new cancer drugs independently. Therefore, developing new anticancer drugs with high activity and low side effects to meet clinical needs is urgent. In particular, the discovery of new antitumor candidate compounds from traditional Chinese medicines is a major focus of current drug development.

[0004] Jasmine root, also known as jasmine flower root, is the dried root of Jasminum sambac (L.) Ait., a dicotyledonous plant of the genus Jasminum in the family Oleaceae. It is warm in nature, bitter in taste, and toxic, native to India. In my country, it is primarily produced in Jiangsu, Zhejiang, Fujian, Taiwan, Guangdong, and Sichuan. According to ancient Chinese texts, jasmine root has been used to relieve pain and provide a certain degree of anesthetic effect for fractures, dislocations, and bone setting. It has the effects of boosting immunity, clearing away heat and detoxifying, and relieving swelling and pain. It has a beneficial therapeutic effect on patients suffering from dysentery, abdominal pain, intestinal diseases, and conjunctivitis. It is commonly used clinically to treat a variety of conditions, including fractures and tendon injuries, dental caries, and vertex pain. It is also recorded that jasmine root is the active ingredient in "Ma Fei San," a remedy developed by Hua Tuo for use in surgery. Summary of the Invention

[0005] The invention separates and obtains a class of novel pentacyclic triterpenoid compounds with novel structure from jasmine roots, and the compounds have strong inhibitory activity on tumors.

[0006] The present invention provides six new pentacyclic triterpenoid compounds as shown below:

[0007]

[0008] The present invention also provides a method for extracting and separating the novel pentacyclic triterpenoid compounds, comprising the following steps:

[0009] (S1) crushing jasmine root, extracting with ethanol and aqueous solution, and concentrating the extract to obtain a total extract;

[0010] (S2) dispersing the total extract obtained in step (S1) with water, extracting with organic solvents of different polarities, and concentrating the extracts to obtain extracts of different polarities;

[0011] (S3) subjecting the extract obtained in step (S2) to column separation to obtain a crude fraction; and

[0012] (S4) separating and purifying the crude fraction obtained in step (S3) to obtain the novel pentacyclic triterpenoid compound shown above.

[0013] According to an embodiment of the present invention, the ethanol aqueous solution extraction in step (S1) can be soaking or reflux extraction.

[0014] According to an embodiment of the present invention, the soaking temperature in the soaking extraction is 15-30° C., preferably room temperature; the soaking time is 20-80 days, preferably 25-65 days, for example 60 days.

[0015] According to an embodiment of the present invention, the reflux time in the reflux extraction may be 10-24 hours, for example, 12-15 hours.

[0016] According to an embodiment of the present invention, the organic solvents of different polarities used in step (S2) are selected from petroleum ether, ethyl acetate, chloroform or n-butanol.

[0017] According to an embodiment of the present invention, the extract used in step (S3) is a chloroform extract.

[0018] According to an embodiment of the present invention, the chromatographic separation in step (S3) includes but is not limited to silica gel column separation, preparative liquid chromatography separation and any combination thereof.

[0019] According to an embodiment of the present invention, the separation and purification in step (S4) is selected from silica gel column separation, gel column separation, preparative plate separation or preparative liquid chromatography separation and any combination thereof.

[0020] According to an embodiment of the present invention, the method for extracting and separating the novel pentacyclic triterpenoid compound comprises the following steps:

[0021] (1) crushing jasmine roots, soaking them in ethanol water solution, filtering and concentrating them to obtain a total extract;

[0022] (2) dispersing the total extract obtained in step (1) with water, extracting with petroleum ether, ethyl acetate and chloroform respectively for 3-7 times, and concentrating the extracts of different polarities to obtain petroleum ether extract, ethyl acetate extract and chloroform extract respectively;

[0023] (3) separating the chloroform extract obtained in step (2) by silica gel column and performing gradient elution with a developing solvent to obtain crude fractions of different polarities;

[0024] (4) First, the crude fractions were detected by TLC, and similar fractions were combined. Then, the crude fractions of different polarities in step (3) were analyzed by HPLC and similar fractions were combined. Then, the crude fractions were separated and purified by silica gel column, preparative plate separation or preparative liquid chromatography, and the R f The same components or components with the same retention time can be recrystallized multiple times to obtain new pentacyclic triterpenoid compounds.

[0025] According to an embodiment of the present invention, in step (1), the mass fraction of ethanol in the ethanol aqueous solution may be 50-80%, for example, 50%, 60%, 70% or 80%.

[0026] According to an embodiment of the present invention, in step (2), the mass volume ratio (g / mL) of the total extract to water is (0.2-3):1, for example (0.5-2):1, exemplified by 1:2.

[0027] According to an embodiment of the present invention, in step (2), the volume ratio of the organic solvent used for extraction to water is 1:(0.2-3), such as 1:(0.5-2), and exemplified by 1:2.

[0028] According to an embodiment of the present invention, in step (3), the developing agent is petroleum ether and / or ethyl acetate, starting from pure petroleum ether, gradually increasing the amount of ethyl acetate while reducing the amount of petroleum ether, and finally becoming pure ethyl acetate. Preferably, the volume ratio of petroleum ether to ethyl acetate is 1:0, 0.9:0.1, 0.8:0.2, 0.7:0.3, 0.6:0.4, 0.5:0.5, 0.4:0.6, 0.3:0.7, 0.2:0.8, 0.1:0.9, 0:1.

[0029] According to an embodiment of the present invention, in step (3), V is collected 石油醚 / V 乙酸乙酯 =2:1 and 1:1 elution fractions, 50 sites with different polarities (Fr1-Fr 50 According to an embodiment of the present invention, in step (4), the HPLC analysis conditions are as follows: mobile phase: V 甲醇 :V 水(含0.3％磷酸) =7:3, column temperature was room temperature, and detection wavelength was 200-400 nm integrated wavelength.

[0030] According to an embodiment of the present invention, in step (4), the separation and purification is performed by selecting a component with a larger polarity difference (for example, selecting R f 3-5 components with a difference of 0.5-1).

[0031] According to an embodiment of the present invention, in step (4), the separation and purification can be repeated.

[0032] According to an embodiment of the present invention, in step (4), the recrystallization is performed using a mixture of methanol, tetrahydrofuran and water.

[0033] According to an embodiment of the present invention, step (4) is performed as follows: the 50 components obtained in step (3) are first subjected to preliminary TLC detection, and similar components are combined to obtain 30 components; then, these 30 components are subjected to HPLC qualitative analysis (chromatographic conditions: V 甲醇 :V 水(含0.3％磷酸) , 7:3, column temperature is room temperature, detection wavelength is 200-400nm), similar components (retention time difference is about 0.01 minutes) are merged; then 5 components with large polarity difference (core substance retention time difference is about 0.5-5 minutes) are selected and then subjected to the first silica gel column separation. After the obtained components are subjected to preliminary TLC analysis, similar components are merged and the components with large polarity difference (R f The components with a difference of about 0.2-0.6) are subjected to a second silica gel column separation, and then the components after the second column separation are subjected to preparative plate separation or preparative high performance liquid chromatography separation, and the same R f The components with the same retention time or the same retention time are combined and then recrystallized to obtain the new pentacyclic triterpenoid compound as described above.

[0034] The present invention also provides the use of at least one of the novel pentacyclic triterpenoid compounds in the preparation of a drug for treating and / or preventing cancer.

[0035] According to an embodiment of the present invention, the cancer is selected from lung cancer, gastric cancer, breast cancer or cervical cancer.

[0036] Beneficial effects

[0037] The present invention separates and obtains a class of novel pentacyclic triterpenoid compounds with novel structure from jasmine root. The compounds have strong inhibitory activity on various cancer cells (such as gastric cancer cell lines, colorectal cancer cell lines, breast cancer cell lines and cervical cancer cell lines).

[0038] The extraction method of the present invention performs preliminary purification of the plant extract (such as column separation, preparative plate separation, preparative liquid chromatography separation, etc.), followed by recrystallization, and quickly and accurately obtains high-purity target compounds (the purity can be as high as 98% by HPLC testing). It is of great significance for the rapid separation and identification of specific compounds, especially chiral enantiomers, in plant extracts containing complex components. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Compound YJP-1 1 H- 1H COSY and HMBC correlation plots.

[0040] Figure 2 Compound YJP-2 1 H- 1 H COSY and HMBC correlation plots.

[0041] Figure 3 Compound YJP-3 1 H- 1 H COSY and HMBC correlation plots.

[0042] Figure 4 Compound YJP-4 1 H- 1 H COSY and HMBC correlation plots.

[0043] Figure 5 Compound YJP-5 1 H- 1 H COSY and HMBC correlation plots.

[0044] Figure 6 Compound YJP-6 1 H- 1 H COSY and HMBC correlation plots.

[0045] Figure 7 This is the spherical crystal structure diagram of compound YJP-1.

[0046] Figure 8 This is the ROESY correlation diagram of compound YJP-1.

[0047] Figure 9 This is the ROESY correlation diagram of compound YJP-2.

[0048] Figure 10 ROESY correlation diagram of compound YJP-3.

[0049] Figure 11 ROESY correlation diagram of compound YJP-4.

[0050] Figure 12 ROESY correlation diagram of compound YJP-5.

[0051] Figure 13 ROESY correlation diagram of compound YJP-6. DETAILED DESCRIPTION

[0052] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0053] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0054] Instruments and reagents:

[0055] Jasmine root (collected from Ningde, Fujian Province in October 2021), other chemical reagents are all domestically produced chemically pure reagents; CCK8 (Shanghai Beibo Biotechnology Co., Ltd.); DMEM high glucose medium (Thermo Fisher Scientific (Suzhou) Instrument Co., Ltd.); EDTA (pancreatin) (gibco); Foetal Bovine Serum (Biological Industries); phosphate buffered saline solution; 96-well cell culture plate; multi-function microplate reader.

[0056] Example 1: Extraction, Isolation and Structural Identification of New Pentacyclic Triterpenoids

[0057] The extraction and separation of the new pentacyclic triterpenoid compounds shown below were carried out according to the following process:

[0058]

[0059] (a) 20 kg of dried jasmine root (collected from Ningde, Fujian Province in October 2021) was crushed and placed in three 20 L plastic barrels. 15 L of 70% ethanol aqueous solution was added to each barrel and soaked at room temperature for 2 months. The extract was filtered and concentrated to obtain the extract.

[0060] (b) Disperse 1 kg of the extract obtained in step (a) in 2 L of water and extract five times with petroleum ether, ethyl acetate, and chloroform, respectively, using 1000 mL of petroleum ether, ethyl acetate, and chloroform each time. Concentrate the extracts of different polarities to obtain extracts of different polarities.

[0061] (c) subjecting the chloroform extract obtained in step (b) to a first silica gel column separation, using V 石油醚 / V 乙酸乙酯 =1:0 to 0:1 for gradient elution, collect V 石油醚 / V 乙酸乙酯 =2:1 and 1:1 elution fractions, 50 sites with different polarities (Fr1-Fr 50 ).

[0062] (d) The 50 fractions obtained in step (c) were first subjected to preliminary TLC analysis, and similar fractions were combined to obtain 30 fractions. These 30 fractions were then subjected to HPLC qualitative analysis (chromatographic conditions: V 甲醇 :V 水(含0.3％磷酸) , 7:3, column temperature is room temperature, detection wavelength is 200-400nm), similar components (retention time difference is about 0.01 minutes) are merged. Then, 5 components with large polarity difference (core substance retention time difference is about 0.5-5 minutes) are selected and then separated by silica gel column for the first time. After preliminary TLC analysis of the obtained components, similar components are merged and the components with large polarity difference (R f The components with a difference of about 0.2-0.6) are subjected to a second silica gel column separation, and then the components after the second column separation are subjected to preparative plate separation or preparative high performance liquid chromatography separation, and the same R f The components with the same or the same retention time are combined, and then recrystallized (recrystallized by a mixture of methanol, tetrahydrofuran and a small amount of water) to obtain the new pentacyclic triterpenoid compound as described above.

[0063] The purity of the isolated new pentacyclic triterpenoids was determined by HPLC (chromatographic conditions: 18 Column; mobile phase: V 甲醇 :V 水 , 7:3; detection wavelength: 254 nm), and the HPLC chromatography detection results are listed in Table 2.

[0064] The structures of the six new pentacyclic triterpenoids were determined by 1D NMR, 2D NMR and high-resolution mass spectrometry, and their absolute configurations were determined by ROESY and XRD analysis. 1 H- 1 H COSY and HMBC correlation diagrams are attached. Figure 1-6 ; The spherical crystal structure of compound YJP-1 is shown in Figure 7 ; ROESY correlation diagrams for YJP-1 to YJP-6 are attached Figure 8-13 .

[0065] The characterization data of the above six new pentacyclic triterpenoid compounds are as follows: the hydrogen and carbon spectrum data of compounds YJP-1 to YJP-4 are shown in Table 1; the hydrogen and carbon spectrum data of compounds YJP-5 to YJP-6 are as follows.

[0066] Table 1 Proton and carbon spectrum data of compounds YJP-1 to YJP-4

[0067]

[0068]

[0069]

[0070] Note: The blank cells in the above table indicate that there is no chemical shift at the corresponding position.

[0071] YJP-5: light yellow solid, 1 H NMR(400MHz,CD3OD):0.93(3H,s,H-12'),1.01(3H,d,J=4.6Hz,H-15),1.13(3H,d,J=4.6Hz,H-17),1.18(3H,s ,H-11'),1.36(3H,s,H-16),1.38(3H,s,H-18),1.71(1H,m,H-10),1.72(1H,m,H-2α),1.82(1H,m,H-2β),2.30 (1H,m,H-7α),2.32(1H,m,H-8'),2.33(1H,m,H-7'α),2.42(1H,d,J=3.7Hz,H-3α),2.45(1H,d,J=2.1Hz,H-7β) ,2.58(1H,d,J=11.7Hz,H-7'β),3.11(1H,d,J=11.4Hz,H-3α),5.59(1H,s,H-2'),6.77(1H,t,J=2.4Hz,H-4').

[0072] 13 C NMR(100MHz):13.8(C-17),14.5(C-15),16.0(C-11'),18.5(C-12'),22.4(C-6'),22.8(C-18),25. 7(C-16),25.9(C-6),29.4(C-5'),33.6(C-2),35.5(C-3),141.1(C-14),38.6(C-9'),38.7(C-7'),3 9.6(C-10),39.9(C-8'),41.6(C-8),43.2(C-1),69.0(C-4),73.9(C-13),123.6(C-2'),126.8(C-5) ,135.8(C-4'),136.4(C-11),141.0(C-10'),158.7(C-1'),186.0(C-3'),189.6(C-9),200.6(C-8).

[0073] YJP-6: light yellow solid, 1H NMR(400MHz,CD3OD):1.09(3H,d,J=6.2Hz,H-12'),1.11(3H,s,H-11'),1.14(3H,d,J=6.4Hz,H-17),1.18(3 H,s,H-15),1.38(3H,s,H-16),1.42(3H,s,H-18),1.86-1.87(2H,m,H-2),1.88-1.90(2H,m,H-3),2.33-2.34 (2H,m,H-5'),2.35-2.37(1H,m,H-10),2.41(1H,d,J=5.2Hz,H-6β),2.45-2.47(2H,m,H-7),2.59-2.60(2H,m ,H-6'),2.62-2.63(1H,m,H-8'),3.11(1H,s,H-6α),5.83(1H,s,H-2'),6.45(1H,s,H-7'),6.47(1H,s,H-5).

[0074] 13 C NMR(100MHz):13.8(C-17),13.9(C-12'),16.1(C-15),16.7(C-11'),22.8(C-18),25.7(C-16),3 1.8(C-2),33.6(C-3),35.4(C-6),38.0(C-6'),39.9(C-8'),40.0(C-10),41.6(C-7),41.8(C-5' ),43.5(C-1),62.7(C-13),65.5(C-10'),69.1(C-4),123.7(C-2'),126.6(C-5),126.9(C-7'),1 36.1(C-11),158.0(C-14),158.6(C-1'),186.1(C-9),187.3(C-3'),200.5(C-4'),200.6(C-8).

[0075] The HPLC retention time and purity test results of the above compounds are shown in Table 2 below:

[0076] Table 2

[0077] Compound Retention time (min) HPLC purity (%) YJP-1 17.067 98.118 YJP-2 18.807 96.406 YJP-3 12.407 98.391 YJP-4 21.400 98.827 YJP-5 26.586 98.865 YJP-6 14.127 98.426 .

[0078] Example 2: In vitro anti-tumor activity test

[0079] The new pentacyclic triterpenoid compound obtained in Example 1 was tested for its in vitro antitumor activity, mainly testing its inhibitory activity against lung cancer cell line A549, breast cancer cell line MCF-7, and cervical cancer cell line Hela. The specific testing process for the breast cancer cell line MCF-7 is as follows:

[0080] 1. Preparation of test sample concentration

[0081] Weigh 10.0 mg of the test compound into a 5 mL plastic centrifuge tube and dilute to 1 mL with DMSO. This gives an initial concentration of 10.0 mg / mL. Then, serially dilute the initial concentration with DMSO to obtain five different concentration gradients: 5.0 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, and 0.3125 mg / mL. Store in a refrigerator at 4°C until use.

[0082] 2. Cultivation of Cancer Cell Lines and Inhibitory Activity Testing

[0083] The breast cancer cell line MCF-7 was cultured in a 37°C, saturated humidity, 5% CO2 incubator for 24 hours. When the cells reached the logarithmic growth phase, the supernatant was aspirated and digested with 0.25% trypsin-EDTA solution. Digestion was terminated using high-glucose medium. The cells were seeded into 96-well plates at a cell density of 5,000 cells per well. The 96-well plates were incubated in an incubator for 24 hours. The cell culture medium in the 96-well plates was then aspirated and discarded. 100 μL of high-glucose medium was added to the 96-well plates, followed by 1 μL of test sample at various concentrations per well (five replicates per well). The plates were then incubated in a 37°C, saturated humidity, 5% CO2 incubator for 48 hours. After that, 10 μL of CCK8 was added to each well and incubated in a 37°C incubator for 1-4 hours. The absorbance of each well was measured at 450 nm on a multifunctional microplate reader. According to the inhibition rate % = [(control cell OD-drug-added cell OD) / (control cell OD-blank OD)] × 100. The negative control is V 高糖培养基 / V DMSO =10:1 mixed solution.

[0084] The test showed that the inhibition rate of compound YJP-4 on breast cancer cell line MCF-7 was 148.3 μM; the inhibition rate of compound YJP-1 on breast cancer cell line MCF-7 was 243.7 μM.

[0085] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Pentacyclic triterpenoid compounds as shown below:

2. The method for extracting and separating pentacyclic triterpenoid compounds according to claim 1, characterized in that: The following steps are involved: (S1) crushing jasmine root, extracting with ethanol and aqueous solution, and concentrating the extract to obtain a total extract; (S2) dispersing the total extract obtained in step (S1) with water, extracting with organic solvents of different polarities, and concentrating the extracts to obtain extracts of different polarities; (S3) subjecting the extract obtained in step (S2) to column separation to obtain a crude fraction; and (S4) separating and purifying the crude fraction obtained in step (S3) to obtain the novel pentacyclic triterpenoid compound shown above; The ethanol aqueous solution extraction in step (S1) is immersion extraction or reflux extraction; The organic solvent of different polarity used in step (S2) is selected from petroleum ether, ethyl acetate, chloroform or n-butanol; The extract used in step (S3) is chloroform extract; The chromatographic separation in step (S3) is selected from silica gel column separation, preparative liquid chromatography separation, and any combination thereof; The separation and purification in step (S4) is selected from silica gel column separation, gel column separation, preparative plate separation or preparative liquid chromatography separation and any combination thereof.

3. The extraction and separation method according to claim 2, wherein The method for extracting and separating the pentacyclic triterpenoid compounds comprises the following steps: (1) crushing jasmine roots, soaking them in ethanol water solution, filtering and concentrating them to obtain a total extract; (2) dispersing the total extract obtained in step (1) with water, extracting with petroleum ether, ethyl acetate and chloroform respectively for 3-7 times, and concentrating the extracts of different polarities to obtain petroleum ether extract, ethyl acetate extract and chloroform extract respectively; (3) separating the chloroform extract obtained in step (2) by silica gel column and performing gradient elution with a developing solvent to obtain crude fractions of different polarities; (4) First, the crude fractions were detected by TLC, and similar fractions were combined. Then, the crude fractions of different polarities in step (3) were analyzed by HPLC and similar fractions were combined. Then, the crude fractions were separated and purified by silica gel column, preparative plate separation or preparative liquid chromatography, and the R f The same components or components with the same retention time can be recrystallized multiple times to obtain pentacyclic triterpenoid compounds.

4. Use of at least one of the pentacyclic triterpenoid compounds according to claim 1 in the preparation of a medicament for treating breast cancer.

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