Unsaturated ketone component in alhagi spirofilae and separation and extraction method and application thereof
By isolating and purifying (7S,3E,5Z)-7,8-dihydroxy-6-methylocta-3,5-dien-2-one unsaturated ketones from camel thorn, the problem of the failure to effectively utilize its medicinal value in existing technologies has been solved, achieving an inhibitory effect on cervical cancer cells and providing a new treatment option.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- PEOPLES HOSPITAL OF XINJIANG UYGUR AUTONOMOUS REGION
- Filing Date
- 2025-01-09
- Publication Date
- 2026-07-03
Smart Images
Figure CN119798063B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of camel thorn separation and purification technology, specifically to an unsaturated ketone component from camel thorn, its separation and extraction method, and its application. The unsaturated ketone component in camel thorn is an abbreviation of (7S,3E,5Z)-7,8-dihydroxy-6-methylocta-3,5-dien-2-one. Background Technology
[0002] Camel thorn (Alhagi sparsifolia Shap) is a semi-shrub belonging to the genus Alhagi Gagneb in the legume family. It is mainly distributed in Xinjiang, and its roots, flowers, stems, and leaves are all used medicinally, commonly for treating abdominal pain, bloating, dysentery, and diarrhea. It is a traditional Uyghur medicinal herb. Camel thorn contains flavonoids, alkaloids, terpenes, lignans, and phenolic acids, exhibiting biological activities such as anti-tumor activity, treatment of diarrhea, cardiovascular disease, and analgesia. Malignant tumors seriously threaten human life and health. Modern pharmacological studies have shown that the ethanol extract of camel thorn can inhibit the proliferation and migration of human cervical cancer HeLa cells, exhibiting strong cytotoxic activity. The strong anti-tumor activity of the chemical components in camel thorn, coupled with its low price and wide availability, has attracted considerable interest from researchers.
[0003] One of the main medicinal effects of camel thorn comes from unsaturated ketone components. Therefore, developing and utilizing the unsaturated ketone monomers of camel thorn to further explore its potential medicinal value, and determining and characterizing the structure and physicochemical properties of its monomers, is of great significance to the development and utilization of camel thorn. Summary of the Invention
[0004] This invention provides an unsaturated ketone component from camel thorn, its separation and extraction method, and its application, overcoming the shortcomings of the prior art. It is the first to disclose that the unsaturated ketone component from camel thorn has a certain inhibitory effect on HeLa cells and can be used in the preparation of drugs for the prevention / treatment of cervical cancer.
[0005] One of the technical solutions of this invention is achieved through the following measures: an unsaturated ketone component from camel thorn, the chemical structural formula of which is:
[0006] .
[0007] The following are further optimizations and / or improvements to one of the above-mentioned technical solutions:
[0008] The unsaturated ketones in camel thorn were obtained by the following method:
[0009] The first step is to crush the above-ground parts of camel thorn and soak them in ethanol, then extract them by heating and reflux to obtain an extract. The extract is then recovered under reduced pressure and concentrated to obtain camel thorn extract.
[0010] The second step involves dispersing the camel thorn extract in water into a suspension and then extracting it with an equal volume of petroleum ether to obtain the petroleum ether fraction extract and the aqueous layer a.
[0011] The third step is to adjust the acidity of aqueous layer a, and then extract it with an equal volume of ethyl acetate to obtain the ethyl acetate extract and aqueous layer b.
[0012] The fourth step is to adjust the aqueous layer b to alkaline, and then extract it with an equal volume of dichloromethane to obtain the dichloromethane extract and the aqueous layer c.
[0013] Fifth step: The dichloromethane fraction extract was separated by gradient elution using silica gel column chromatography to obtain 10 fractions;
[0014] Step 6: The fifth fraction of the 10 fractions was purified and separated by high performance liquid chromatography gradient elution, and the eluent was collected. At 14.7 minutes, the unsaturated ketone components of camel thorn were obtained.
[0015] In the first step above, soaking specifically refers to soaking at room temperature for 3 to 4 hours.
[0016] In the first step above, 8 ml to 12 ml of ethanol is added for every 1 g of camel thorn, and the mass concentration of ethanol is 95% to 98%.
[0017] In the first step above, the heating temperature is 50°C to 60°C, and the reflux extraction is performed 3 to 4 times.
[0018] In the third step above, hydrochloric acid is used to adjust water layer a to acidity, with a pH value of 1 to 2.
[0019] In the fourth step above, sodium hydroxide is used to adjust water layer b to alkalinity, with a pH value of 9 to 10.
[0020] In the fifth step above, the eluent for silica gel column chromatography is a mixture of methane and methanol, and the volume ratio of methane to methanol in the mixture is 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1, respectively.
[0021] In step six above, the high performance liquid chromatography eluent is a mixture of methanol and water, with a volume ratio of methanol to water of 20:80.
[0022] The second technical solution of the present invention is achieved through the following measures: a method for separating and extracting unsaturated ketone components from camel thorn, which is carried out according to the following method:
[0023] The first step is to crush the above-ground parts of camel thorn and soak them in ethanol, then extract them by heating and reflux to obtain an extract. The extract is then recovered under reduced pressure and concentrated to obtain camel thorn extract.
[0024] The second step involves dispersing the camel thorn extract in water into a suspension and then extracting it with an equal volume of petroleum ether to obtain the petroleum ether fraction extract and the aqueous layer a.
[0025] The third step is to adjust the acidity of aqueous layer a, and then extract it with an equal volume of ethyl acetate to obtain the ethyl acetate extract and aqueous layer b.
[0026] The fourth step is to adjust the aqueous layer b to alkaline, and then extract it with an equal volume of dichloromethane to obtain the dichloromethane extract and the aqueous layer c.
[0027] Fifth step: The dichloromethane fraction extract was separated by gradient elution using silica gel column chromatography to obtain 10 fractions;
[0028] Step 6: The fifth fraction of the 10 fractions was purified and separated by high performance liquid chromatography gradient elution, and the eluent was collected. At 14.7 minutes, the unsaturated ketone components of camel thorn were obtained.
[0029] The third technical solution of the present invention is achieved through the following measures: the application of unsaturated ketone components from camel thorn in the preparation of drugs for the prevention / treatment of cervical cancer.
[0030] This invention discloses for the first time the unsaturated ketone components in camel thorn, and conducts in vitro antitumor pharmacodynamic experiments on the unsaturated ketone components in camel thorn of this invention. The experiments show that the unsaturated ketone components in camel thorn of this invention have a certain inhibitory effect on human HeLa cervical cancer cells, thereby enabling the unsaturated ketone components in camel thorn to be used in the preparation of drugs for the prevention / treatment of cervical cancer. Attached Figure Description
[0031] Figure 1 This is the chemical structural formula of the unsaturated ketone components in camel thorn of the present invention.
[0032] Figure 2 This is the 1H-NMR spectrum of the unsaturated ketone components in camel thorn of the present invention.
[0033] Figure 3 This is the 13C-APT spectrum of the unsaturated ketone components in camel thorn of the present invention.
[0034] Figure 4 This is the 1H-1H COSY spectrum of the unsaturated ketone components in camel thorn of the present invention.
[0035] Figure 5This is the HMBC spectrum of unsaturated ketone components in camel thorn according to the present invention.
[0036] Figure 6 This is the HSQC spectrum of unsaturated ketone components in camel thorn according to the present invention.
[0037] Figure 7 This is the NOESY spectrum of unsaturated ketone components in camel thorn according to the present invention. Detailed Implementation
[0038] This invention is not limited to the following embodiments, and specific implementation methods can be determined according to the technical solutions and actual conditions of this invention. Unless otherwise specified, all chemical reagents and chemicals mentioned in this invention are well-known and commonly used chemical reagents and chemicals in the prior art; unless otherwise specified, all percentages in this invention are mass percentages; unless otherwise specified, all solutions in this invention are aqueous solutions with water as the solvent, for example, hydrochloric acid solution is an aqueous solution of hydrochloric acid; room temperature in this invention generally refers to a temperature between 15°C and 25°C, generally defined as 25°C.
[0039] The present invention will be further described below with reference to embodiments:
[0040] Example 1: The unsaturated ketone components in this camel thorn have the following chemical structural formula:
[0041] .
[0042] Example 2: As an optimization of the above examples, the unsaturated ketone components in camel thorn were obtained by the following method:
[0043] The first step is to crush the above-ground parts of camel thorn and soak them in ethanol, then extract them by heating and reflux to obtain an extract. The extract is then recovered under reduced pressure and concentrated to obtain camel thorn extract.
[0044] The second step involves dispersing the camel thorn extract in water into a suspension and then extracting it with an equal volume of petroleum ether to obtain the petroleum ether fraction extract and the aqueous layer a.
[0045] The third step is to adjust the acidity of aqueous layer a, and then extract it with an equal volume of ethyl acetate to obtain the ethyl acetate extract and aqueous layer b.
[0046] The fourth step is to adjust the aqueous layer b to alkaline, and then extract it with an equal volume of dichloromethane to obtain the dichloromethane extract and the aqueous layer c.
[0047] Fifth step: The dichloromethane fraction extract was separated by gradient elution using silica gel column chromatography to obtain 10 fractions;
[0048] Step 6: The fifth fraction of the 10 fractions was purified and separated by high performance liquid chromatography gradient elution, and the eluent was collected. At 14.7 minutes, the unsaturated ketone components of camel thorn were obtained.
[0049] Example 3: As an optimization of the above example, in the first step, the soaking is specifically performed at room temperature for 3 to 4 hours.
[0050] Example 4: As an optimization of the above example, in the first step, 8 ml to 12 ml of ethanol is added for every 1 g of camel thorn, and the mass concentration of ethanol is 95% to 98%.
[0051] Example 5: As an optimization of the above example, in the first step, the heating temperature is 50°C to 60°C, and the reflux extraction is performed 3 to 4 times.
[0052] Example 6: As an optimization of the above example, in the third step, hydrochloric acid is used to adjust water layer a to acidity, with a pH value of 1 to 2.
[0053] Example 7: As an optimization of the above example, in the fourth step, sodium hydroxide is used to adjust water layer b to alkalinity, with a pH value of 9 to 10.
[0054] Example 8: As an optimization of the above example, in the fifth step, the eluent for silica gel column chromatography is a mixture of methane and methanol, and the volume ratio of methane to methanol in the mixture is 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1, respectively.
[0055] Example 9: As an optimization of the above example, in step 6, the high performance liquid chromatography eluent is a mixture of methanol and water, and the volume ratio of methanol to water in the mixture is 20:80.
[0056] Example 10: Application of unsaturated ketone components in camel thorn in the preparation of drugs for the prevention / treatment of cervical cancer.
[0057] Example 11:
[0058] The unsaturated ketones in camel thorn were obtained by the following method:
[0059] The first step is to crush camel thorn and add 95% ethanol. After soaking at room temperature for 3 hours, the mixture is heated and refluxed at 50°C for 3 times. The extracts from each reflux extraction are combined. The extracts are then recovered under reduced pressure and concentrated to obtain camel thorn extract. 8 ml of 95% ethanol is added for every 1 g of camel thorn.
[0060] The second step involves dispersing the camel thorn extract in water into a suspension and then extracting it with an equal volume of petroleum ether to obtain the petroleum ether fraction extract and the aqueous layer a.
[0061] The third step is to adjust the pH of aqueous layer a to 1 with hydrochloric acid, and then extract it with an equal volume of ethyl acetate to obtain the ethyl acetate extract and aqueous layer b.
[0062] The fourth step is to adjust the pH of aqueous layer b to alkaline (pH 9) with sodium hydroxide, and then extract it with an equal volume of dichloromethane to obtain the dichloromethane extract and aqueous layer c.
[0063] Fifth step: After separating the dichloromethane fraction extract by gradient elution using silica gel column chromatography, 10 fractions were obtained. The silica gel column chromatography eluent was a mixture of methane and methanol, and the volume ratio of methane to methanol in the mixture was 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1, respectively.
[0064] Step 6: The fifth fraction of the obtained 10 fractions was purified and separated by high performance liquid chromatography gradient elution, and the eluent was collected. At 14.7 minutes, the unsaturated ketone components of camel thorn were obtained. The high performance liquid chromatography eluent was a mixture of methanol and water with a volume ratio of methanol to water of 20:80.
[0065] Example 12:
[0066] The unsaturated ketones in camel thorn were obtained by the following method:
[0067] The first step is to crush camel thorn and add 95% ethanol. After soaking at room temperature for 4 hours, the mixture is heated and refluxed at 60°C for 4 extractions. The extracts from each reflux extraction are combined. The extracts are then recovered under reduced pressure and concentrated to obtain camel thorn extract. 12 ml of 95% ethanol is added for every 1 g of camel thorn.
[0068] The second step involves dispersing the camel thorn extract in water into a suspension and then extracting it with an equal volume of petroleum ether to obtain the petroleum ether fraction extract and the aqueous layer a.
[0069] The third step is to adjust the pH of aqueous layer a to 2 with hydrochloric acid, and then extract it with an equal volume of ethyl acetate to obtain the ethyl acetate extract and aqueous layer b.
[0070] The fourth step is to adjust the pH of aqueous layer b to alkaline (pH 10) with sodium hydroxide, and then extract it with an equal volume of dichloromethane to obtain the dichloromethane extract and aqueous layer c.
[0071] Fifth step: After separating the dichloromethane fraction extract by gradient elution using silica gel column chromatography, 10 fractions were obtained. The silica gel column chromatography eluent was a mixture of methane and methanol, and the volume ratio of methane to methanol in the mixture was 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1, respectively.
[0072] Step 6: The fifth fraction of the obtained 10 fractions was purified and separated by high performance liquid chromatography gradient elution, and the eluent was collected. At 14.7 minutes, the unsaturated ketone components of camel thorn were obtained. The high performance liquid chromatography eluent was a mixture of methanol and water with a volume ratio of methanol to water of 20:80.
[0073] Example 13:
[0074] The unsaturated ketones in camel thorn were obtained by the following method:
[0075] The first step is to crush camel thorn and add 95% ethanol. After soaking at room temperature for 3.5 hours, heat and reflux at 55°C for 3 extractions. Combine the extracts from each reflux extraction, and then recover and concentrate the extracts under reduced pressure to obtain camel thorn extract. 10 ml of 95% ethanol is added for every 1 g of camel thorn.
[0076] The second step involves dispersing the camel thorn extract in water into a suspension and then extracting it with an equal volume of petroleum ether to obtain the petroleum ether fraction extract and the aqueous layer a.
[0077] The third step is to adjust the pH of aqueous layer a to 1.5 with hydrochloric acid, and then extract it with an equal volume of ethyl acetate to obtain the ethyl acetate extract and aqueous layer b.
[0078] The fourth step involves adjusting the aqueous layer b to alkaline (pH 9.5) with sodium hydroxide, followed by extraction with an equal volume of dichloromethane to obtain the dichloromethane extract and the aqueous layer c.
[0079] Fifth step: After separating the dichloromethane fraction extract by gradient elution using silica gel column chromatography, 10 fractions were obtained. The silica gel column chromatography eluent was a mixture of methane and methanol, and the volume ratio of methane to methanol in the mixture was 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1, respectively.
[0080] Step 6: The fifth fraction of the obtained 10 fractions was purified and separated by high performance liquid chromatography gradient elution, and the eluent was collected. At 14.7 minutes, the unsaturated ketone components of camel thorn were obtained. The high performance liquid chromatography eluent was a mixture of methanol and water with a volume ratio of methanol to water of 20:80.
[0081] Experimental Example 1: Structural characterization analysis of unsaturated ketone components in camel thorn of the present invention.
[0082] Experimental method: Nuclear magnetic resonance spectroscopy analysis was performed on the unsaturated ketone components in camel thorn prepared in Example 13 of this invention.
[0083] Experimental results: The 1H-NMR spectrum of the unsaturated ketone components in camel thorn prepared in Example 13 of this invention is as follows: Figure 2 As shown, the 13C-APT spectrum of the unsaturated ketone components in camel thorn prepared in Example 13 of this invention is as follows. Figure 3 As shown, the 1H-1H COSY spectrum of the unsaturated ketone components in camel thorn prepared in Example 13 of this invention is as follows. Figure 4 As shown, the HMBC spectrum of the unsaturated ketone components in camel thorn prepared in Example 13 of this invention is as follows. Figure 5 As shown, the HSQC spectrum of the unsaturated ketone components in camel thorn prepared in Example 13 of this invention is as follows. Figure 6 As shown, the NOESY spectrum of the unsaturated ketone components in camel thorn prepared in Example 13 of this invention is as follows. Figure 7 As shown, for Figures 2 to 7 Perform spectral analysis, and Figures 2 to 3 Each peak is assigned a location. Figures 2 to 3 The peak assignments are shown in Table 1. From the data in Table 1, it can be seen that the chemical structural formulas of the unsaturated ketone components in camel thorn prepared in Example 13 of this invention are as follows: Figure 1 As shown, the unsaturated ketone components in this camel thorn are readily soluble in methanol.
[0084] Experimental Example 2: In vitro antitumor pharmacodynamic experiment of unsaturated ketone components in camel thorn according to the present invention
[0085] Experimental Methods: The unsaturated ketone components from camel thorn prepared in Example 13 of this invention were subjected to in vitro antitumor pharmacodynamic experiments using the MTT assay. Specific procedures were as follows: The unsaturated ketone components from camel thorn prepared in Example 13 of this invention were used as the experimental group, and cisplatin was used as the control group. HeLa (human cervical cancer cells) cells were selected as the experimental subjects for both groups. After dilution of the culture medium, 100 μL of the culture medium was seeded into 96-well plates at a density of 6 × 10⁴ cells per well. After normal incubation for 24 hours, the corresponding drugs were added to each group, resulting in final drug concentrations of 6.25 μM (Group 1), 12.5 μM (Group 2), 25 μM (Group 3), 50 μM (Group 4), and 100 μM (Group 5), for a total of 5 concentrations, with 3 replicates per concentration. After 48 hours of incubation, 10 μL of MTT was added to each well for staining. After another 4 hours of incubation, the original culture medium was discarded, and DMSO was added to each well. 150 μL was placed on a shaker and shaken at low speed for 10 min to fully dissolve the crystals. The optical density was measured at 570 nm wavelength using an enzyme-linked immunosorbent assay (ELISA) reader. The 50% inhibition concentration (IC50, μM) was calculated based on the optical density value. The method for calculating IC50 based on optical density value is a known technique.
[0086] Experimental results: The IC50 results of the unsaturated ketone components in camel thorn of the present invention on HeLa cells are shown in Table 2. As can be seen from Table 2, the unsaturated ketone components in camel thorn of the present invention have a certain inhibitory effect on HeLa cells.
[0087] In summary, this invention discloses for the first time the unsaturated ketone components in camel thorn, and conducts in vitro antitumor pharmacodynamic experiments on the unsaturated ketone components in camel thorn of this invention. The experiments show that the unsaturated ketone components in camel thorn of this invention have a certain inhibitory effect on human HeLa cervical cancer cells, thereby enabling the unsaturated ketone components in camel thorn to be used in the preparation of drugs for the prevention / treatment of cervical cancer.
[0088] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.
[0089]
Claims
1. An unsaturated ketone component from camel thorn, characterized in that... Its chemical structural formula is: 。 2. A method for separating and extracting unsaturated ketone components from camel thorn according to claim 1, characterized in that... Perform the following steps: The first step is to crush the above-ground parts of camel thorn and soak them in ethanol, then extract them by heating and reflux to obtain an extract. The extract is then recovered under reduced pressure and concentrated to obtain camel thorn extract. The second step involves dispersing the camel thorn extract in water into a suspension and then extracting it with an equal volume of petroleum ether to obtain the petroleum ether fraction extract and the aqueous layer a. The third step is to adjust the acidity of aqueous layer a, and then extract it with an equal volume of ethyl acetate to obtain the ethyl acetate extract and aqueous layer b. The fourth step is to adjust the aqueous layer b to alkaline, and then extract it with an equal volume of dichloromethane to obtain the dichloromethane extract and the aqueous layer c. Fifth step: The dichloromethane fraction extract was separated by gradient elution using silica gel column chromatography to obtain 10 fractions; Step 6: The fifth fraction of the 10 fractions was purified and separated by high performance liquid chromatography gradient elution, and the eluent was collected. At 14.7 minutes, the unsaturated ketone components of camel thorn were obtained.
3. The method for separating and extracting unsaturated ketone components from camel thorn according to claim 2, characterized in that... In the first step, the soaking is specifically performed at room temperature for 3 to 4 hours; or / and, in the first step, 8 to 12 ml of ethanol is added for every 1 g of camel thorn, with the ethanol having a mass concentration of 95% to 98%.
4. The method for separating and extracting unsaturated ketone components from camel thorn according to claim 2 or 3, characterized in that... In the first step, the heating temperature is 50℃ to 60℃, and the reflux extraction is performed 3 to 4 times.
5. The method for separating and extracting unsaturated ketone components from camel thorn according to claim 3, characterized in that... In the third step, hydrochloric acid is used to adjust water layer a to acidity, with a pH value of 1 to 2.
6. The method for separating and extracting unsaturated ketone components from camel thorn according to claim 2, 3, or 5, characterized in that... In the fourth step, sodium hydroxide is used to adjust water layer b to alkalinity, with a pH value of 9 to 10.
7. The method for separating and extracting unsaturated ketone components from camel thorn according to claim 6, characterized in that... In step six, the high-performance liquid chromatography eluent is a mixture of methanol and water, with a volume ratio of methanol to water of 20:
80.
8. The use of the unsaturated ketone components in camel thorn according to claim 1 in the preparation of drugs for the prevention and / or treatment of cervical cancer.