Unsaturated ketone component in alhagi sparsifolia as well as separation and extraction method and application thereof

Abstract

The invention relates to the technical field of separation and purification of alhagi sparsifolia, in particular to an unsaturated ketone component in alhagi sparsifolia and a separation and extraction method and application thereof, the unsaturated ketone component in alhagi sparsifolia is abbreviated as (7S, 3E, 5Z)-7, 8-dihydroxy-6-methylocta-3, 5-dien-2-one, the invention discloses the unsaturated ketone component in alhagi sparsifolia for the first time, and the unsaturated ketone component in alhagi sparsifolia can be used for separating and purifying alhagi sparsifolia. In addition, in-vitro anti-tumor pharmacodynamic experiments are carried out on the unsaturated ketone components in the alhagi sparsifolia, and the experiments show that the unsaturated ketone components in the alhagi sparsifolia have a certain inhibition effect on human Hela cervical cancer cells, so that the unsaturated ketone components in the alhagi sparsifolia can be applied to preparation of drugs for preventing / treating cervical cancer.

Description

Technical Field

[0001] The invention relates to the technical field of camel thorn separation and purification, and is an unsaturated ketone component in camel thorn and a separation and extraction method and application thereof. The unsaturated ketone component in camel thorn is the abbreviation of (7S, 3E, 5Z)-7,8-dihydroxy-6-methylocta-3,5-dien-2-one. Background Art

[0002] Camel thorn (Alhagi sparsifolia Shap) is a semi-shrub of the genus Alhagi Gagneb in the Leguminosae family. It is mainly distributed in Xinjiang. Its roots, flowers, stems, leaves and other parts can be used as medicine. It is often used to treat abdominal pain, bloating, dysentery and diarrhea. It is a traditional Uyghur medicinal material. Camel thorn contains chemical components such as flavonoids, alkaloids, terpenes, lignans and phenolic acids, which have biological activities such as anti-tumor, treatment of diarrhea, cardiovascular disease and analgesia. Malignant tumors seriously threaten human life and health. Modern pharmacological studies have shown that camel thorn ethanol extract has the effect of inhibiting the proliferation and migration of human cervical cancer HeLa cells and has strong cytotoxic activity. The chemical components in camel thorn have strong anti-tumor activity, and the price is cheap and the source is wide, which has attracted the interest of many researchers.

[0003] One of the medicinal effects of camel thorn mainly comes from its unsaturated ketone components. Therefore, the development and utilization of unsaturated ketone monomer compounds of camel thorn, further exploring its potential medicinal value, and determining and characterizing the structure and physicochemical properties of its monomer compounds are of great significance to the development and utilization of camel thorn. Summary of the invention

[0004] The present invention provides an unsaturated ketone component in camel thorn and a separation and extraction method and application thereof, which overcomes the shortcomings of the above-mentioned prior art. It discloses for the first time that the unsaturated ketone component in camel thorn has a certain inhibitory effect on Hela cells and can be used as an application in preparing a drug for preventing / treating cervical cancer.

[0005] One of the technical solutions of the present invention is achieved by the following measures: an unsaturated ketone component in camel thorn, whose chemical structural formula is: .

[0006] The following is a further optimization and / or improvement of one of the above-mentioned technical solutions: The unsaturated ketone components in the camel thorn are obtained by the following method: The first step is to crush the aerial part of camel thorn and soak it in ethanol, and then extract it by heating and refluxing to obtain an extract, and then recover and concentrate the extract by decompression to obtain camel thorn extract; The second step is to disperse the camel thorn extract into a suspension with water, and then extract it with an equal volume of petroleum ether to obtain a petroleum ether extract and a water layer a; The third step is to adjust the water layer a to be acidic, and then extract it with ethyl acetate in an equal volume to the water layer a to obtain an ethyl acetate extract and a water layer b; Step 4, adjusting the water layer b to alkaline, and then extracting with dichloromethane of the same volume as the water layer b to obtain a dichloromethane extract and a water layer c; Step 5, taking the extract of the dichloromethane part and separating it by silica gel column chromatography with gradient elution to obtain 10 fractions; In the sixth step, the fifth fraction among the 10 fractions obtained was purified and separated by high performance liquid chromatography gradient elution, and the eluate was collected to obtain the unsaturated ketone components in camel thorn at 14.7 minutes.

[0007] In the first step, the soaking is specifically soaking at room temperature for 3 to 4 hours.

[0008] In the first step, 8 ml to 12 ml of ethanol is added to every 1 g of camel thorn, and the mass concentration of ethanol is 95% to 98%.

[0009] In the first step, the heating temperature is 50°C to 60°C, and the number of reflux extractions is 3 to 4 times.

[0010] In the third step, hydrochloric acid is used to adjust the aqueous layer a to be acidic, with a pH value of 1 to 2.

[0011] In the fourth step, sodium hydroxide is used to adjust the aqueous layer b to alkalinity, with a pH value of 9 to 10.

[0012] In the fifth step, the silica gel column chromatography eluent is a mixture of methane and methanol, and the volume ratios of methane and methanol in the mixture are 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1, respectively.

[0013] In the sixth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water in the mixture is 20:80.

[0014] The second technical solution of the present invention is achieved by the following measures: A method for separating and extracting unsaturated ketone components in camel thorn is carried out according to the following method: The first step is to crush the aerial part of camel thorn and soak it in ethanol, and then extract it by heating and refluxing to obtain an extract, and then recover and concentrate the extract by decompression to obtain camel thorn extract; The second step is to disperse the camel thorn extract into a suspension with water, and then extract it with an equal volume of petroleum ether to obtain a petroleum ether extract and a water layer a; The third step is to adjust the water layer a to be acidic, and then extract it with ethyl acetate in an equal volume to the water layer a to obtain an ethyl acetate extract and a water layer b; Step 4, adjusting the water layer b to alkaline, and then extracting with dichloromethane of the same volume as the water layer b to obtain a dichloromethane extract and a water layer c; Step 5, taking the extract of the dichloromethane part and separating it by silica gel column chromatography with gradient elution to obtain 10 fractions; In the sixth step, the fifth fraction among the 10 fractions obtained was purified and separated by high performance liquid chromatography gradient elution, and the eluate was collected to obtain the unsaturated ketone components in camel thorn at 14.7 minutes.

[0015] The third technical solution of the present invention is achieved through the following measures: an application of unsaturated ketone components in camel thorn in the preparation of drugs for preventing / treating cervical cancer.

[0016] The present invention discloses unsaturated ketone components in camel thorn for the first time, and conducts in vitro antitumor pharmacodynamic experiments on the unsaturated ketone components in the camel thorn of the present invention. The experiments show that the unsaturated ketone components in the camel thorn of the present invention have a certain inhibitory effect on human Hela cervical cancer cells, thereby enabling the unsaturated ketone components in the camel thorn to be used in the preparation of drugs for preventing / treating cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the chemical structural formula of the unsaturated ketone component in the camel thorn of the present invention.

[0018] Figure 2 The 1H-NMR spectrum of the unsaturated ketone components in the camel thorn of the present invention.

[0019] Figure 3 The 13C-APT spectrum of the unsaturated ketone components in the camel thorn of the present invention.

[0020] Figure 4 The 1H-1H COSY spectrum of the unsaturated ketone components in Camelus thorni of the present invention.

[0021] Figure 5 The figure is the HMBC spectrum of the unsaturated ketone components in Camelus thorn of the present invention.

[0022] Figure 6 The HSQC spectrum of the unsaturated ketone components in Camel thorn of the present invention is shown in FIG.

[0023] Figure 7 The NOESY spectrum of the unsaturated ketone components in Camel thorn of the present invention. DETAILED DESCRIPTION

[0024] The present invention is not limited by the following embodiments, and the specific implementation method can be determined according to the technical scheme of the present invention and the actual situation. Unless otherwise specified, the various chemical reagents and chemicals mentioned in the present invention are all chemical reagents and chemicals known and used in the prior art; unless otherwise specified, the percentages in the present invention are all mass percentages; unless otherwise specified, the solutions in the present invention are all aqueous solutions with water as the solvent, for example, hydrochloric acid solution is a hydrochloric acid aqueous solution; the normal temperature and room temperature in the present invention generally refer to a temperature of 15°C to 25°C, and are generally defined as 25°C.

[0025] The present invention will be further described below in conjunction with embodiments: Example 1: The unsaturated ketone component in the camel thorn has the chemical structural formula: .

[0026] Example 2: As an optimization of the above example, the unsaturated ketone components in camel thorn are obtained by the following method: The first step is to crush the aerial part of camel thorn and soak it in ethanol, and then extract it by heating and refluxing to obtain an extract, and then recover and concentrate the extract by decompression to obtain camel thorn extract; The second step is to disperse the camel thorn extract into a suspension with water, and then extract it with an equal volume of petroleum ether to obtain a petroleum ether extract and a water layer a; The third step is to adjust the water layer a to be acidic, and then extract it with ethyl acetate in an equal volume to the water layer a to obtain an ethyl acetate extract and a water layer b; Step 4, adjusting the water layer b to alkaline, and then extracting with dichloromethane of the same volume as the water layer b to obtain a dichloromethane extract and a water layer c; Step 5, taking the extract of the dichloromethane part and separating it by silica gel column chromatography with gradient elution to obtain 10 fractions; In the sixth step, the fifth fraction among the 10 fractions obtained was purified and separated by high performance liquid chromatography gradient elution, and the eluate was collected to obtain the unsaturated ketone components in camel thorn at 14.7 minutes.

[0027] Example 3: As an optimization of the above example, in the first step, the soaking is specifically soaking at room temperature for 3 to 4 hours.

[0028] Example 4: As an optimization of the above example, in the first step, 8 ml to 12 ml of ethanol is added to every 1 g of camel thorn, and the mass concentration of ethanol is 95% to 98%.

[0029] Example 5: As an optimization of the above example, in the first step, the heating temperature is 50°C to 60°C, and the number of reflux extractions is 3 to 4 times.

[0030] Example 6: As an optimization of the above example, in the third step, hydrochloric acid is used to adjust the water layer a to be acidic, with a pH value of 1 to 2.

[0031] Example 7: As an optimization of the above example, in the fourth step, sodium hydroxide is used to adjust the water layer b to alkalinity, with a pH value of 9 to 10.

[0032] Example 8: As an optimization of the above example, in the fifth step, the silica gel column chromatography eluent is a mixture of methane and methanol, and the volume ratios of methane and methanol in the mixture are 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1, respectively.

[0033] Example 9: As an optimization of the above example, in the sixth step, the HPLC eluent is a mixture of methanol and water, and the volume ratio of methanol to water in the mixture of methanol and water is 20:80.

[0034] Example 10: Application of the unsaturated ketone components in Camelus thorn in the preparation of drugs for preventing / treating cervical cancer.

[0035] Embodiment 11: The unsaturated ketone components in the camel thorn are obtained by the following method: The first step is to crush the camel thorn and add ethanol with a mass concentration of 95%, soak it at room temperature for 3 hours, heat it under reflux extraction at 50°C for 3 times, combine the extracts from each reflux extraction, and recover and concentrate the extracts under reduced pressure to obtain a camel thorn extract, wherein 8 ml of ethanol with a mass concentration of 95% is added to every 1g of camel thorn; The second step is to disperse the camel thorn extract into a suspension with water, and then extract it with an equal volume of petroleum ether to obtain a petroleum ether extract and a water layer a; The third step is to adjust the aqueous layer a to acidity (pH value is 1) with hydrochloric acid, and then extract it with ethyl acetate in an equal volume to the aqueous layer a to obtain an ethyl acetate extract and an aqueous layer b; Step 4: adjust the aqueous layer b to alkaline (pH 9) with sodium hydroxide, and then extract with dichloromethane in an equal volume to the aqueous layer b to obtain a dichloromethane extract and an aqueous layer c; The fifth step is to take the extract of the dichloromethane part and separate it by gradient elution with silica gel column chromatography to obtain 10 fractions, wherein the silica gel column chromatography eluent is a mixture of methane and methanol, and the volume ratios of methane and methanol in the mixture of methane and methanol are 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1, respectively; In the sixth step, the fifth fraction among the 10 fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the unsaturated ketone components in camel thorn at 14.7 minutes, wherein the high performance liquid chromatography eluent is a mixture of methanol and water, and the volume ratio of methanol to water in the mixture of methanol and water is 20:80.

[0036] Embodiment 12: The unsaturated ketone components in the camel thorn are obtained by the following method: The first step is to crush the camel thorn and add 95% ethanol, soak it at room temperature for 4 hours, heat it under reflux extraction at 60°C for 4 times, combine the extracts from each reflux extraction, recover the extracts under reduced pressure and concentrate them to obtain a camel thorn extract, wherein 12 ml of 95% ethanol is added to every 1g of camel thorn; The second step is to disperse the camel thorn extract into a suspension with water, and then extract it with an equal volume of petroleum ether to obtain a petroleum ether extract and a water layer a; The third step is to adjust the aqueous layer a to acidity (pH value is 2) with hydrochloric acid, and then extract with ethyl acetate of equal volume to the aqueous layer a to obtain ethyl acetate extract and aqueous layer b; Step 4: adjust the aqueous layer b to alkaline (pH 10) with sodium hydroxide, and then extract with dichloromethane in an equal volume to the aqueous layer b to obtain a dichloromethane extract and an aqueous layer c; The fifth step is to take the extract of the dichloromethane part and separate it by gradient elution with silica gel column chromatography to obtain 10 fractions, wherein the silica gel column chromatography eluent is a mixture of methane and methanol, and the volume ratios of methane and methanol in the mixture of methane and methanol are 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1, respectively; In the sixth step, the fifth fraction among the 10 fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the unsaturated ketone components in camel thorn at 14.7 minutes, wherein the high performance liquid chromatography eluent is a mixture of methanol and water, and the volume ratio of methanol to water in the mixture of methanol and water is 20:80.

[0037] Embodiment 13: The unsaturated ketone components in the camel thorn are obtained by the following method: The first step is to crush the camel thorn and add 95% ethanol, soak it at room temperature for 3.5 hours, and then heat it under reflux extraction at 55°C for 3 times, combine the extracts from each reflux extraction, and recover and concentrate the extracts under reduced pressure to obtain a camel thorn extract, wherein 10 ml of 95% ethanol is added to every 1g of camel thorn; The second step is to disperse the camel thorn extract into a suspension with water, and then extract it with an equal volume of petroleum ether to obtain a petroleum ether extract and a water layer a; The third step is to adjust the aqueous layer a to acidity (pH value is 1.5) with hydrochloric acid, and then extract with ethyl acetate of equal volume to the aqueous layer a to obtain ethyl acetate extract and aqueous layer b; Step 4: adjust the aqueous layer b to alkaline (pH 9.5) with sodium hydroxide, and then extract with dichloromethane in an equal volume to the aqueous layer b to obtain a dichloromethane extract and an aqueous layer c; The fifth step is to take the extract of the dichloromethane part and separate it by gradient elution with silica gel column chromatography to obtain 10 fractions, wherein the silica gel column chromatography eluent is a mixture of methane and methanol, and the volume ratios of methane and methanol in the mixture of methane and methanol are 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1, respectively; In the sixth step, the fifth fraction among the 10 fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the unsaturated ketone components in camel thorn at 14.7 minutes, wherein the high performance liquid chromatography eluent is a mixture of methanol and water, and the volume ratio of methanol to water in the mixture of methanol and water is 20:80.

[0038] Experimental Example 1: Structural characterization analysis of unsaturated ketone components in Camel thorn of the present invention.

[0039] Experimental method: The unsaturated ketone components in the camel thorn prepared in Example 13 of the present invention were analyzed by nuclear magnetic resonance spectroscopy.

[0040] Experimental results: The 1H-NMR spectrum of the unsaturated ketone components in the camel thorn prepared in Example 13 of the present 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 the present 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 the present invention is as follows Figure 4 As shown, the HMBC spectrum of the unsaturated ketone components in the camel thorn prepared in Example 13 of the present invention is as follows Figure 5 As shown, the HSQC spectrum of the unsaturated ketone components in camel thorn prepared in Example 13 of the present invention is as follows Figure 6 As shown, the NOESY spectrum of the unsaturated ketone components in the camel thorn prepared in Example 13 of the present invention is as follows Figure 7 As shown, Figures 2 to 7 Perform graph analysis and Figures 2 to 3 Each peak is assigned, Figures 2 to 3 The peak attribution is shown in Table 1. From the data in Table 1, it can be seen that the chemical structural formula of the unsaturated ketone component in the camel thorn prepared in Example 13 of the present invention is as follows: Figure 1As shown, the unsaturated ketone components in the camel thorn are easily soluble in methanol.

[0041] Experimental Example 2: In vitro antitumor pharmacodynamics experiment of unsaturated ketone components in Camel thorn of the present invention Experimental method: The unsaturated ketone components in camel thorn prepared in Example 13 of the present invention were subjected to an in vitro antitumor pharmacodynamics experiment, and the in vitro antitumor pharmacodynamics experiment used the MTT colorimetric method. The specific operation is as follows: the unsaturated ketone components in camel thorn prepared in Example 13 of the present invention were used as the experimental group, and cisplatin was used as the control group. Hela (human cervical cancer cell) cells were selected as the experimental objects in the experimental group and the control group. After the culture medium was diluted, it was inoculated in a 96-well plate at a density of 6×104, 100 μL per well. After normal culture in the incubator for 24 hours, the corresponding drugs were added to each group, so that the final concentrations of the drugs in each group were 6.25 μM (Group 1), 12.5 μM (Group 2), 25 μM (Group 3), 50 μM (Group 4), and 100 μM (Group 5), respectively. There were 5 concentrations in total, and 3 replicates for each concentration; after 48 hours of culture, 10 μL of MTT was added to each well for staining; after continuing to culture for four hours, the original culture medium was aspirated and discarded, and DMSO was added to each well 150 μL was placed on a shaker and oscillated at a low speed for 10 min to fully dissolve the crystals. The optical density value was detected at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) and the 50% inhibition concentration (IC50, μM) was calculated based on the optical density value. The method for calculating IC50 based on the optical density value is a well-known technique.

[0042] Experimental results: The IC50 results of the unsaturated ketone components in the camel thorn of the present invention on Hela cells are shown in Table 2. It can be seen from Table 2 that the unsaturated ketone components in the camel thorn of the present invention have a certain inhibitory effect on Hela cells.

[0043] In summary, the present invention discloses the unsaturated ketone components in camel thorn for the first time, and conducts in vitro antitumor pharmacodynamic experiments on the unsaturated ketone components in camel thorn of the present invention. The experiments show that the unsaturated ketone components in camel thorn of the present 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 preventing / treating cervical cancer.

[0044] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Non-essential technical features can be added or reduced according to actual needs to meet the requirements of different situations.

Claims

1. An unsaturated ketone component in camel thorn, characterized in that Its chemical structure is: 。 2. The unsaturated ketone component in camel thorn according to claim 1, characterized in that Obtained as follows: The first step is to crush the aerial part of camel thorn and soak it in ethanol, and then extract it by heating and refluxing to obtain an extract, and then recover and concentrate the extract by decompression to obtain camel thorn extract; The second step is to disperse the camel thorn extract into a suspension with water, and then extract it with an equal volume of petroleum ether to obtain a petroleum ether extract and a water layer a; The third step is to adjust the water layer a to be acidic, and then extract it with ethyl acetate in an equal volume to the water layer a to obtain an ethyl acetate extract and a water layer b; Step 4, adjusting the water layer b to alkaline, and then extracting with dichloromethane of the same volume as the water layer b to obtain a dichloromethane extract and a water layer c; Step 5, taking the extract of the dichloromethane part and separating it by silica gel column chromatography with gradient elution to obtain 10 fractions; In the sixth step, the fifth fraction among the 10 fractions obtained was purified and separated by high performance liquid chromatography gradient elution, and the eluate was collected to obtain the unsaturated ketone components in camel thorn at 14.7 minutes.

3. The unsaturated ketone component in camel thorn according to claim 2, characterized in that In the first step, the soaking is specifically soaking 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, and the mass concentration of ethanol is 95% to 98%.

4. The unsaturated ketone component in camel thorn according to claim 2 or 3, characterized in that In the first step, the heating temperature is 50° C. to 60° C., and the number of reflux extractions is 3 to 4 times.

5. The unsaturated ketone component in camel thorn according to claim 2, 3 or 4, characterized in that In the third step, the aqueous layer a is adjusted to be acidic with a pH value of 1 to 2 using hydrochloric acid.

6. The unsaturated ketone component in camel thorn according to any one of claims 2 to 5, characterized in that In the fourth step, the aqueous layer b is adjusted to be alkaline with a pH value of 9 to 10 using sodium hydroxide.

7. The unsaturated ketone component in camel thorn according to any one of claims 2 to 6, characterized in that In the fifth step, the silica gel column chromatography eluent is a mixture of methane and methanol, and the volume ratios of methane to methanol in the mixture are 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1, respectively.

8. The unsaturated ketone component in camel thorn according to any one of claims 2 to 7, characterized in that In the sixth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water in the mixture is 20:

80.

9. A method for separating and extracting unsaturated ketone components in camel thorn according to any one of claims 1, 3 to 8, characterized in that Proceed as follows: The first step is to crush the aerial part of camel thorn and soak it in ethanol, and then extract it by heating and refluxing to obtain an extract, and then recover and concentrate the extract by decompression to obtain camel thorn extract; The second step is to disperse the camel thorn extract into a suspension with water, and then extract it with an equal volume of petroleum ether to obtain a petroleum ether extract and a water layer a; The third step is to adjust the water layer a to be acidic, and then extract it with ethyl acetate in an equal volume to the water layer a to obtain an ethyl acetate extract and a water layer b; Step 4, adjusting the water layer b to alkaline, and then extracting with dichloromethane of the same volume as the water layer b to obtain a dichloromethane extract and a water layer c; Step 5, taking the extract of the dichloromethane part and separating it by gradient elution on a silica gel column chromatography to obtain 10 fractions; In the sixth step, the fifth fraction among the 10 fractions obtained was purified and separated by high performance liquid chromatography gradient elution, and the eluate was collected to obtain the unsaturated ketone components in camel thorn at 14.7 minutes.

10. Use of the unsaturated ketone components in Camelus thorni according to any one of claims 1 to 8 in the preparation of a drug for preventing and / or treating cervical cancer.

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