Phenylpropanoid component in alhagi sparsifolia as well as extraction and separation method and application of phenylpropanoid component
Through a multi-step extraction and isolation method, the phenylened component was successfully extracted and isolated from camel pricks, and its inhibitory effect on cervical cancer cells was demonstrated, which solved the problem of insufficient application of this component in the prior art and achieved its potential application in cervical cancer drugs.
Patent Information
- Application Number
- CN202510034404.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-04-11
AI Technical Summary
The prior art is difficult to effectively utilize the phenylened ingredients in camel pricks, especially in their use as a preparation of preventive or anti-cervical cancer drugs.
A multi-step extraction and separation method, including ethanol soaking and heating reflux extraction of the above-ground portion of the dried camel spines, followed by extraction using petroleum ether, ethyl acetate and dichloromethane, and gradient elution and purification by silica gel column chromatography, ODS column chromatography, gel column chromatography and high performance liquid chromatography, finally obtaining the target product styrene-like components.
Effective extraction and isolation of phenylened components in camel auger was achieved, and it was shown that this component has a strong inhibitory effect on human cervical cancer cells (Hela cells), so it can be used to prepare preventive or anti-cervical cancer drugs.
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Abstract
Description
[0001] The invention relates to the technical field of camel thorn separation and purification, and discloses a phenylpropanoid component in camel thorn and an extraction and separation method and application thereof. Background Art
[0002] Camel thorn Alhagi sparsifolia Camel thorn is a semi-shrub of the genus Camelus in the Leguminosae family. It is mainly distributed in plain deserts and semi-desert areas in Xinjiang, Inner Mongolia, Gansu and other provinces and regions. It is used by the people to treat rheumatism and cancer. Camel thorn contains flavonoids, lignans, organic acids, alkaloids, terpenes and other ingredients, and has good biological activity in treating diarrhea and anti-tumor. Malignant tumors seriously threaten human life and health. Modern pharmacological studies have shown that camel thorn also has potential application value in cancer treatment. Camel thorn is rich in chemical components, has strong anti-tumor activity, is cheap and widely available, which has aroused the interest of many researchers.
[0003] One of the medicinal effects of camel thorn mainly comes from phenylpropanoid components. Therefore, the development and utilization of phenylpropanoid 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 a phenylpropanoid component in camel thorn and a method for extracting and separating the component and application thereof, which overcomes the deficiencies of the above-mentioned prior art and discloses for the first time the phenylpropanoid component in camel thorn and its application in preparing a drug for preventing cervical cancer or / and preparing an anti-cervical cancer drug.
[0005] One of the technical solutions of the present invention is achieved by the following measures: a phenylpropanoid component in camel thorn, whose chemical structural formula is: Among them, the phenylpropanoid component in camel thorn is the abbreviation of the compound (1S,2R)-3-ethoxy-1-(4-hydroxy-3,5-dimethoxyphenyl)propane-1,2-diol.
[0006] The following is a further optimization and / or improvement of one of the above-mentioned technical solutions: The phenylpropanoid components in the camel thorn are obtained according to the following steps: The first step is to grind the dried aerial part of Camel thorn and add ethanol, soak it at room temperature for 3.5 hours to 4 hours, and then heat and reflux extract it at 50° C. to 60° C. for 3 times, each time for 1.5 hours to 3 hours, combine the reflux extracts of each time, recover them under reduced pressure, and concentrate them to obtain Camel thorn extract; The second step is to disperse the camel thorn extract with water, and extract it with petroleum ether, ethyl acetate and dichloromethane in sequence to obtain extracts of petroleum ether part, ethyl acetate part and dichloromethane part; The third 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 gradient eluent of the silica gel column chromatography includes dichloromethane and methanol; In the fourth step, the fourth fraction of the obtained 10 fractions is subjected to ODS column chromatography gradient elution separation to obtain 8 fractions, wherein the ODS column chromatography gradient eluent includes methanol and water; Step 5, the second fraction among the eight fractions obtained is further separated by gel column chromatography to obtain nine fractions; In the sixth step, the fourth fraction of the nine fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the target product phenylpropanoid component at 16.9 minutes, wherein the high performance liquid chromatography eluent is a mixture of methanol and water.
[0007] In the first step, 8 ml to 12 ml of 95% ethanol is added to every 1 g of dried camel thorn.
[0008] In the above second step, when ethyl acetate is used for extraction, 0.01 mol / L hydrochloric acid solution is added to adjust the pH value of the aqueous phase to 1 to 2. When dichloromethane is used for extraction, 0.1 mol / L sodium hydroxide solution is added to adjust the pH value of the aqueous phase to 9 to 10.
[0009] In the above third step, the volume ratios of dichloromethane and methanol are 1:0, 100:1, 50:1, 25:1, 10:1, 8:1, 5:1, 3:1, 1:1, and 0:1, respectively.
[0010] In the above fourth step, the volume ratios of methanol and water are 10:90, 20:80, 30:70, 40:60, 50:50, 80:20, 90:10 and 100:0 respectively.
[0011] In the sixth step, the volume ratio of methanol to water is 30:70.
[0012] The second technical solution of the present invention is achieved by the following measures: A method for extracting and separating phenylpropanoid components from camel thorn is carried out according to the following steps: The first step is to grind the dried aerial part of Camel thorn and add ethanol, soak it at room temperature for 3.5 hours to 4 hours, and then heat and reflux extract it at 50° C. to 60° C. for 3 times, each time for 1.5 hours to 3 hours, combine the reflux extracts of each time, recover them under reduced pressure, and concentrate them to obtain Camel thorn extract; The second step is to disperse the camel thorn extract with water, and extract it with petroleum ether, ethyl acetate and dichloromethane in sequence to obtain extracts of petroleum ether part, ethyl acetate part and dichloromethane part; The third 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 gradient eluent of the silica gel column chromatography includes dichloromethane and methanol; In the fourth step, the fourth fraction of the obtained 10 fractions is subjected to ODS column chromatography gradient elution separation to obtain 8 fractions, wherein the ODS column chromatography gradient eluent includes methanol and water; Step 5, the second fraction among the eight fractions obtained is further separated by gel column chromatography to obtain nine fractions; In the sixth step, the fourth fraction of the nine fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the target product phenylpropanoid component at 16.9 minutes, wherein the high performance liquid chromatography eluent is a mixture of methanol and water.
[0013] The third technical solution of the present invention is achieved through the following measures: an application of phenylpropanoid components in camel thorn as a drug for preparing a drug for preventing cervical cancer.
[0014] The fourth technical solution of the present invention is achieved through the following measures: an application of phenylpropanoid components in camel thorn as a preparation of anti-cervical cancer drugs.
[0015] The present invention discloses for the first time the phenylpropanoid components in camel thorn, and conducts an in vitro anticancer pharmacodynamics experiment on Hela (human cervical cancer cell) cells with the compound. The experiment clearly shows that the compound has a strong inhibitory effect on Hela (human cervical cancer cell) cells, thereby enabling the compound to be used for preparing drugs for preventing cervical cancer and / or preparing drugs for fighting cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Attached Figure 1 The figure is a chemical structure diagram of the phenylpropanoid component described in the present invention.
[0017] Attached Figure 2 The phenylpropanoid component of the present invention 1 H-NMR spectrum.
[0018] Attached Figure 3 The phenylpropanoid component of the present invention 13 C-APT spectrum.
[0019] Attached Figure 4 The phenylpropanoid component of the present invention 1 H- 1 H COSY spectrum.
[0020] Attached Figure 5 It is the HMBC spectrum of the phenylpropanoid components described in the present invention.
[0021] Attached Figure 6 It is the HSQC spectrum of the phenylpropanoid components described in the present invention.
[0022] Attached Figure 7 It is the NOESY spectrum of the phenylpropanoid components described in the present invention. DETAILED DESCRIPTION
[0023] 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 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.
[0024] The present invention will be further described below in conjunction with embodiments: Example 1: The chemical structure of the phenylpropanoid component in the camel thorn is: Among them, the phenylpropanoid component in camel thorn is the abbreviation of the compound (1S,2R)-3-ethoxy-1-(4-hydroxy-3,5-dimethoxyphenyl)propane-1,2-diol.
[0025] The phenylpropanoid components in camel thorn described in the present invention are subjected to nuclear magnetic resonance spectroscopy analysis. 1 H-NMR spectrum Figure 2 As shown in Figure 2, the phenylpropanoid components in camel thorn 13 C-APT spectrum Figure 3 As shown in Figure 2, the phenylpropanoid components in camel thorn 1 H- 1 H COSY spectrum Figure 4 As shown in Figure 2, the HMBC spectrum of phenylpropanoid components in camel thorn is as follows Figure 5 As shown in the figure, the HSQC spectrum of phenylpropanoid components in camel thorn is as follows Figure 6 As shown in Figure 2, the NOESY spectrum of phenylpropanoid components in camel thorn is as follows: Figure 7 As shown, Figures 2 to 7 Perform spectrum analysis and compare spectrum 2 and Figure 3 Each peak is assigned, Figure 2 and Figure 3The peak attribution is shown in Table 1. From the data in Table 1, it can be seen that the chemical structural formula of the phenylpropanoid component in the camel thorn of the present invention is as follows Figure 1 As shown, it is easily soluble in methanol.
[0026] Embodiment 2: As an optimization of the above embodiment, the following steps are performed: The first step is to grind the dried aerial part of Camel thorn and add ethanol, soak it at room temperature for 3.5 hours to 4 hours, and then heat and reflux extract it at 50° C. to 60° C. for 3 times, each time for 1.5 hours to 3 hours, combine the reflux extracts of each time, recover them under reduced pressure, and concentrate them to obtain Camel thorn extract; The second step is to disperse the camel thorn extract with water, extract it with petroleum ether, ethyl acetate and dichloromethane in sequence, and obtain extracts of petroleum ether part, ethyl acetate part and dichloromethane part; The third 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 gradient eluent of the silica gel column chromatography includes dichloromethane and methanol; In the fourth step, the fourth fraction of the obtained 10 fractions is subjected to ODS column chromatography gradient elution separation to obtain 8 fractions, wherein the ODS column chromatography gradient eluent includes methanol and water; Step 5, the second fraction among the eight fractions obtained is further separated by gel column chromatography to obtain nine fractions; In the sixth step, the fourth fraction of the nine fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the target product phenylpropanoid component at 16.9 minutes, wherein the high performance liquid chromatography eluent is a mixture of methanol and water.
[0027] Example 3: As an optimization of the above example, in the first step, 8 ml to 12 ml of 95% ethanol is added to every 1 g of dried camel thorn.
[0028] Example 4: As an optimization of the above example, in the second step, when ethyl acetate is used for extraction, 0.01 mol / L hydrochloric acid solution is added to adjust the pH value of the aqueous phase to 1 to 2. When dichloromethane is used for extraction, 0.1 mol / L sodium hydroxide solution is added to adjust the pH value of the aqueous phase to 9 to 10.
[0029] Example 5: As an optimization of the above example, in the third step, the volume ratios of dichloromethane and methanol are 1:0, 100:1, 50:1, 25:1, 10:1, 8:1, 5:1, 3:1, 1:1, and 0:1, respectively.
[0030] Example 6: As an optimization of the above example, in the fourth step, the volume ratios of methanol and water are 10:90, 20:80, 30:70, 40:60, 50:50, 80:20, 90:10, and 100:0, respectively.
[0031] Example 7: As an optimization of the above example, in the sixth step, the volume ratio of methanol to water is 30:70.
[0032] Embodiment 8: The method for extracting and separating phenylpropanoid components from Camel thorn is characterized by following the steps below: The first step is to grind the dried aerial part of Camel thorn and add ethanol, soak it at room temperature for 3.5 hours to 4 hours, and then heat and reflux extract it at 50° C. to 60° C. for 3 times, each time for 1.5 hours to 3 hours, combine the reflux extracts of each time, recover them under reduced pressure, and concentrate them to obtain Camel thorn extract; The second step is to disperse the camel thorn extract with water, extract it with petroleum ether, ethyl acetate and dichloromethane in sequence, and obtain extracts of petroleum ether part, ethyl acetate part and dichloromethane part; The third 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 gradient eluent of the silica gel column chromatography includes dichloromethane and methanol; In the fourth step, the fourth fraction of the obtained 10 fractions is subjected to ODS column chromatography gradient elution separation to obtain 8 fractions, wherein the ODS column chromatography gradient eluent includes methanol and water; Step 5, the second fraction among the eight fractions obtained is further separated by gel column chromatography to obtain nine fractions; In the sixth step, the fourth fraction of the nine fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the target product phenylpropanoid component at 16.9 minutes, wherein the high performance liquid chromatography eluent is a mixture of methanol and water.
[0033] Example 9: Application of the phenylpropanoid components in Camel thorn as a drug for the prevention of cervical cancer.
[0034] Example 10: Application of the phenylpropanoid components in Camelus thorn as a preparation of anti-cervical cancer drugs.
[0035] Example 11: The phenylpropanoid component in the camel thorn is obtained according to the following method: The phenylpropanoid component in the camel thorn is obtained according to the following method: The first step is to crush 50 g of the dried aerial part of Camel thorn and add 400 mL of ethanol, soak at room temperature for 3 hours, and then heat and reflux for extraction at 50°C for 3 times, each time for 2 hours, combine the reflux extracts of each time, recover under reduced pressure, and concentrate to obtain Camel thorn extract; The second step is to disperse the camel thorn extract with water, extract with petroleum ether, ethyl acetate and dichloromethane in sequence, add 0.01 mol / L hydrochloric acid solution when ethyl acetate is used for extraction, and adjust the pH value of the aqueous phase to 1.8, and add 0.1 mol / L sodium hydroxide solution when dichloromethane is used for extraction, and adjust the pH value of the aqueous phase to 9, and finally obtain 43.0 g of dichloromethane extract; The third step is to obtain 10 fractions by gradient elution of the extract of the dichloromethane part by silica gel column chromatography, wherein the gradient eluent of the silica gel column chromatography includes dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 1:0, 100:1, 50:1, 25:1, 10:1, 8:1, 5:1, 3:1, 1:1, and 0:1, respectively; In the fourth step, the fourth fraction of the 10 fractions is subjected to gradient elution separation by ODS column chromatography to obtain 8 fractions, wherein the gradient eluent of the ODS column chromatography comprises methanol and water, and the volume ratio of methanol to water is 10:90, 20:80, 30:70, 40:60, 50:50, 80:20, 90:10, and 100:0, respectively; Step 5, the second fraction among the eight fractions obtained is further separated by gel column chromatography to obtain nine fractions; In the sixth step, the fourth fraction of the nine fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the target product phenylpropanoid component at 16.9 minutes, wherein the high performance liquid chromatography eluent is a mixture of methanol and water, and the volume ratio of methanol to water is 30:70.
[0036] The phenylpropanoid components in the camel thorn described in the present invention are subjected to an in vitro anti-tumor pharmacodynamics experiment, and the in vitro anti-tumor pharmacodynamics experiment utilizes the MTT colorimetric method.
[0037] The phenylpropanoid components in camel thorn 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 both the experimental and control groups. After the culture medium was diluted, 6×10 4 The density of the cells was inoculated in a 96-well plate, with 100 μL per well. After normal culture in an 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 culture for 48 hours, 10 μL of MTT was added to each well for staining. After further culture for four hours, the original culture medium was discarded, 150 μL of DMSO was added to each well, and the cells were shaken at a low speed on a shaker for 10 minutes to fully dissolve the crystals. The optical density value was detected at a wavelength of 570 nm using an enzyme-linked immunosorbent assay, and the 50% inhibitory concentration (IC) was calculated based on the optical density value. 50, μM), optical density value was used to calculate IC 50 The calculation method of the IC of the experimental group and the control group on Hela cells is known in the art. 50 The numerical values are shown in Table 2. It can be seen from the data in Table 2 that the phenylpropanoid components in the camel thorn described in the present invention have a certain inhibitory effect on Hela cells.
[0038] In summary, the present invention discloses for the first time the phenylpropanoid components in camel thorn, and the phenylpropanoid components in camel thorn described in the present invention have a certain inhibitory effect on Hela cells, so that the phenylpropanoid components in camel thorn of the present invention can be used for preparing drugs for preventing cervical cancer and / or preparing anti-cervical cancer drugs.
[0039] The above technical features respectively constitute various 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. A phenylpropanoid component in camel thorn, characterized in that Its chemical structure is: 。 2. The phenylpropanoid component in camel thorn according to claim 1, characterized in that Follow the steps below to get: The first step is to grind the dried aerial part of Camel thorn and add ethanol, soak it at room temperature for 3.5 hours to 4 hours, and then heat and reflux extract it at 50° C. to 60° C. for 3 times, each time for 1.5 hours to 3 hours, combine the reflux extracts of each time, recover them under reduced pressure, and concentrate them to obtain Camel thorn extract; The second step is to disperse the camel thorn extract with water, and extract it with petroleum ether, ethyl acetate and dichloromethane in sequence to obtain extracts of petroleum ether part, ethyl acetate part and dichloromethane part; The third 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 gradient eluent of the silica gel column chromatography includes dichloromethane and methanol; In the fourth step, the fourth fraction of the obtained 10 fractions is subjected to ODS column chromatography gradient elution separation to obtain 8 fractions, wherein the ODS column chromatography gradient eluent includes methanol and water; Step 5, the second fraction among the eight fractions obtained is further separated by gel column chromatography to obtain nine fractions; In the sixth step, the fourth fraction of the nine fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the target product phenylpropanoid component at 16.9 minutes, wherein the high performance liquid chromatography eluent is a mixture of methanol and water.
3. The phenylpropanoid component in camel thorn according to claim 2, characterized in that In the first step, 8 ml to 12 ml of 95% ethanol was added per 1 g of dried camel thorn.
4. The phenylpropanoid component in camel thorn according to claim 2 or 3, characterized in that In the second step, when ethyl acetate is used for extraction, 0.01 mol / L hydrochloric acid solution is added to adjust the pH value of the aqueous phase to 1 to 2. When dichloromethane is used for extraction, 0.1 mol / L sodium hydroxide solution is added to adjust the pH value of the aqueous phase to 9 to 10.
5. The phenylpropanoid component in camel thorn according to claim 2, 3 or 4, characterized in that In the third step, the volume ratios of dichloromethane and methanol are 1:0, 100:1, 50:1, 25:1, 10:1, 8:1, 5:1, 3:1, 1:1, and 0:1, respectively.
6. The phenylpropanoid component in camel thorn according to claim 2, 3, 4 or 5, characterized in that In the fourth step, the volume ratios of methanol and water are 10:90, 20:80, 30:70, 40:60, 50:50, 80:20, 90:10, and 100:0, respectively.
7. The phenylpropanoid component in camel thorn according to claim 2, 3, 4, 5 or 6, characterized in that In the sixth step, the volume ratio of methanol to water is 30:
70.
8. A method for extracting and separating phenylpropanoid components from camel thorn according to any one of claims 1 to 7, characterized in that Follow the steps below: The first step is to grind the dried aerial part of Camel thorn and add ethanol, soak it at room temperature for 3.5 hours to 4 hours, and then heat and reflux extract it at 50° C. to 60° C. for 3 times, each time for 1.5 hours to 3 hours, combine the reflux extracts of each time, recover them under reduced pressure, and concentrate them to obtain Camel thorn extract; The second step is to disperse the camel thorn extract with water, and extract it with petroleum ether, ethyl acetate and dichloromethane in sequence to obtain extracts of petroleum ether part, ethyl acetate part and dichloromethane part; The third 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 gradient eluent of the silica gel column chromatography includes dichloromethane and methanol; In the fourth step, the fourth fraction of the obtained 10 fractions is subjected to ODS column chromatography gradient elution separation to obtain 8 fractions, wherein the ODS column chromatography gradient eluent includes methanol and water; Step 5, the second fraction among the eight fractions obtained is further separated by gel column chromatography to obtain nine fractions; In the sixth step, the fourth fraction of the nine fractions obtained is purified and separated by high performance liquid chromatography gradient elution, and the eluate is collected to obtain the target product phenylpropanoid component at 16.9 minutes, wherein the high performance liquid chromatography eluent is a mixture of methanol and water.
9. Use of the phenylpropanoid component in camel thorn according to any one of claims 1 to 7 as a drug for preventing cervical cancer.
10. Use of the phenylpropanoid component in Camelus thorn according to any one of claims 1 to 7 as a drug for preparing an anti-cervical cancer drug.