Ornithine alkaloids from camel thorn and their extraction method and application

Abstract

The present invention relates to the technical field of camel thorn separation and purification, and discloses an ornithine alkaloid from camel thorn, an extraction method thereof, and an application thereof. The method comprises: crushing the dried aerial part of camel thorn, adding methanol, and heating under reflux for extraction. The obtained total extract of camel thorn is dispersed with water, extracted with petroleum ether, ethyl acetate, and dichloromethane, and concentrated. The dichloromethane extract is separated by gradient elution on a silica gel column, and the fifth fraction is purified and separated by gradient elution on a high-performance liquid chromatography (HPLC), and the eluate is collected to obtain the ornithine alkaloid from camel thorn at 7.3 minutes. The present invention discloses for the first time 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one from camel thorn, which has a certain inhibitory effect on human HeLa cervical cancer cells, thereby enabling the application of such compounds from camel thorn as potential anti-cervical cancer drugs.

Description

Technical Field

[0001] The invention relates to the technical field of camel thorn separation and purification, and discloses ornithine alkaloids in camel thorn, an extraction method and application thereof, wherein the ornithine alkaloids in camel thorn are 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in the camel thorn. Background Art

[0002] Camel thorn (Alhagis parsifolia Shap) is a subshrub of the genus Alhagi Gagneb, subfamily Papilionoideae, Leguminosae family. It stands 25 to 40 cm tall, with an erect, finely striated stem and alternate, ovate, obovate, or obovate-rounded, glabrous leaves with a short petiole. Camel thorn grows primarily in arid and semi-arid regions, including northwest my country, Turkmenistan, and Pakistan, with Xinjiang being the primary production area. Due to its unique habitat, research on its active ingredients has been limited. Alkaloids, one of its primary biological activities, possess numerous benefits, including anti-tumor and diarrhea treatment. Clinically, it is primarily used for a variety of conditions, including cervical cancer and irritable bowel syndrome. Malignant tumors pose a serious threat to human life and health. Given the high cost of many new anticancer drugs and the potential for drug resistance and side effects with long-term use, the search for highly effective and low-toxic anticancer drugs is crucial for cancer treatment. The alkaloid components in camel thorn have strong anti-tumor activity, are cheap and widely available, and have attracted the interest of many researchers.

[0003] One of the medicinal effects of camel thorn mainly comes from alkaloid compounds. Therefore, the development and utilization of ornithine alkaloid monomer compounds of camel thorn, further exploring their potential medicinal value, and determining and characterizing the structure and physicochemical properties of their monomer compounds are of great significance to the development and utilization of camel thorn. Summary of the Invention

[0004] The present invention provides an ornithine alkaloid in camel thorn, an extraction method and an application thereof, overcoming the deficiencies of the above-mentioned prior art. The invention discloses for the first time an ornithine alkaloid in camel thorn, namely 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in camel thorn, which can be used for preparing drugs for preventing cervical cancer and anti-tumor drugs.

[0005] One of the technical solutions of the present invention is achieved by the following measures: 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one from camel thorn, i.e., an ornithine alkaloid from camel thorn, has the chemical formula:

[0006] .

[0007] The second technical solution of the present invention is achieved by the following measures: A method for extracting 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one from camel spur is carried out according to the following method:

[0008] The first step is to grind the required amount of dried aerial part of camel thorn and add methanol, soak at room temperature, heat and reflux to extract, combine the reflux extracts, recover under reduced pressure, and concentrate to obtain the camel thorn total extract;

[0009] The second step is to disperse the camel thorn total extract into a suspension with water, extract it with petroleum ether, ethyl acetate and dichloromethane in sequence, and concentrate the extract to obtain petroleum ether extract, ethyl acetate extract and dichloromethane extract respectively;

[0010] The third step is to take the extract of the dichloromethane part and separate it by silica gel column chromatography with gradient elution to obtain 10 fractions;

[0011] In the fourth step, the fifth fraction among the 10 fractions was purified and separated by high performance liquid chromatography gradient elution, and the eluate was collected to obtain 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in camel thorn at 7.3 minutes, that is, the ornithine alkaloid in camel thorn.

[0012] The following is a further optimization and / or improvement of the second technical solution of the above invention:

[0013] In the first step, 8 ml to 12 ml of methanol was added per 1 g of the dried aerial parts of Altissima thorn.

[0014] In the first step, the soaking time is 3 to 4 hours, and the conditions for heating reflux extraction are: heating temperature of 50° C. to 60° C., reflux extraction 3 times, and each extraction time of 1 to 3 hours.

[0015] In the third step, 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, 20:1, 10:1, 5:1, 1:1, and 0:1.

[0016] In the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 20:80.

[0017] The third technical solution of the present invention is achieved through the following measures: an application of 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in camel thorn as a drug for the prevention of cervical cancer.

[0018] The fourth technical solution of the present invention is achieved through the following measures: an application of 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in camel thorn as a drug for the preparation of a drug for treating cervical cancer.

[0019] The fifth technical solution of the present invention is achieved through the following measures: an application of 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in camel spur as a health product for the prevention and treatment of cervical cancer.

[0020] The present invention discloses for the first time 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in camel thorn, which has a certain inhibitory effect on human Hela cervical cancer cells, thereby enabling the application of such compounds in camel thorn as potential anti-cervical cancer tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Attachment Figure 1 The present invention is 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in camel spur 1 H-NMR spectrum.

[0022] Attachment Figure 2 The present invention is 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in camel spur 13 C-APT spectrum.

[0023] Attachment Figure 3 The present invention is 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in camel spur 1 H- 1 HCOSY spectrum.

[0024] Attachment Figure 4 The figure is the HMBC spectrum of 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in the camel thorn of the present invention.

[0025] Attachment Figure 5 The HSQC spectrum of 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in the camel thorn of the present invention is shown in FIG.

[0026] Attachment Figure 6 The NOESY spectrum of 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in the camel thorn of the present invention is shown in FIG. DETAILED DESCRIPTION

[0027] The present invention is not limited to the following embodiments, and specific implementation methods can be determined based on the technical solutions of the present invention and actual conditions. Unless otherwise specified, the various chemical reagents and chemicals mentioned in the present invention are all chemical reagents and chemicals commonly known in the prior art; unless otherwise specified, the percentages in the present invention are all percentages by mass; unless otherwise specified, the solutions in the present invention are all aqueous solutions with water as the solvent, for example, hydrochloric acid solution is an aqueous hydrochloric acid solution; normal temperature and room temperature in the present invention generally refer to temperatures between 15°C and 25°C, and are generally defined as 25°C.

[0028] The present invention will be further described below in conjunction with the embodiments:

[0029] Example 1: 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in Camelus thorni, i.e., an ornithine alkaloid in Camelus thorni, has the chemical structural formula:

[0030] .

[0031] Example 2: 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one from Camelus spur was extracted according to the following method:

[0032] The first step is to grind the required amount of dried aerial part of camel thorn and add methanol, soak at room temperature, heat and reflux to extract, combine the reflux extracts, recover under reduced pressure, and concentrate to obtain the camel thorn total extract;

[0033] The second step is to disperse the camel thorn total extract into a suspension with water, extract it with petroleum ether, ethyl acetate and dichloromethane in sequence, and concentrate the extract to obtain petroleum ether extract, ethyl acetate extract and dichloromethane extract respectively;

[0034] The third step is to take the extract of the dichloromethane part and separate it by silica gel column chromatography with gradient elution to obtain 10 fractions;

[0035] In the fourth step, the fifth fraction among the 10 fractions was purified and separated by high performance liquid chromatography gradient elution, and the eluate was collected to obtain 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in camel thorn at 7.3 minutes, that is, the ornithine alkaloid in camel thorn.

[0036] Example 3: As an optimization of the above example, in the first step, 8 ml to 12 ml of methanol was added to every 1 g of the dried aerial parts of Altissima thorn.

[0037] Example 4: As an optimization of the above example, in the first step, the soaking time is 3 to 4 hours, and the conditions for heating reflux extraction are: heating temperature is 50°C to 60°C, reflux extraction is performed 3 times, and each extraction is 1 to 3 hours.

[0038] Example 5: As an optimization of the above example, in the third step, the silica gel column chromatography gradient eluent includes dichloromethane and methanol, and the volume ratios of dichloromethane and methanol are 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1, respectively.

[0039] Example 6: As an optimization of the above example, in the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 20:80.

[0040] Example 7: Use of 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in Camelus thorn as a drug for preventing cervical cancer.

[0041] Example 8: Use of 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in Camelus thorn as a drug for treating cervical cancer.

[0042] Example 9: Application of 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in Camel thorn as a health product for preventing and treating cervical cancer.

[0043] Example 10: 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one from Camelus spur was extracted according to the following method:

[0044] The first step is to grind the dried aerial part of camel thorn and add methanol, adding 8ml to 12ml of methanol for every 1g of camel thorn, soaking at room temperature for 3 hours, heating and refluxing at 50°C for 3 times, each extraction time for 3 hours, combining the reflux extracts, recovering under reduced pressure, and concentrating to obtain the camel thorn total extract;

[0045] The second step is to disperse the camel thorn total extract into a suspension with water, extract it with petroleum ether, ethyl acetate and dichloromethane in sequence, and concentrate the extract to obtain petroleum ether extract, ethyl acetate extract and dichloromethane extract respectively;

[0046] The third step is to take the dichloromethane extract and separate it by silica gel column chromatography gradient elution to obtain 10 fractions, wherein the silica gel column chromatography gradient eluent includes dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1 respectively;

[0047] In the fourth step, the fifth fraction among the 10 fractions was purified and separated by high performance liquid chromatography gradient elution, and the eluate was collected to obtain 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in camel thorn at 7.3 minutes, that is, the ornithine alkaloid in camel thorn, wherein the high performance liquid chromatography eluent is a mixture of methanol and water in a volume ratio of 20:80.

[0048] The 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one (i.e., the ornithine alkaloid in camel thorn) obtained in Example 10 was subjected to nuclear magnetic resonance spectroscopy analysis.

[0049] The 1H-NMR spectrum of 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in the camel spur of the present invention is as follows: Figure 1 As shown, the 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in camel spur of the present invention 13 C-APT spectrum Figure 2 As shown, the 1H-1HCOSY spectrum of 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in the camel thorn of the present invention is as follows Figure 3 As shown, the HMBC spectrum of 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in the camel thorn of the present invention is as follows Figure 4As shown, the HSQC spectrum of 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in the camel thorn of the present invention is as follows Figure 5 As shown, the NOESY spectrum of 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in the camel thorn of the present invention is as follows Figure 6 As shown, Figures 1 to 6 Perform spectrum analysis and compare spectrum 1 and Figure 2 Each peak is assigned, Figure 1 and Figure 2 The peak assignments are shown in Table 1. From the data in Table 1, it can be seen that the chemical structure of 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in the camel spur of the present invention is as follows, and it is easily soluble in methanol:

[0050] .

[0051] The 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in the camel spur of the present invention is subjected to an in vitro anti-tumor pharmacodynamics experiment, and the in vitro anti-tumor pharmacodynamics experiment utilizes an MTT colorimetric method.

[0052] 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one was used as the experimental group, 5-FU was used as the control group, and a blank group was set up. Hela (human cervical cancer cell) cells were selected as experimental objects in the experimental group, the control group and the blank group. After the culture medium was diluted, 6×10 4 The cells were inoculated at a density of 100 μL per well in a 96-well plate. 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 12.5 μg / mL (group 1), 25 μg / mL (group 2), 50 μg / mL (group 3), 100 μg / mL (group 4), and 200 μg / mL (group 5), respectively. There were 5 concentrations in total, with 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 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 (ELISA) and the 50% inhibitory concentration (IC50, μg / mL) was calculated based on the optical density value. The method for calculating IC50 by optical density value is a well-known technique.

[0053] The IC50 values ​​of the experimental group and the control group on Hela cells are shown in Table 2. As can be seen from the data in Table 2, 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one in the camel thorn extract of the present invention has a certain inhibitory effect on Hela cells.

[0054] In summary, the present invention discloses for the first time 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one from camel thorn, which has a certain inhibitory effect on human HeLa cervical cancer cells, thereby enabling the application of such compounds from camel thorn as potential anti-cervical cancer drugs.

[0055] 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 removed according to actual needs to meet the requirements of different situations.

[0056]

[0057]

Claims

1. A method for extracting 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one from camel spur, characterized in that Extracted as follows: The first step is to grind the required amount of dried aerial part of camel thorn and add methanol, soak at room temperature, heat and reflux to extract, combine the reflux extracts, recover under reduced pressure, and concentrate to obtain the camel thorn total extract; The second step is to disperse the camel thorn total extract into a suspension with water, extract it with petroleum ether, ethyl acetate and dichloromethane in sequence, and concentrate the extract to obtain petroleum ether extract, ethyl acetate extract and dichloromethane extract respectively; The third step is to take the extract of the dichloromethane part and separate it by silica gel column chromatography with gradient elution to obtain 10 fractions; In the fourth step, the fifth fraction of the ten fractions was purified and separated by high performance liquid chromatography gradient elution, and the eluate was collected to obtain 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one from camel thorn at 7.3 minutes, i.e., the ornithine alkaloid from camel thorn, with the chemical formula: 。 2. The method for extracting 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one from camel spur according to claim 1, characterized in that In the first step, 8 ml to 12 ml of methanol was added per 1 g of the dried aerial parts of Altha thorn.

3. The method for extracting 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one from camel spur according to claim 1 or 2, characterized in that In the first step, the soaking time is 3 to 4 hours, and the conditions for heating reflux extraction are: heating temperature is 50° C. to 60° C., reflux extraction is performed 3 times, and each extraction time is 1 to 3 hours.

4. The method for extracting 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one from camel spur according to claim 3, characterized in that In the third step, the silica gel column chromatography gradient eluent includes dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:

1.

5. The method for extracting 2-methylhexahydro-3H-pyrrolo[1,2-a]imidazol-3-one from camel spur according to claim 1, 2 or 4, characterized in that In the fourth step, the HPLC eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 20:80.

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