CN117447478A - Ornithine alkaloid in alhagi sparsifolia, and extraction method and application thereof - Google Patents
Ornithine alkaloid in alhagi sparsifolia, and extraction method and application thereof Download PDFInfo
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- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical class NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 title claims abstract description 16
- 238000000605 extraction Methods 0.000 title claims abstract description 11
- 241001626384 Alhagi sparsifolia Species 0.000 title claims description 49
- 241000214034 Alhagi Species 0.000 claims abstract description 44
- 206010008342 Cervix carcinoma Diseases 0.000 claims abstract description 18
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims abstract description 18
- 201000010881 cervical cancer Diseases 0.000 claims abstract description 18
- 239000003814 drug Substances 0.000 claims abstract description 12
- 238000000926 separation method Methods 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 72
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 39
- 238000010992 reflux Methods 0.000 claims description 14
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000000284 extract Substances 0.000 claims description 11
- 238000010828 elution Methods 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- -1 2-methylhexahydro-3H-pyrrolo [1,2-a ] imidazol-3-one Chemical compound 0.000 claims description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 8
- 239000003208 petroleum Substances 0.000 claims description 8
- 238000002791 soaking Methods 0.000 claims description 7
- 239000002027 dichloromethane extract Substances 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 5
- 238000010898 silica gel chromatography Methods 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 239000012259 ether extract Substances 0.000 claims description 4
- 239000002024 ethyl acetate extract Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000000725 suspension Substances 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000002265 prevention Effects 0.000 claims 2
- 206010028980 Neoplasm Diseases 0.000 abstract description 10
- 150000001875 compounds Chemical class 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 6
- 230000005764 inhibitory process Effects 0.000 abstract description 4
- 230000009982 effect on human Effects 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 10
- 238000001228 spectrum Methods 0.000 description 7
- 238000005457 optimization Methods 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000220485 Fabaceae Species 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 229930013930 alkaloid Natural products 0.000 description 3
- 239000002246 antineoplastic agent Substances 0.000 description 3
- 229940041181 antineoplastic drug Drugs 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 241000255952 Papilio Species 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 1
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 1
- 230000005918 in vitro anti-tumor Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000002551 irritable bowel syndrome Diseases 0.000 description 1
- 235000021374 legumes Nutrition 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000002733 pharmacodynamic assay Methods 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
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Abstract
The invention relates to the technical field of separation and purification of alhagi, in particular to an ornithine alkaloid in alhagi, and an extraction method and application thereof. The invention discloses 2-methylhexahydroxy-3H-pyrrrolo [1,2-a ] imidazol-3-one in alhagi for the first time, which has a certain inhibition effect on human Hela cervical cancer cells, so that the compounds in alhagi can be used as potential anti-cervical cancer tumor drugs.
Description
Technical Field
The invention relates to the technical field of separation and purification of alhagi, in particular to ornithine alkaloid in alhagi as well as an extraction method and application thereof, wherein the ornithine alkaloid in alhagi is 2-methylhexahydroo-3H-pyrrolo [1,2-a ] imidazol-3-one in alhagi.
Background
The alhagi (alhagi sparsifolia shape) is a half shrub of alhagi genus (alhagi gagneb) of the subfamily fabaceae (Papilio idae) of the family Leguminosae (Leguminosae), and has a height of 25 cm to 40 cm, a stem upright, a fine streak, a She Husheng, an oval, an inverted oval or a rounded oval, no hair, and a short stalk. The alhagi is mainly grown in arid and semiarid areas, and is distributed in northwest, tukuman, pakistan and other places in China, and the main production area is Xinjiang. Because of the unique growth environment, people have little research on the functional components of the alhagi sparsifolia, one of the main biological activities of the alhagi sparsifolia is alkaloid compound, and the alhagi sparsifolia has various effects of resisting tumors, treating diarrhea and the like, and is mainly used for treating various diseases such as cervical cancer, irritable bowel syndrome and the like clinically. Malignant tumors seriously threaten the life and health of human beings, and because many novel anticancer drugs are expensive, the long-term application is easy to cause drug resistance and side effects, so that the search for high-efficiency low-toxicity anticancer drugs is a key for tumor treatment. The alkaloid component in the alhagi has stronger anti-tumor activity, is low in price and wide in source, thereby attracting the interests of a plurality of researchers.
One of the drug effects of the alhagi sparsifolia mainly comes from alkaloid compounds, so that the ornithine alkaloid monomer compounds of the alhagi sparsifolia are developed and utilized, the potential medicinal value of the alhagi sparsifolia is further excavated, the structure and physicochemical properties of the monomer compounds of the alhagi sparsifolia are determined and characterized, and the alhagi sparsifolia has important significance in development and utilization of the alhagi sparsifolia.
Disclosure of Invention
The invention provides ornithine alkaloid in alhagi, and an extraction method and application thereof, overcomes the defects of the prior art, and firstly discloses application of ornithine alkaloid in alhagi, namely 2-methylhexahydroo-3H-pyrroo [1,2-a ] imidozol-3-one in alhagi, in preparation of drugs for preventing cervical cancer and anti-tumor drugs.
One of the technical schemes of the invention is realized by the following measures: 2-methylhexahydro-3H-pyrrolo [1,2-a ] imidazol-3-one of Alhagi, namely ornithine alkaloid of Alhagi, has the chemical structural formula:
。
the second technical scheme of the invention is realized by the following measures: a method for extracting 2-methylhexahydro-3H-pyrrrolo [1,2-a ] imidazol-3-one from Alhagi sparsifolia comprises the following steps:
firstly, crushing the overground parts of the required amount of dry alhagi sparsifolia, adding methanol into the overground parts, soaking the overground parts at room temperature, heating and reflux-extracting, combining the reflux extracting solutions each time, recovering the reflux extracting solutions under reduced pressure, and concentrating the reflux extracting solutions to obtain alhagi sparsifolia total extractum;
dispersing the alhagi sparsifolia total extract into suspension by using water, sequentially extracting with petroleum ether, ethyl acetate and dichloromethane, and concentrating the extract to obtain petroleum ether extract, ethyl acetate extract and dichloromethane extract respectively;
thirdly, taking the extract of the dichloromethane part, and performing gradient elution and separation by using a silica gel column chromatography to obtain 10 fractions;
and fourthly, purifying and separating the 5 th fraction in the 10 fractions after high performance liquid chromatography gradient elution, collecting the eluate, and obtaining the 2-methylhexahydroo-3H-pyrrrolo [1,2-a ] imidazol-3-one in the alhagi in 7.3 minutes, namely the ornithine alkaloid in the alhagi.
The following is a further optimization and/or improvement of the second technical scheme of the invention:
in the first step, 8ml to 12ml of methanol is added to 1g of the dried alhagi sparsifolia aerial parts.
In the first step, the soaking time is 3 to 4 hours, and the conditions of heating reflux extraction are as follows: reflux-extracting at 50-60deg.C for 3 times and 1-3 hr each time.
In the third step, the silica gel column chromatographic gradient eluent comprises dichloromethane and methanol, wherein the volume ratio of the dichloromethane to the methanol is 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1 and 0:1 in sequence.
In the fourth step, the high performance liquid chromatography eluent is a mixed solution of methanol and water, wherein the volume ratio of the methanol to the water is 20:80.
The third technical scheme of the invention is realized by the following measures: application of 2-methylhexahydro-3H-pyrrrolo [1,2-a ] imidazol-3-one in alhagi sparsifolia in preparing medicine for preventing cervical cancer tumor is provided.
The fourth technical scheme of the invention is realized by the following measures: application of 2-methylhexahydro-3H-pyrrrolo [1,2-a ] imidazol-3-one in alhagi sparsifolia in preparing medicine for treating cervical cancer tumor is provided.
The fifth technical scheme of the invention is realized by the following measures: application of 2-methylhexahydro-3H-pyrrrolo [1,2-a ] imidazol-3-one in alhagi sparsifolia as health product for preventing and treating cervical cancer is provided.
The invention discloses 2-methylhexahydroxy-3H-pyrrrolo [1,2-a ] imidazol-3-one in alhagi for the first time, which has a certain inhibition effect on human Hela cervical cancer cells, so that the compounds in alhagi can be used as potential anti-cervical cancer tumor drugs.
Drawings
FIG. 1 is a schematic diagram of 2-methylhexahydroo-3H-pyrrrolo [1,2-a ] of the present invention in alhagi sparsifolia]imidazol-3-one 1 H-NMR spectrum.
FIG. 2 is a schematic diagram of 2-methylhexahydroo-3H-pyrrrolo [1,2-a ] of the present invention in alhagi sparsifolia]imidazol-3-one 13 C-APT spectrogram。
FIG. 3 is a schematic diagram of 2-methylhexahydroo-3H-pyrrrolo [1,2-a ] of the present invention in alhagi sparsifolia]imidazol-3-one 1 H- 1 HCOSY profile.
FIG. 4 is a HMBC pattern of 2-methylhexahydro-3H-pyrrrolo [1,2-a ] imidazol-3-one of the present invention in alhagi sparsifolia.
FIG. 5 is a HSQC spectrum of 2-methylhexahydro-3H-pyrrrolo [1,2-a ] imidazol-3-one in alhagi of the present invention.
FIG. 6 is a NOESY spectrum of 2-methylhexahydro-3H-pyrrrolo [1,2-a ] imidazol-3-one in alhagi of the present invention.
Detailed Description
The present invention is not limited by the following examples, and specific embodiments can be determined according to the technical scheme and practical situations of the present invention. The various chemical reagents and chemical supplies mentioned in the invention are all commonly known and used in the prior art unless specified otherwise; the percentages in the invention are mass percentages unless specified otherwise; the solutions in the invention are aqueous solutions in which the solvent is water unless otherwise specified, for example, the hydrochloric acid solution is hydrochloric acid aqueous solution; the room temperature and the room temperature in the present invention generally refer to temperatures ranging from 15 ℃ to 25 ℃, and are generally defined as 25 ℃.
The invention is further described below with reference to examples:
example 1: the chemical structural formula of the ornithine alkaloid in the alhagi is 2-methylhexahydroo-3H-pyrrrolo [1,2-a ] imidazol-3-one:
。
example 2: the 2-methylhexahydroo-3H-pyrrrolo [1,2-a ] imidazol-3-one in the alhagi is extracted according to the following method:
firstly, crushing the overground parts of the required amount of dry alhagi sparsifolia, adding methanol into the overground parts, soaking the overground parts at room temperature, heating and reflux-extracting, combining the reflux extracting solutions each time, recovering the reflux extracting solutions under reduced pressure, and concentrating the reflux extracting solutions to obtain alhagi sparsifolia total extractum;
dispersing the alhagi sparsifolia total extract into suspension by using water, sequentially extracting with petroleum ether, ethyl acetate and dichloromethane, and concentrating the extract to obtain petroleum ether extract, ethyl acetate extract and dichloromethane extract respectively;
thirdly, taking the extract of the dichloromethane part, and performing gradient elution and separation by using a silica gel column chromatography to obtain 10 fractions;
and fourthly, purifying and separating the 5 th fraction in the 10 fractions after high performance liquid chromatography gradient elution, collecting the eluate, and obtaining the 2-methylhexahydroo-3H-pyrrrolo [1,2-a ] imidazol-3-one in the alhagi in 7.3 minutes, namely the ornithine alkaloid in the alhagi.
Example 3: as an optimization of the above example, in the first step, 8ml to 12ml of methanol was added per 1g of the dry aerial parts of the alhagi sparsifolia.
Example 4: as an optimization of the above examples, in the first step, the soaking time was 3 to 4 hours, and the conditions for the heat reflux extraction were: reflux-extracting at 50-60deg.C for 3 times and 1-3 hr each time.
Example 5: as an optimization of the above embodiment, in the third step, the silica gel column chromatography gradient eluent includes dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is sequentially 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1.
Example 6: as an optimization of the above embodiment, in the fourth step, the high performance liquid chromatography eluent is a mixed solution of methanol and water, wherein the volume ratio of methanol to water is 20:80.
Example 7: the application of 2-methylhexahydro-3H-pyrrrolo [1,2-a ] imidazol-3-one in the alhagi sparsifolia as the preparation of the medicine for preventing cervical cancer tumor.
Example 8: the application of 2-methylhexahydro-3H-pyrrrolo [1,2-a ] imidazol-3-one in the alhagi sparsifolia in preparing the medicine for treating cervical cancer tumor is provided.
Example 9: the application of 2-methylhexahydro-3H-pyrrrolo [1,2-a ] imidazol-3-one in the alhagi sparsifolia as a health care product for preventing and treating cervical cancer tumor is provided.
Example 10: the 2-methylhexahydroo-3H-pyrrrolo [1,2-a ] imidazol-3-one in the alhagi is extracted according to the following method:
firstly, crushing the overground parts of the dried alhagi sparsifolia, adding methanol into the overground parts, adding 8ml to 12ml of methanol into each 1g of alhagi sparsifolia, soaking the mixture for 3 hours at room temperature, carrying out heating reflux extraction for 3 times at 50 ℃ for 3 hours each time, combining the reflux extraction solutions each time, carrying out decompression recovery and concentration to obtain alhagi sparsifolia total extractum;
dispersing the alhagi sparsifolia total extract into suspension by using water, sequentially extracting with petroleum ether, ethyl acetate and dichloromethane, and concentrating the extract to obtain petroleum ether extract, ethyl acetate extract and dichloromethane extract respectively;
step three, taking dichloromethane extract, and performing gradient elution and separation on the dichloromethane extract by using a silica gel column to obtain 10 fractions, wherein the gradient elution liquid of the silica gel column comprises dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is sequentially 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1 and 0:1;
and fourthly, purifying and separating the 5 th fraction in the 10 fractions after high performance liquid chromatography gradient elution, collecting the eluate, and obtaining 2-methylhexahydroo-3H-pyrrool [1,2-a ] imidazol-3-one in the alhagi in 7.3 minutes, namely ornithine alkaloid in the alhagi, wherein the high performance liquid chromatography eluate is a mixture of methanol and water in a volume ratio of 20:80.
Nuclear magnetic resonance spectroscopy was performed on 2-methylhexahydro-3H-pyrrrolo [1,2-a ] imidazol-3-one (i.e., ornithine alkaloid in Alhagi) obtained in example 10.
2-methylhexahydroo-3H-pyrrrolo [1,2-a ] in Alhagi spars of the invention]imidazol-3-one 1 The H-NMR spectrum is shown in FIG. 1, 2-methylhexahydroo-3H-pyrrolo [1,2-a ] in the alhagi sparsifolia of the present invention]imidazol-3-one 13 The C-APT spectrum is shown in FIG. 2, 2-methylhexahydroo-3H-pyrrolo [1,2-a ] in the alhagi sparsifolia of the present invention]The 1H-1HCOSY spectrum of imidazol-3-one is shown in FIG. 3, 2-methylhexahydroo-3H-pyrrrolo [1,2-a ] in the alhagi sparsifolia of the present invention]HMBC spectra of imidazol-3-one are shown in FIG. 4, and the present invention is directed to Alhagi sparsifolia2-methylhexahydroo-3H-pyrrrolo [1,2-a ]]The HSQC spectrum of imidozol-3-one is shown in FIG. 5, 2-methylhexahydroo-3H-pyrroo [1,2-a ] in the alhagi sparsa of the present invention]The NOESY spectra of imidazol-3-one are shown in FIG. 6, the spectra of FIG. 1 to FIG. 6 were analyzed, the peaks of the spectra 1 and FIG. 2 were assigned, and the peak assignment of the peaks of FIG. 1 and FIG. 2 is shown in Table 1. As can be seen from the data of Table 1, 2-methylhexahydroo-3H-pyrrolo [1,2-a ] in the alhagi sparsifolia of the present invention]The chemical structural formula of the imidazol-3-one is shown below, and is readily soluble in methanol:
。
2-methylhexahydro-3H-pyrrolo [1,2-a ] imidazol-3-one of the present invention was subjected to an in vitro antitumor pharmacodynamic assay using MTT colorimetric method.
With 2-methylhexahydroo-3H-pyrroo [1,2-a ]]Taking imidozol-3-one as experimental group, taking 5-FU as control group, setting blank group, selecting Hela (human cervical cancer cell) cells as experimental object, diluting culture medium, and taking 6×10 4 After the culture is normally carried out in an incubator for 24 hours, the corresponding medicines are added into each group, so that the final concentration of each group of medicines is respectively 12.5 mug/mL (1 group), 25 mug/mL (2 group), 50 mug/mL (3 group), 100 mug/mL (4 group) and 200 mug/mL (5 group), 5 concentrations are set in total, and 3 compound holes are arranged in each concentration; after 48 hours of incubation, 10 μl of MTT was added to each well for staining; after the culture is continued for four hours, the original culture solution is sucked and removed, 150 mu L of DMSO is added into each hole, the mixture is placed on a shaking table and oscillated for 10 minutes at a low speed to enable crystals to be fully dissolved, an optical density value is detected at a wavelength of 570nm of an ELISA (enzyme-linked immunosorbent assay) instrument, 50% inhibition concentration (IC 50, mu g/mL) is calculated according to the optical density value, and the calculation method for calculating the IC50 of the optical density value is a known technology.
The IC50 of the experimental group and the control group against Hela cells is shown in Table 2. As can be seen from the data in Table 2, 2-methylhexahydroo-3H-pyrrolo [1,2-a ] imidazol-3-one in alhagi according to the present invention has some inhibitory effect on Hela cells.
In conclusion, the invention discloses 2-methylhexahydroo-3H-pyrrool-3-one in alhagi for the first time, which has a certain inhibition effect on human Hela cervical cancer cells, so that the compounds in alhagi can be used as potential anti-cervical cancer tumor drugs.
The technical characteristics form the embodiment of the invention, have stronger adaptability and implementation effect, and can increase or decrease unnecessary technical characteristics according to actual needs so as to meet the requirements of different situations.
Claims (9)
1. 2-methylhexahydro-3H-pyrrolo [1,2-a ] imidazol-3-one of alhagi, namely ornithine alkaloid of alhagi, is characterized by having a chemical structural formula:
。
2. a method of extracting 2-methylhexahydroo-3H-pyrrolo [1,2-a ] imidazol-3-one from alhagi sparsifolia according to claim 1, wherein the method comprises the steps of:
firstly, crushing the overground parts of the required amount of dry alhagi sparsifolia, adding methanol into the overground parts, soaking the overground parts at room temperature, heating and reflux-extracting, combining the reflux extracting solutions each time, recovering the reflux extracting solutions under reduced pressure, and concentrating the reflux extracting solutions to obtain alhagi sparsifolia total extractum;
dispersing the alhagi sparsifolia total extract into suspension by using water, sequentially extracting with petroleum ether, ethyl acetate and dichloromethane, and concentrating the extract to obtain petroleum ether extract, ethyl acetate extract and dichloromethane extract respectively;
thirdly, taking the extract of the dichloromethane part, and performing gradient elution and separation by using a silica gel column chromatography to obtain 10 fractions;
and fourthly, purifying and separating the 5 th fraction in the 10 fractions after high performance liquid chromatography gradient elution, collecting the eluate, and obtaining the 2-methylhexahydroo-3H-pyrrrolo [1,2-a ] imidazol-3-one in the alhagi in 7.3 minutes, namely the ornithine alkaloid in the alhagi.
3. The method for extracting 2-methylhexa-3H-pyrrrolo [1,2-a ] imidazol-3-one from alhagi sparsifolia according to claim 2, wherein in the first step, 8ml to 12ml of methanol is added per 1g of the dried alhagi sparsifolia aerial parts.
4. A process for the extraction of 2-methylhexahydroxy-3H-pyrrolo [1,2-a ] imidazol-3-one from alhagi sparsifolia according to claim 2 or 3, wherein in the first step the soaking time is from 3 hours to 4 hours and the conditions for heat reflux extraction are: reflux-extracting at 50-60deg.C for 3 times and 1-3 hr each time.
5. The method for extracting 2-methylhexahydroxy-3H-pyrrolo [1,2-a ] imidazol-3-one from alhagi sparsifolia according to claim 2, 3 or 4, wherein in the third step, the silica gel column chromatography gradient eluent comprises dichloromethane and methanol, and the volume ratio of dichloromethane and methanol is 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, 0:1 in order.
6. The method for extracting 2-methylhexahydroxy-3H-pyrrool [1,2-a ] imidazol-3-one from alhagi sparsifolia according to claim 2, 3, 4 or 5, wherein in the fourth step, the high performance liquid chromatography eluent is a mixture of methanol and water, wherein the volume ratio of methanol to water is 20:80.
7. Use of 2-methylhexahydroo-3H-pyrrolo [1,2-a ] imidazol-3-one in alhagi as claimed in claim 1 for the manufacture of a medicament for the prevention of cervical cancer.
8. Use of 2-methylhexahydroo-3H-pyrrolo [1,2-a ] imidazol-3-one in alhagi as claimed in claim 1 for the manufacture of a medicament for the treatment of cervical cancer.
9. Use of 2-methylhexahydro-3H-pyrrolo [1,2-a ] imidazol-3-one in alhagi sparsifolia according to claim 1 for the preparation of a health product for the prevention and treatment of cervical cancer.
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