CN116003371B - Terpenoid, and extraction method and application thereof - Google Patents
Terpenoid, and extraction method and application thereof Download PDFInfo
- Publication number
- CN116003371B CN116003371B CN202310021651.8A CN202310021651A CN116003371B CN 116003371 B CN116003371 B CN 116003371B CN 202310021651 A CN202310021651 A CN 202310021651A CN 116003371 B CN116003371 B CN 116003371B
- Authority
- CN
- China
- Prior art keywords
- ethyl acetate
- petroleum ether
- terpenoid
- separating
- extract
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 150000003505 terpenes Chemical class 0.000 title claims abstract description 22
- 238000000605 extraction Methods 0.000 title claims abstract description 15
- 238000000926 separation method Methods 0.000 claims abstract description 25
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 10
- 201000011510 cancer Diseases 0.000 claims abstract description 8
- 239000003814 drug Substances 0.000 claims abstract description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 46
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 32
- 239000003208 petroleum Substances 0.000 claims description 18
- 239000000284 extract Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 11
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000000741 silica gel Substances 0.000 claims description 9
- 229910002027 silica gel Inorganic materials 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 8
- 238000002791 soaking Methods 0.000 claims description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 206010006187 Breast cancer Diseases 0.000 claims description 5
- 208000026310 Breast neoplasm Diseases 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 239000002024 ethyl acetate extract Substances 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 4
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 4
- 230000014759 maintenance of location Effects 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- 238000011282 treatment Methods 0.000 claims description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 239000002026 chloroform extract Substances 0.000 claims description 2
- 239000012259 ether extract Substances 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 230000010354 integration Effects 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 238000010298 pulverizing process Methods 0.000 claims description 2
- 238000011321 prophylaxis Methods 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 description 15
- 241000207840 Jasminum Species 0.000 description 11
- 235000010254 Jasminum officinale Nutrition 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 206010008342 Cervix carcinoma Diseases 0.000 description 4
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 4
- 239000002246 antineoplastic agent Substances 0.000 description 4
- 229940041181 antineoplastic drug Drugs 0.000 description 4
- 201000010881 cervical cancer Diseases 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 208000010392 Bone Fractures Diseases 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000010992 reflux Methods 0.000 description 3
- 238000001896 rotating frame Overhauser effect spectroscopy Methods 0.000 description 3
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 2
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 2
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 2
- 206010017076 Fracture Diseases 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 2
- 238000004896 high resolution mass spectrometry Methods 0.000 description 2
- 230000005918 in vitro anti-tumor Effects 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000419 plant extract Substances 0.000 description 2
- 238000004262 preparative liquid chromatography Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 201000011549 stomach cancer Diseases 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- -1 terpenoid compound Chemical class 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 1
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 206010010741 Conjunctivitis Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000007356 Fracture Dislocation Diseases 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 240000005385 Jasminum sambac Species 0.000 description 1
- 235000007457 Jasminum sambac Nutrition 0.000 description 1
- 102000006833 Multifunctional Enzymes Human genes 0.000 description 1
- 108010047290 Multifunctional Enzymes Proteins 0.000 description 1
- 241000207834 Oleaceae Species 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000003444 anaesthetic effect Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 208000002925 dental caries Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 208000001848 dysentery Diseases 0.000 description 1
- 208000037902 enteropathy Diseases 0.000 description 1
- 238000002481 ethanol extraction Methods 0.000 description 1
- 241001233957 eudicotyledons Species 0.000 description 1
- 239000012909 foetal bovine serum Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 208000028774 intestinal disease Diseases 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 201000005249 lung adenocarcinoma Diseases 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000036457 multidrug resistance Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 238000000238 one-dimensional nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000008363 phosphate buffer Chemical class 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 238000004237 preparative chromatography Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a terpenoid shown in a formula (I), and an extraction method and application thereof. The invention also discloses a separation method of the terpenoid and application of the terpenoid in preparing a medicament for treating cancer.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a terpenoid, an extraction method and application thereof.
Background
Cancer has become the leading fatal disease worldwide, and can occur in various organs and tissues at any age.
Therefore, development of new anticancer drugs is a challenge to be solved.
However, most cancer patients usually find the disease to be middle to late stage, and the overall clinical treatment effect is poor, especially the continuous occurrence of multi-drug resistance, so that the treatment difficulty of the cancer is serious. Although some small molecule anticancer chemotherapeutics and antibody drugs have entered the clinic. However, most of the new anticancer drugs used in clinic are basically imported and have high treatment cost. The number of new anticancer drugs independently developed in China is relatively small. Therefore, development of a novel anticancer drug with high activity and low side effect is urgent to meet clinical demands. Especially, the discovery of new antitumor candidate compounds from traditional Chinese medicines is the mainstream of current drug development.
The jasmine root is a dry root of a dicotyledon, jasminum (Jasminum sambac (l.) ait.) plant belonging to the genus jasminum of the family Oleaceae. Warm nature, bitter taste, toxic and original in India. The Chinese is mainly produced in Jiangsu, zhejiang, fujian, taiwan, guangdong, sichuan and other places. According to the ancient book records in China, jasmine roots are used for fracture, dislocation and bone fracture, and have analgesic effect and certain anesthetic effect. The jasmine root has the effects of improving immunity, clearing heat and detoxicating, and relieving swelling and pain. Has good therapeutic effect on patients with dysentery, abdominal pain, enteropathy, and conjunctivitis. It is used for treating fracture, injury of tendons, dental caries, and parietal headache.
The invention extracts and separates jasmine root growing in Fujian , so as to find out new drug candidate compound or lead compound with anti-tumor activity.
Disclosure of Invention
The terpenoid with novel structure is separated from jasmine roots, and has strong inhibitory activity on tumors.
The invention provides a terpenoid shown in a formula (I):
the invention also provides an extraction and separation method of the terpenoid shown in the formula (I), which comprises the following steps:
(1) Crushing jasmine roots, extracting with ethanol water solution, and concentrating the extract to obtain a total extract;
(2) Dispersing the total extract obtained in the step (1) with water, extracting with organic solvents with different polarities, and concentrating the extract to obtain extracts with different polarities;
(3) Carrying out column separation on the extractum with different polarities obtained in the step (2) to obtain a crude component; and
(4) And (3) separating and purifying the crude component obtained in the step (3) to obtain the terpenoid shown in the formula (I).
In an embodiment according to the present invention, the aqueous ethanol extraction in step (1) may be a soaking or reflux extraction.
According to an embodiment of the invention, the soaking extraction: the soaking temperature is 15-30deg.C, preferably room temperature; the soaking time is 20-40 days, preferably 25-35 days, such as 28 days, 29 days, 30 days, 31 days, and 32 days.
According to an embodiment of the invention, the reflux extraction: the reflux time may be 10-24 hours, for example 12-15 hours.
According to an embodiment of the present invention, the organic solvents of different polarity used in step (2) are selected from petroleum ether, ethyl acetate, chloroform or n-butanol.
According to an embodiment of the present invention, the chromatographic separation in step (3) includes, but is not limited to, silica gel column separation, preparative liquid chromatography separation, and any combination thereof.
According to an embodiment of the present invention, the separation and purification in step (4) is selected from the group consisting of silica gel column separation, preparative plate separation, or preparative liquid chromatography separation, and any combination thereof.
According to an embodiment of the present invention, the method for extracting and separating the compound represented by formula (I) comprises the steps of:
(1) Pulverizing radix Jasmini sambac, soaking in ethanol water solution, filtering, and concentrating to obtain total extract;
(2) Dispersing the total extract obtained in the step (1) with water, sequentially extracting with petroleum ether, ethyl acetate and chloroform for 3-5 times, and concentrating the extractive solutions with different polarities to obtain petroleum ether extract, ethyl acetate extract and chloroform extract;
(3) Separating the ethyl acetate extract obtained in the step (2) by a silica gel column, and performing gradient elution by using a developing agent to obtain coarse components with different polarities;
(4) Analyzing the crude components with different polarities in the step (3) by HPLC, combining, separating by a silica gel column, separating by a preparation plate or separating and purifying by a preparation liquid chromatograph, combining the components with the same R f or the same retention time, and recrystallizing for multiple times to obtain the terpenoid shown in the formula (I).
According to an embodiment of the present invention, in the step (1), the mass fraction of ethanol in the aqueous ethanol solution may be 50-80%, for example 50%, 60%, 70% or 80%.
According to an embodiment of the invention, in step (2), the mass to volume ratio (g/mL) of the total extract to water is (0.2-3): 1, for example (0.5-2): 1, exemplary 1:1.
According to an embodiment of the invention, in said step (2), the volume ratio of organic solvent to water used for extraction is 1 (0.2-3), for example 1 (0.5-2), and is exemplified by 1:2.
According to an embodiment of the present invention, the developing agent in step (3) is petroleum ether and/or ethyl acetate, and starting from pure petroleum ether, the amount of ethyl acetate is gradually increased while the amount of petroleum ether is reduced, and finally pure ethyl acetate is obtained. Preferably, the volume ratio of petroleum ether to ethyl acetate is 1:0, 0.9:0.1, 0.8:0.2, 0.7:0.3, 0.6:0.4, 0.5:0.5, 0.4:0.6, 0.3:0.7, 0.2:0.8, 0.1:0.9, 0:1.
According to an embodiment of the invention, in step (4), the HPLC analysis conditions are as follows: mobile phase: v Methanol :V Water and its preparation method ( Containing 0.3 Phosphoric acid ) = 7:3, column temperature at room temperature, detection wavelength at 200-400nm integration wavelength.
According to an embodiment of the present invention, in the step (4), the separation and purification are performed by selecting a component having a larger polarity difference (for example, selecting 3 to 5 components having a difference of R f to 0.5 to 1).
According to an embodiment of the present invention, in step (4), the separation and purification may be repeated.
The invention also provides application of the terpenoid shown in the formula (I) in preparing a medicament for treating and/or preventing cancers.
According to an embodiment of the invention, the cancer is selected from lung cancer, stomach cancer, breast cancer or cervical cancer.
Advantageous effects
The terpenoid compound with novel structure is separated from jasmine roots, and has strong inhibitory activity on various cancer cells (such as gastric cancer cell lines, colorectal cancer cell lines, breast cancer cell lines and cervical cancer cell lines).
The extraction method provided by the invention has the advantages that the plant extract is subjected to primary purification (such as column separation, preparation plate separation, preparation liquid chromatography separation and the like), then recrystallization is carried out, and the target compound with high purity (the purity can be up to 98%) is rapidly and accurately obtained, so that the extraction method has important significance in rapid separation and identification of specific compounds, particularly chiral enantiomers, in the plant extract containing complex components.
Drawings
FIG. 1 is an HPLC chromatogram of a compound of formula (I);
FIG. 2 is a HMBC correlation diagram of a compound of formula (I);
FIG. 3 is a ROESY correlation diagram of a compound of formula (I);
FIG. 4 is a diagram showing the structure of spherical crystals of the compound represented by formula (I).
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.
Instrument and reagent:
Jasmine root (2021, 10 months from Fujian province ), other chemical reagents are all domestic chemical pure reagents; CCK8 (Shanghai Bei Bo biotechnology limited); DMEM high sugar medium (sameifer's instruments limited); EDTA (pancreatin) (gibco); foetal Bovine Serum (Biological Industries); phosphate buffer salt solution; 96-well cell culture plates; multifunctional enzyme labeling instrument.
Example 1: terpenoid extraction separation and structural identification shown in formula (I)
The extraction and separation of the compounds are carried out according to the following procedures:
(a) 20 kg of dried jasmine roots (2021, 10 months from Fujian province) are crushed, respectively filled into 3 plastic barrels of 20L, respectively added with 15L of 70% ethanol/water solution for soaking for 2 months at room temperature, and filtered and concentrated to obtain extract.
(B) Dispersing 1 kg of the extract obtained in the step a in 2L of water, and extracting with petroleum ether, ethyl acetate and chloroform respectively for 5 times in sequence, wherein the dosage of petroleum ether, ethyl acetate and chloroform is 1000 ml each time. Concentrating the extractive solutions of different polarities to obtain extract of different polarities.
(C) And c, performing primary silica gel column separation on the ethyl acetate extract obtained in the step b, performing gradient elution by using V Petroleum ether /V Acetic acid ethyl ester =1:0 to 0:1, and collecting the eluting components of V Petroleum ether /V Acetic acid ethyl ester =2:1 and 1:1 to obtain 50 parts (Fr 1-Fr50) (I) with different polarities.
(D) The 50 fractions obtained in step (c) were first examined by preliminary TLC and similar fractions were combined to obtain 30 fractions (II). Then, HPLC qualitative analysis (chromatographic conditions: V Methanol :V Water and its preparation method ( Containing 0.3 Phosphoric acid ), 7:3, column temperature: room temperature, detection wavelength: 200-400 nm) was performed on the component (II), respectively, and the similar components were combined. Then selecting 5 components with larger polarity difference (the difference of the retention time of the core substances is about 0.5-5 minutes), then respectively carrying out primary silica gel column separation, combining the approximate components after carrying out primary TLC analysis on the obtained components, selecting the component with larger polarity difference (the difference of R f is about 0.2-0.6) to carry out secondary silica gel column separation, then carrying out preparative plate separation or preparative high performance liquid chromatography separation on the component separated by the secondary column, combining the components with the same R f value or the same retention time obtained by the preparative chromatography separation, and then recrystallizing to obtain the terpenoid shown in the formula (I).
For isolated = terpenoid, its purity was determined by HPLC: purity 94.34%, rt= 18.880min; (chromatographic conditions: C 18 column; mobile phase: V Methanol :V Water and its preparation method , 7:3; detection wavelength: 254 nm), HPLC chromatogram of which is shown in FIG. 1.
The structure was determined by 1D NMR and 2D NMR, high resolution mass spectrometry, and the absolute configuration was determined by ROESY and XRD analysis methods. The HMBC of the compound shown in formula (I) is shown in figure 2, the ROESY is shown in figure 3, and the XRD spherical pattern is shown in figure 4.
The characterization data of the terpenoids shown in formula (I) are as follows: yellow crystals, melting point: 147-148 ; HR-MS for "C 30H32O6 +Na": 511.2091 Experimental values 511.2091;1H NMR(400MHz,CD3OD,ppm,J/Hz):0.95(3H,d,J7.1Hz,H-13),1.29(2H,s,H-8),1.35(2H,m,H-7),1.40(1H,m,H-9),1.61(1H,m,H-5'),1.70(3H,s,H-14'),2.21(1H,m,H-5'),2.27(3H,s,H-17'),2.41(2H,m,H-6),2.88(1H,m,H-6'),3.05(1H,m,H-6'),3.96(1H,d,J10.6Hz,H-15'),4.47(1H,d,J10.6Hz,H-15'),4.77(1H,d,J2.3Hz,H-12),4.97(1H,d,J2.3Hz,H-12),5.15(1H,s,H-4),5.25(2H,d,J19.7Hz,H-18'),6.11(1H,s,H-2'),6.80(1H,s,H-7').13C NMR(100MHz):17.7(C-7),18.6(C-13),23.6(C-6),24.0(C-6'),24.87(C-5'),24.89(C-14'),24.94(C-17'),29.5(C-8),34.7(C-9),47.8(C-13'),55.4(C-4'),64.6(C-15'),102.62(C-12),108.1(C-4),113.7(C-10'),117.3(C-18'),121.7(C-7'),126.7(C-2'),127.0(C-12'),136.7(C-11'),140.1(C-11),142.5(C-10),143.66(C-8'),143.68(C-16'),147.69(C-9'),147.71(C-3'),156.6(C-1),171.3(C-1'),172.4(C-3),190.3(C-5).
Example 2: in vitro antitumor Activity test
The in vitro antitumor activity test is carried out on the terpenoid shown in the formula (I), and the inhibition activity of the terpenoid on lung cancer cell strains A549 and cervical cancer cell strains Hela is mainly studied.
1. Concentration preparation of test sample
13.0Mg of the compound represented by formula (I) was weighed into a 5mL plastic centrifuge tube, and diluted to 1mL with DMSO. The initial concentration of 13.0mg/mL was obtained. Then the initial concentration is diluted by DMSO in a multiple ratio to obtain 6.5mg/mL,3.25mg/mL,1.625mg/mL,0.8125mg/mL and 0.40625mg/mL of 5 different concentration gradients in sequence, and the mixture is placed in a refrigerator at 4 for later use.
2. Culture of cancer cell lines and test of inhibitory Activity
The lung adenocarcinoma cell line (a 549) was placed in an incubator containing 5% CO 2 at 37 and saturated humidity for 24 hours, when the cells were in logarithmic growth phase, the supernatant culture was aspirated and after digestion with 0.25% trypsin-EDTA solution, the digestion was stopped using high sugar medium. And cells were seeded in 96-well plates such that the cell density was 5000 cells/well. The 96-well plate was placed in an incubator for 24 hours. With consequent pipetting of the cell culture broth in the 96-well plate. And 100. Mu.L of high sugar culture medium is added into a 96-well plate, then 1. Mu.L of test samples with different concentrations are added into each well (5 compound wells are arranged at each concentration), then the mixture is placed into an incubator with saturated humidity of 37 and 5% CO 2 for continuous culture for 48 hours, 10. Mu.L of CCK8 is added into each well, and incubation is continued for 1-4 hours in the incubator with 37 . The absorbance value per well at a wavelength of 450nm was measured on a multifunctional microplate reader. Inhibition = [ (control cell OD-dosing cell OD)/(control cell OD-blank OD) ]x100. The negative control is a mixed solution of V High sugar culture medium /VDMSO:10:1.
The test procedure of the compound shown in the formula (I) on the culture of cervical cancer cell strain Hela and breast cancer cell strain MCF-7 is the same as that of the test procedure of the inhibition activity.
The IC 50 value of the compound shown in the formula (I) on the breast cancer cell line MCF-7 is 75.44 mug/mL.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A terpenoid represented by formula (I):
2. The method for extracting and separating a terpenoid represented by the formula (I) according to claim 1, comprising the steps of:
(1) Pulverizing radix Jasmini sambac, soaking in ethanol water solution, filtering, and concentrating to obtain total extract;
(2) Dispersing the total extract obtained in the step (1) with water, sequentially extracting with petroleum ether, ethyl acetate and chloroform for 3-5 times, and concentrating the extractive solutions with different polarities to obtain petroleum ether extract, ethyl acetate extract and chloroform extract;
(3) Separating the ethyl acetate extract obtained in the step (2) by a silica gel column, and performing gradient elution by using a developing agent to obtain coarse components with different polarities;
The developing agent in the step (3) is petroleum ether and/or ethyl acetate, and the amount of the ethyl acetate is gradually increased from pure petroleum ether while the amount of the petroleum ether is reduced, so that the pure ethyl acetate is finally obtained;
(4) Analyzing the crude components with different polarities in the step (3) by HPLC, combining, separating by a silica gel column, separating by a preparation plate or separating and purifying by a preparation liquid chromatograph, combining the components with the same R f or the same retention time, and recrystallizing for multiple times to obtain the terpenoid shown in the formula (I).
3. The extraction and separation method according to claim 2, wherein,
In the step (1), the mass fraction of the ethanol in the ethanol water solution is 50-80%;
And/or in the step (2), the mass-volume ratio of the total extract to the water is (0.2-3) g to 1mL;
and/or in the step (2), the volume ratio of the organic solvent used for extraction to water is 1 (0.2-3);
And/or, in the step (3), the developing agent is petroleum ether and/or ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 1:0, 0.9:0.1, 0.8:0.2, 0.7:0.3, 0.6:0.4, 0.5:0.5, 0.4:0.6, 0.3:0.7, 0.2:0.8, 0.1:0.9, 0:1;
and/or, in step (4), HPLC analysis conditions are as follows: mobile phase: v Methanol :V Containing 0.3 Phosphoric acid water = 7:3, column temperature at room temperature, detection wavelength at 200-400nm integration wavelength;
and/or, in the step (4), separating and purifying the components with larger polarity difference;
and/or, in the step (4), the separation and purification may be repeated.
4. Use of a terpenoid represented by formula (I) according to claim 1 for the preparation of a medicament for the treatment and/or prophylaxis of a cancer selected from breast cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310021651.8A CN116003371B (en) | 2023-01-04 | 2023-01-04 | Terpenoid, and extraction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310021651.8A CN116003371B (en) | 2023-01-04 | 2023-01-04 | Terpenoid, and extraction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116003371A CN116003371A (en) | 2023-04-25 |
| CN116003371B true CN116003371B (en) | 2024-04-16 |
Family
ID=86031630
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310021651.8A Active CN116003371B (en) | 2023-01-04 | 2023-01-04 | Terpenoid, and extraction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116003371B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115466248A (en) * | 2022-02-08 | 2022-12-13 | 厦门稀土材料研究所 | Diterpenoid compound and extraction method and application thereof |
-
2023
- 2023-01-04 CN CN202310021651.8A patent/CN116003371B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115466248A (en) * | 2022-02-08 | 2022-12-13 | 厦门稀土材料研究所 | Diterpenoid compound and extraction method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116003371A (en) | 2023-04-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111704544B (en) | Labdane diterpenoid compound and separation method and application thereof | |
| CN113754533A (en) | Oxidized labdane diterpenoid compounds and separation method and application thereof | |
| CN110818669A (en) | Aquilaria sinensis tetrahydro 2- (2-phenethyl) chromone compound and separation method and application thereof | |
| CN108689851B (en) | Tiglic alkane type diterpene compound and preparation method and application thereof | |
| CN115466248B (en) | Diterpenoid compound and extraction method and application thereof | |
| CN110746421B (en) | Extraction method and application of indole monoterpene compound | |
| CN116003371B (en) | Terpenoid, and extraction method and application thereof | |
| CN116003238B (en) | Sesquiterpenoids in jasmine roots, and extraction method and application thereof | |
| CN107698510A (en) | The alkaloid compound and extracting method extracted from blue or green bamboo mark | |
| CN114957362B (en) | Method for separating anti-inflammatory active ingredients of coreopsis tinctoria | |
| CN112939737B (en) | Compound extracted from burdock leaves and having effect of protecting alcoholic liver injury and preparation method and application thereof | |
| CN113105522B (en) | Oriental water plantain triterpenes compound, preparation method, structure characterization method and application thereof | |
| CN109651233B (en) | Alkaloid compound, and method for extracting and separating alkaloid compound from dendrobium clavatum and application of alkaloid compound | |
| CN117756621A (en) | New pentacyclic triterpene compound and its extraction method and application | |
| CN114933602A (en) | High-oxidation germacrane type sesquiterpene lactone compounds in elephantopus scaber and preparation method and application thereof | |
| CN103183597A (en) | Diaryl neptanone compound having antineoplastic activity, preparing method and application | |
| CN114213497B (en) | Steroid compound in herba Ajugae, and extraction method and application thereof | |
| CN113149820A (en) | Monocyclic heteroterpene structural compound, preparation method and application thereof | |
| CN109206392B (en) | Coumarin compound and preparation method and application thereof | |
| CN111606801A (en) | Split-ring labdane diterpenoid compound and separation method and application thereof | |
| CN100434419C (en) | Compound of monocyclic polysubstitution saturated cyclohexanones, prepartion method and usage | |
| CN112402412B (en) | Application of inner ester compound of jingdao in preparing medicine for treating inflammation-caused diseases | |
| CN114315870B (en) | Dimeric alkaloid and preparation and application thereof | |
| CN112125804B (en) | Diterpenoid of euphorbia lathyris, preparation method and anti-leukemia application thereof | |
| CN114349808B (en) | Separation and purification method of rabdosia amethystoides saponin A and B monomers and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |