CN115433068A - Method for separating and extracting chrysophanol, luteolin, abrine and protocatechuic acid from herba abri and application thereof - Google Patents
Method for separating and extracting chrysophanol, luteolin, abrine and protocatechuic acid from herba abri and application thereof Download PDFInfo
- Publication number
- CN115433068A CN115433068A CN202210678255.8A CN202210678255A CN115433068A CN 115433068 A CN115433068 A CN 115433068A CN 202210678255 A CN202210678255 A CN 202210678255A CN 115433068 A CN115433068 A CN 115433068A
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- CN
- China
- Prior art keywords
- luteolin
- chrysophanol
- compound
- extraction
- extracting
- Prior art date
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- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 title claims abstract description 56
- IQPNAANSBPBGFQ-UHFFFAOYSA-N luteolin Chemical compound C=1C(O)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(O)C(O)=C1 IQPNAANSBPBGFQ-UHFFFAOYSA-N 0.000 title claims abstract description 56
- LRDGATPGVJTWLJ-UHFFFAOYSA-N luteolin Natural products OC1=CC(O)=CC(C=2OC3=CC(O)=CC(O)=C3C(=O)C=2)=C1 LRDGATPGVJTWLJ-UHFFFAOYSA-N 0.000 title claims abstract description 56
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- YQUVCSBJEUQKSH-UHFFFAOYSA-N 3,4-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 title claims abstract description 38
- NZPQWZZXRKZCDU-UHFFFAOYSA-N chrysophanol Natural products Cc1cc(O)c2C(=O)c3c(O)cccc3Oc2c1 NZPQWZZXRKZCDU-UHFFFAOYSA-N 0.000 title claims abstract description 35
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- CZCIKBSVHDNIDH-UHFFFAOYSA-N Nalpha-methyl-DL-tryptophan Natural products C1=CC=C2C(CC(NC)C(O)=O)=CNC2=C1 CZCIKBSVHDNIDH-UHFFFAOYSA-N 0.000 title claims abstract description 15
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Images
Classifications
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a method for separating and extracting chrysophanol, luteolin, abrine and protocatechuic acid from abrus cantoniensis hance and application thereof.
Description
Technical Field
The invention belongs to the technical field of extraction of traditional Chinese medicinal materials, and particularly relates to a method for separating and extracting chrysophanol, luteolin, abrin and protocatechuic acid from abrus cantoniensis hance and application thereof.
Background
Abrus cantoniensis (Abrus cantonensis) is a dry whole plant of Abrus in Leguminosae, is mainly produced in south of Ling of China, is mainly distributed in Guangdong, guangxi and the like, and belongs to one of strong medicinal materials. The abrus cantoniensis has a long history of medication in China, is a Chinese herbal medicine with homology of medicine and food, is rich in various bioactive components, and can be used as a medicine by whole herbs. In folk, because abrus herb has wide pharmacological action, is cheap and easy to cultivate, people often eat abrus herb in a way of cooking herbal tea, cooking soup and the like, and the abrus herb is used for relieving summer heat and clearing damp and has the effects of soothing liver and relieving pain, clearing damp and removing jaundice, clearing heat and detoxifying and the like. The abrus cantoniensis hance is rich in various biological activities, such as anti-tumor, anti-oxidation, antibacterial and antiviral, anti-inflammatory and analgesic, lipid-lowering and liver-protecting, immunity-enhancing and wound healing-promoting effects, is derived from various chemical components such as polysaccharides, flavonoids, alkaloids, triterpenes, anthraquinone compounds, trace elements and other types, is clinically and frequently used for treating diseases such as hepatitis, damp-heat jaundice, uncomfortable ribs, gastral cavity distending pain, acute mastitis, internal injury from falls and the like, and has good application potential in the industries such as food, health care products, medicines, cosmetics and the like.
Although abrus cantoniensis hance has various biological activities, the abrus cantoniensis hance is widely researched mainly in terms of pharmacological actions of tumor resistance and oxidation resistance. For example: the invention discloses application of abrus herb extract in preparing anticancer drugs, wherein the application of abrus herb petroleum ether extract, abrus herb ethyl acetate extract, abrus herb n-butyl alcohol extract and/or abrus herb water extract in preparing anticancer drugs (especially gastric cancer, liver cancer and breast cancer) is respectively researched, and bax protein can be promoted and bcl-2 expression can be inhibited to enable tumor cells to undergo apoptosis. For another example, the invention patent with publication number CN107334688A discloses abrus herb flavone with antioxidant activity and an extraction method and application thereof, wherein abrus herb flavone is mainly extracted by adopting enzymatic catalysis and ethanol ultrasonic extraction, can be used in various skin care products, and has antioxidant function.
The existing research shows that the abrus herb not only has various biological activities, but also contains rich chemical components and has rich pharmacological actions, but the research aiming at the abrus herb at present only stays at the pharmacodynamic action of a certain compound, such as: the invention discloses a method for extracting an amic acid component from Chinese elder herb leaves or abrus cantoniensis leaves, a method for extracting abrus cantoniensis total saponins, a method for extracting abrus cantoniensis free amino acid, and the like, which are disclosed in the invention patent with the publication number of CN106265865A, the invention patent with the publication number of CN104069154A, and the invention patent with the publication number of CN103989733A, and the like, and pharmacodynamic researches aiming at specific monomer compounds can not be deeply carried out. As is well known, the active monomer compound extracted and separated from the Chinese medicinal materials has important significance for researching the action mechanism and the relation between the action mechanism and the drug effect, so the invention is developed in response to further promoting the popularization and the application of the abrus herb in foods, health care products, medicines and even clinics.
Disclosure of Invention
The invention aims to obtain unknown active monomer compounds in abrus herb and research the drug effect of the compounds, and provides a method for separating and extracting chrysophanol, luteolin, abrine and protocatechuic acid from abrus herb.
The invention is realized by the following technical scheme: a method for separating and extracting chrysophanol, luteolin, abrine and protocatechuic acid from herba abri comprises the following steps:
s1, drying and crushing whole herb of abrus cantoniensis hance, soaking the herb in 8-10 times of 95% ethanol, performing reflux extraction at 70-80 ℃ for 1h, repeating the reflux extraction for 2-3 times, combining extracting solutions, filtering, and removing ethanol to obtain an extracted concentrated solution;
s2, diluting the extraction concentrated solution with water, extracting with ethyl acetate for 2-3 times at an extraction ratio of 1-2: 2-4, combining an extraction phase and an extraction liquid respectively, and performing reduced pressure concentration on the extraction phase and the extraction liquid respectively to obtain an ethyl acetate part extract and a water part extract;
s3, dissolving the ethyl acetate part extract by using methanol, separating by using a silica gel column chromatography to obtain a Fr.1-3 component, a Fr.4-7 component and a Fr.8-10 component,
s4, dissolving the Fr.1-3 component with ethyl acetate, filtering, concentrating, freezing, crystallizing and drying to obtain chrysophanol;
s5, preparing and separating the Fr.4-7 component by using a C18 column, and then concentrating and drying to obtain luteolin;
s6, preparing and separating the Fr.8-10 components by using a C18 column, and concentrating and drying to obtain abrine;
s7, dissolving the water-part extract, separating by using a C18 column, concentrating and drying to obtain the protocatechuic acid.
Further, in the step S3, the silica gel column chromatography satisfies the following conditions:
sample mixing silica gel: 200-300 meshes;
removing the lotion: dichloromethane-methanol;
gradient elution: 40: 1 → 30: 1 → 20: 1 → 10: 1.
Further, in the step S5, the C18 column preparation and separation satisfies the following conditions:
mobile phase: acetonitrile-0.1% formic acid water with the volume ratio of 30: 70;
wavelength: 350nm.
Further, in the step S6, the C18 column preparation separation satisfies the following condition:
mobile phase: 0.4 percent of triethylamine-0.2 percent of phosphoric acid, and the volume ratio is 18: 82;
wavelength: 280nm.
Further, in the step S7, the C18 column preparation separation satisfies the following condition:
mobile phase: methanol-0.1% by volume of PA, in a volume ratio of 15: 85;
wavelength: 260nm.
And the application of the method in preparing anti-tumor drugs.
And the application of the method in preparing a DNA Topo I inhibitor medicine.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) The invention separates and extracts four active monomer compounds in the abrus herb for the first time, wherein the four active monomer compounds are respectively chrysophanol, luteolin, abrin and protocatechuic acid, and the application of the abrus herb in the preparation of anti-tumor and inhibitor medicines is realized.
(2) The invention takes the whole herb of the abrus herb as a raw material, takes ethyl acetate as an extracting agent, and extracts and separates chrysophanol, luteolin, abrin and protocatechuic acid respectively aiming at an ethyl acetate extraction part and a water part thereof by combining a silica gel column chromatography and a C18 column preparation separation mode, thereby realizing the pharmacological research of specific chemical components in the abrus herb and laying a foundation for the clinical application of the abrus herb.
Drawings
FIG. 1 is a drawing of Compound 1 1 H-NMR spectrum.
FIG. 2 is a drawing of Compound 1 13 C-NMR spectrum.
FIG. 3 is an infrared spectrum of Compound 1.
FIG. 4 is a UV spectrum of Compound 1.
FIG. 5 is a mass spectrum of Compound 1.
FIG. 6 is a drawing of Compound 2 1 H-NMR spectrum.
FIG. 7 is a drawing of Compound 2 13 C-NMR spectrum.
FIG. 8 is an infrared spectrum of Compound 2.
FIG. 9 is a UV spectrum of Compound 2.
FIG. 10 is a mass spectrum of Compound 2.
Fig. 11 is a mass spectrum of compound 3.
FIG. 12 shows a scheme for preparing Compound 3 1 H-NMR spectrum.
FIG. 13 shows Compound 4 1 H-NMR spectrum.
FIG. 14 is a drawing of Compound 4 13 C-NMR spectrum.
FIG. 15 is a mass spectrum (1) of Compound 4.
Fig. 16 is a mass spectrum (2) of compound 4.
FIG. 17 shows the flow results of 48h of luteolin effect on MCF-7 cells (Comp-FITC-A: annexin-V-FITC-A).
FIG. 18 shows the flow-through results of the action of luteolin on SGC-7901 cells for 48h (Comp-FITC-A: annexin-V-FITC-A).
FIG. 19 shows the flow results of 48h of luteolin effect on BEL-7404 cells (Comp-FITC-A: annexin-V-FITC-A).
FIG. 20 shows the results of the inhibitory effect of camptothecin on DNA Topo I.
FIG. 21 shows the result of the inhibitory effect of luteolin on DNA Topo I
In fig. 17 to 19, control refers to the flow result of the blank Control group without drug; 0.1% DMSO refers to the flow assay of 0.1% dimethylsulfoxide; reagent refers to the flow results of 50. Mu.L of 0.1M NaCl.
In fig. 20, the sequence from left to right is: m: lambda-Hind III marker, lane 1: pBR322 was added, and DNA Topo I was not added, lane 2: pBR322 and DNA Topo I were added, lanes 3, 4, 5, 6, 7, 8, 9: the drug concentrations of added camptothecin were 200, 100, 50, 25, 10, 5, 2.5 μ M, respectively.
In fig. 21, from left to right: m: lambda-Hind III marker, lane 1: pBR322 was added, and DNA Topo I was not added, lane 2: adding 200 μ M of positive control drug camptothecin; lane 3: pBR322 and DNA Topo I were added, lanes 4, 5, 6, 7, 8: the concentrations of luteolin added are 240, 120, 60, 30 and 15 μ M respectively.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1:
the embodiment is a method for separating and extracting chrysophanol, luteolin, abrine and protocatechuic acid from abrus cantoniensis hance, which comprises the following steps:
firstly, soaking about 44Kg of dried and crushed abrus herb in 10 times of 95% ethanol, performing reflux extraction at 75 ℃ for 1 time, repeating the extraction for 2 times, combining extracting solutions, filtering by using filter cloth, and performing reduced pressure concentration to obtain an extracted concentrated solution.
And step two, diluting the extract concentrated solution with pure water, extracting with ethyl acetate for 3 times at an extraction ratio of 1: 2, respectively combining an extract phase and an extract liquid, concentrating the extract phase under reduced pressure to obtain an ethyl acetate part extract, and preparing a water part extract from the extract liquid by the same method.
Step three, dissolving the ethyl acetate part extract by using methanol, stirring the extract by using a 200-mesh silica gel dry method, loading the extract on a column by using a silica gel wet method, performing gradient elution according to the ratio of 40: 1 → 30: 1 → 20: 1 → 10: 1 by using dichloromethane-methanol as a eluent, collecting components, performing tracking detection on a compound by using TLC, and combining the same components of Rf values to obtain a Fr.1-component, a Fr.4-7 component and a Fr.8-10 component.
Further, dissolving Fr.1-3 component with ethyl acetate, filtering, concentrating, freeze crystallizing, and drying to obtain compound 1 (500 mg); separating Fr.4-7 with acetonitrile-0.1% formic acid water (volume ratio 30: 70) as mobile phase at 350nm wavelength by C18 column, concentrating, and drying to obtain compound 2 (500 mg); the Fr.8-10 components are prepared and separated by a C18 column at the wavelength of 280nm by taking 0.4% triethylamine-0.2% phosphoric acid (volume ratio is 18: 82) as a mobile phase, and then the compound 3 (300 mg) is obtained after concentration and drying.
And step four, dissolving the water part extract, using methanol-0.1% phosphoric acid (volume ratio 15: 85) as a mobile phase, preparing and separating by using a C18 column at the wavelength of 260nm, concentrating and drying to obtain a compound 4 (300 mg).
Example 2:
the embodiment is a method for separating and extracting chrysophanol, luteolin, abrine and protocatechuic acid from abrus cantoniensis hance, which comprises the following steps:
firstly, soaking about 44Kg of dried and crushed whole herb of abrus cantoniensis hance in 8 times of 95% ethanol, performing reflux extraction at 70 ℃ for 1h, repeating the extraction for 2 times, combining the extracting solutions, filtering the extracting solutions by using filter cloth, and performing reduced pressure concentration to obtain an extracted concentrated solution.
And step two, diluting the extract concentrated solution with pure water, extracting with ethyl acetate for 2 times at an extraction ratio of 1: 3, respectively combining an extract phase and an extract liquid, concentrating the extract phase under reduced pressure to obtain an ethyl acetate part extract, and preparing a water part extract from the extract liquid by the same method.
Dissolving the ethyl acetate part extract with methanol, mixing the extract with 300-mesh silica gel by a dry method, loading the mixture on a column by a wet method, performing gradient elution according to the ratio of 40: 1 → 30: 1 → 20: 1 → 10: 1 by taking dichloromethane-methanol as a eluent, collecting components, performing tracking detection on the compound by TLC, and combining Rf values with the same components to obtain a Fr.1-component, a Fr.4-7 component and a Fr.8-10 component.
Further, dissolving Fr.1-3 component with ethyl acetate, filtering, concentrating, freeze crystallizing, and drying to obtain compound 1 (500 mg); separating Fr.4-7 with acetonitrile-0.1% formic acid water (volume ratio 30: 70) as mobile phase at 350nm wavelength by C18 column, concentrating, and drying to obtain compound 2 (500 mg); the Fr.8-10 components are prepared and separated by a C18 column under the wavelength of 280nm by taking 0.4% triethylamine-0.2% phosphoric acid (volume ratio is 18: 82) as a mobile phase, and then the compound 3 (300 mg) is obtained after concentration and drying.
And step four, dissolving the water part extract, using methanol-0.1% phosphoric acid (volume ratio 15: 85) as a mobile phase, preparing and separating by using a C18 column at the wavelength of 260nm, concentrating and drying to obtain a compound 4 (300 mg).
Example 3:
the embodiment is a method for separating and extracting chrysophanol, luteolin, abrine and protocatechuic acid from abrus cantoniensis hance, which comprises the following steps:
step one, soaking about 44Kg of dried and crushed abrus herb in 10 times of 95% ethanol, performing reflux extraction at 75 ℃ for 1h, repeating for 3 times, combining extracting solutions, filtering twice by using filter cloth, performing reduced pressure concentration on filtrate at 55 ℃ by using a rotary evaporator to obtain an extract which is nearly in an alcohol-free semi-flowing state, enriching the extract obtained by rotary evaporation, continuously evaporating residual ethanol in a water bath until no alcohol smell exists, and finally obtaining a dark brown viscous extraction concentrated solution.
Step two, diluting the extract concentrated solution with pure water, extracting with ethyl acetate for 3 times at an extraction ratio of 1: 1, respectively combining an extraction phase and an extraction liquid, concentrating the extraction phase under reduced pressure to obtain an ethyl acetate part extract, and preparing a water part extract from the extraction liquid by the same method.
Step three, dissolving the ethyl acetate part extract by using methanol, then mixing the sample by using a 300-mesh silica gel dry method, taking a 15-time silica gel wet method to put on a column, taking dichloromethane-methanol as a eluent, performing gradient elution according to the ratio of 40: 1 → 30: 1 → 20: 1 → 10: 1, collecting components, performing tracking detection on a compound by using TLC, and combining Rf values with same flow components to obtain a Fr.1-component, a Fr.4-7 component and a Fr.8-10 component.
Further, dissolving Fr.1-3 component with ethyl acetate, filtering, concentrating, freeze crystallizing, air drying at 50 deg.C, and drying under reduced pressure at 40 deg.C to obtain compound 1 (500 mg); taking the Fr.4-7 components and acetonitrile-0.1% formic acid water (volume ratio 30: 70) as a mobile phase, separating by using a C18 column at the wavelength of 350nm, concentrating a product preparation solution at 50 ℃ until a solid is separated out, drying by blowing at 50 ℃ and drying under reduced pressure at 40 ℃ to obtain a compound 2 (500 mg); the Fr.8-10 components are prepared and separated by a C18 column under the wavelength of 280nm by taking 0.4% triethylamine-0.2% phosphoric acid (volume ratio is 18: 82) as a mobile phase, and a product preparation solution is concentrated to be dry at 45 ℃, dried by air blowing at 40 ℃ and dried under reduced pressure at 40 ℃ to obtain a compound 3 (300 mg).
And step four, dissolving the water part extract, using methanol-0.1% phosphoric acid (volume ratio 15: 85) as a mobile phase, preparing and separating by using a C18 column at the wavelength of 260nm, concentrating a product preparation solution at 50 ℃, and performing freeze drying and reduced pressure drying at 40 ℃ to obtain a compound 4 (300 mg).
Example 4: structural characterization of Compound 1
The compound 1 is extracted and separated by the method of example 3, and the compound 1 is orange yellow solid with a melting point of 196.0-198.0 ℃. Dissolved in DMSO, and insoluble in methanol, acetonitrile and water. Petroleum ether-ethyl acetate-formic acid (10: 1: 0.2), cyclohexane-acetone-formic acid (8: 0.5: 0.1) and cyclohexane-ethyl acetate-formic acid (8: 1: 0.1) were spread on silica gel G thin layer plates, respectively, and fluorescence was observed under 365nm ultraviolet light, and the spot of compound 1 was clear and single.
From compound 1 1 H-NMR(400MHz，CDCl 3 ) The spectrum (see FIG. 1) shows delta H 12.07 (1H, s) and delta H 11.96 (1H, s) is the 2 active hydrogen signals conjugated to carbonyl groups, delta H 7.80～7.75(1H,m)，δ H 7.64(1H,t,J＝ 8.0Hz)，δ H 7.60(1H,s)，δ H 7.28～7.25(1H,m)，δ H 7.06 (1H, s) is the 5 benzene ring hydrogen signal, delta H 2.44 (3H, s) is a methyl radical having a chemical shift up to a value of delta H 2.44, indicating that it is attached to a benzene ring.
From compound 1 13 C-NMR(100MHz，CDCl 3 ) The spectrum (see FIG. 2) shows delta C 192.4，δ C 181.8 Is the 2 carbonyl carbon signal, delta C 162.3，δ C 124.5，δ C 149.3，δ C 121.3，δ C 119.8，δ C 136.9，δ C 124.3，δ C 162.6，δ C 133.2，δ C 133.5，δ C 115.8，δ C 113.6 is a 12-benzene ring carbon signal indicating that compound 1 has 2 benzene rings, delta C 22.2 carbon signal for methyl on phenyl ring. From the combination of hydrogen spectrum and carbon spectrum, the compound has 2 benzene rings, 2 carbonyl groups and 1 methyl group.
Further, the presence of hydroxyl groups (3448 cm) in the molecular structure is shown in the infrared spectrum (see FIG. 3) -1 ) Double bond (1676 cm) -1 ) Aromatic ring (1605 cm) -1 ) An equal characteristic peak signal; its UV absorption peaks λ max =224, 256, 287, 430nm (see fig. 4) show a quinone-like structure with a chromophore. ESI-MS m/z (see FIG. 5): 253.2[ 2 ] M-H] ─ Molecular weight M =254.24.
Bonding with 1 H-NMR、 13 C-NMR, IR, UV and other data, and the molecular formula of the compound 1 is deduced to be: c 15 H 10 O 4 The calculated unsaturation is Ω =11. In conclusion, the monomeric compound 1 was identified as Chrysophanol (Chrysophanol). The chemical structure is shown in the following formula (1), and the hydrogen signal and the carbon signal of the compound are respectively shown in tables 1 and 2.
The compounds of Table 1Nuclear magnetic resonance hydrogen spectrum of object 1 ( 1 H-NMR) data to reference data
Note: actual test conditions were: 1 H NMR(400MHz,CDCl 3 )；
literature test conditions: 1 H NMR(400MHz,CDCl 3 )。
TABLE 2 preparation of Compound 1 nuclear magnetic resonance carbon spectrum ( 13 C-NMR) data to reference data
Note: actual test conditions were: 13 C-NMR(100MHz,CDCl 3 )；
literature test conditions: 13 C-NMR(100MHz,CDCl 3 )。
example 5: structural characterization of Compound 2
From compound 2 1 H-NMR(400MHz，DMSO-d 6 ) The spectrum (see FIG. 6) shows delta H 6.19(1H,d,J＝ 2.0Hz)，δ H 6.44(1H,d,J＝2.0Hz)，δ H 6.67(1H,s)，δ H 6.89(1H,d,J＝8.3Hz)，δ H 7.44-7.37 (2H, m) are 6 benzene ring hydrogen signals, delta H 12.97 (1H, s) is 1 active hydrogen signal conjugated with carbonyl and is a characteristic hydroxyl hydrogen signal of 5-site of flavone, and the compound 2 is a flavonoid compound.
From compound 2 13 C-NMR(100MHz，DMSO-d 6 ) The spectrum (see FIG. 7) shows delta C 164.0，δ C 103.0，δ C 181.7，δ C 161.6，δ C 98.9，δ C 164.2，δ C 93.9，δ C 157.4，δ C 103.8，δ C 121.6，δ C 113.4，δ C 145.8，δ C 149.7，δ C 116.1，δ C 119.0 is a 15 benzene ring carbon or alkene carbon signal, which indicates that the compound 2 is a flavonoid compound.
Furthermore, the presence of hydroxyl groups (3418 cm) in the structure is shown in the infrared spectrum (see FIG. 8) -1 ) Carbonyl group (1655 cm) -1 ) Aromatic group (1501 cm) -1 ) And (4) waiting for characteristic signal peaks. Its UV absorption peak λ max =207, 253, 351nm (see fig. 9) shows the presence of a carbon-carbon double bond (207 nm) in the structure. ESI-MS m/z (see FIG. 10): 285.0[ M-H ]] - Molecular weight M =286.24.
Bonding with 1 H-NMR、 13 C-NMR, IR and UV data, and the molecular formula of the compound 2 is deduced to be: c 15 H 10 O 6 The calculated unsaturation is Ω =11. In summary, the monomeric compound 2 was identified as Luteolin (Luteolin), and the chemical structure thereof is shown in the following formula (2), and the hydrogen signal and the carbon signal of the compound 2 are shown in tables 3 and 4, respectively.
TABLE 3 NMR spectrum of Compound 2: ( 1 H-NMR) data to reference data
Note: actual test conditions: 1 H NMR(400MHz,DMSO-d6)；
literature test conditions: 1 H NMR(600MHz,DMSO-d6)。
TABLE 4 NMR C spectra of Compound 2: ( 13 C-NMR) data to reference data
Note: actual measurement conditions: 13 C-NMR(100MHz,DMSO-d6)；
literature test conditions: 13 C-NMR(150MHz,DMSO-d6)。
example 6: structural characterization of Compound 3
From compound 3 1 H-NMR structural data show: presence of delta H 10.87(1H,s,1-NH)，δ H 7.58(1H,m,H-4)，δ H 7.33(1H,m,H-7)，δ H 7.22(1H,s,H-2)，δ H 7.05(1H,m,H-6)，δ H 6.98(1H,m,H-5)，δ H 3.33～3.50(3H,m,H-10,H-11)，δ H 2.36(3H,s,N-CH 3 ). In that 1 The H-NMR spectrum (see FIG. 12) shows. Delta H 10.87 (1H, s) is an active hydrogen signal, presumably the NH signal, delta H 7.58(1H,m)，δ H 7.33(1H,m)，δ H 7.22(1H,s)，δ H 7.05(1H,m)，δ H 6.98 (1H, m) is a 5-phenyl ring or double bond hydrogen signal, delta H 3.33-3.50 (3H, m, H-10, H-11) are hydrogen signals on aliphatic or azino carbons,. Delta. H 2.36 (3H, s) is the methyl hydrogen signal.
Bonding with 1 The molecular formula of the compound 3 is deduced to be shown by H-NMR and MS data: c 12 H 14 N 2 O 2 As described above, monomeric compound 3 was identified as abrin (Abrine), and its chemical structure is shown in the following formula (3).
Example 7: structural characterization of Compound 4
From compound 4 1 H-NMR(400MHz，DMSO-d 6 ) The spectrum (see FIG. 13) shows δ H 7.35(1H,d,J ＝2.0Hz)，δ H 7.30(1H,dd,J＝8.2,2.1Hz)，δ H 6.79 (1h, d, j = 8.2hz) is 3 benzene ring hydrogen signals, and from the peak pattern and coupling constant of hydrogen, it can be known that the 3 hydrogens are ABX coupled systems.
From compound 4 13 C-NMR(100M Hz，DMSO-d 6 ) Spectrum (see fig. 14) is in 13 Delta in C-NMR C 169.2 Is a carboxylic acid or ester carbonyl carbon signal, δ C 117.0，δ C 118.4，δ C 123.5，δ C 123.7，δ C 146.7，δ C 151.8 is a 6 benzene ring carbon signal indicating that compound 4 has a benzene ring and a carboxyl group.
Bonding with 1 H-NMR、 13 C-NMR and MS data show that the compound 4 has a molecular formula: c 7 H 6 O 4 The calculated unsaturation is Ω =5. As described above, monomeric compound 4 was identified as Protocatechuic acid (Protocatechuic acid), and its chemical structure was shown in the following formula (4), and the hydrogen signal and carbon signal of compound 4 were shown in tables 5 and 6, respectively.
TABLE 5 NMR spectrum of Compound 4 (NMR) 1 H-NMR) data to reference data
Note: actual test conditions were: 1 H NMR(400MHz,DMSO-d6)；
literature test conditions: 1 H NMR(300MHz,DMSO-d6)。
TABLE 6 NMR C spectra of Compound 4: ( 13 C-NMR) data to reference data
Note: actual test conditions were: 13 C-NMR(100MHz,DMSO-d6)；
literature test conditions: 13 C-NMR(75MHz,DMSO-d6)。
example 8: experiment on antitumor Activity
1. Inhibition of tumor cell proliferation by monomeric compounds
(1) Cell culture and plating
Human breast cancer MCF-7 cells, human gastric cancer SGC-7901 cells, human liver cancer BEL-7404 cells, 3 kinds of cancer cells: MCF-7 and BEL-7404 cell strains: P/S containing DMEM +10% by FBS + 1; SGC-7901 cell line: the RPMI-1640+10% of FBS +1% of the cancer cells were each placed in 5% CO 2 And culturing in a 37 ℃ saturated humidity incubator. 3 cell strains which are normally cultured and are in logarithmic phase are taken, rinsed for 2-3 times by PBS buffer solution, 0.8-1mL of 0.25% trypsin is taken for digestion, digestion is stopped by complete culture medium, cell suspension is sucked and transferred to a centrifuge tube, andcentrifuging at 1000rpm for 3min, discarding supernatant, collecting cells, counting cells, and adjusting cell concentration to 3 × 10 3 And inoculating the cells/well into a 96-well plate, and placing 90 mu L of each well into an incubator for standing culture for 24 hours.
(2) Dissolution and dilution of the drug
Chrysophanol (molecular weight: 254.24), mother liquor: DMSO 5mg/mL (19.67 mM). 1mM:1.867mL of complete cell culture medium + 10. Mu.L of mother liquor;
abrine (molecular weight: 218.25), mother liquor: 0.1M NaOH dissolved and neutralized with 1M HCl to pH =7, 8.73mg/mL (40 mM);
(3) Pharmaceutical intervention
Setting zero-adjusting group (only adding culture medium), control group (containing cell sap and culture solution, without medicine) as 0-adding medicine group, DMSO group (containing cell sap, culture solution and medicine solvent DMSO, without medicine), experimental group (containing cell sap, culture solution and medicine), wherein the medicine is chrysophanol and luteolin, after cell adherent culture for 24h, changing into DMEM/RPMI-1640 complete culture medium containing medicines with different concentrations, and continuously placing in 5 CO 2 And culturing in a 37 ℃ saturated humidity incubator. The time for intervention of the drug in the 3 tumor cells is 24h, 48h and 72h respectively, wherein the concentration of the drug chrysophanol in the 3 tumor cells in the 3 time periods is as follows: 5. 10, 50, 100, 200, 300 μ M; the concentration of the drug luteolin intervening in 3 tumor cells in 24h, 48h and 72h time periods is 5, 10, 20, 40, 60, 80 and 100 mu M. 100 μ L of medium containing different concentrations of drug was added to each well, with 6 replicates per concentration.
(4) Colorimetric method detection by MTT method
After 3 different time points of drug intervention, 100 μ L of MTT solution was added to each well before termination of the experiment, and the incubation was continued in an incubator for 4h at 37 ℃ in the dark. And (3) throwing the culture solution when the incubation is finished, adding 150 mu L of DMSO into each hole to terminate the reaction, then putting the mixture into a shaking table, shaking the mixture evenly to fully dissolve crystals, and determining the dissolving time according to the size and dissolving condition of the crystals. Measuring absorbance (OD) of each well at 490nm with a multifunctional microplate reader, calculating the experimental result according to the following formula A, and measuring the data with average number ±Standard deviation of
IC's representing, and calculating, drug intervention in 3 cell lines at 3 time points 50 Values, with P <0.05, indicate that the difference is statistically significant.
Formula A: cell survival rate (%) = (experimental group-zero set group)/(0 additional drug group-zero set group) × 100%.
(5) Results of the experiment
The results of the influence of chrysophanol with different concentrations on the activities of the MCF-7, SGC-7901 and BEL-7404 three tumor cells 24, 48 and 72h are shown in Table 7, and experimental data shows that chrysophanol has an inhibitory effect on the in vitro proliferation of 3 tumor cells at 3 time points, and the survival rate of the tumor cells is increased along with the reduction of the drug concentration; the inhibition increases with increasing drug concentration, and is concentration dependent. IC of 3 time points by calculating chrysophanol to intervene on 3 tumor cells 50 Value, finding the IC of chrysophanol interfering 3 kinds of tumor cells for 72 hours 50 The values were 108.5. Mu.M, 39.21. Mu.M and 46.30. Mu.M, respectively. IC comparing effect of chrysophanol on 3 cells for 72h 50 Values, chrysophanol was found to be sensitive to 3 cells: SGC-7901 > BEL-7404 > MCF-7. Therefore, chrysophanol is sensitive to SGC-7901 cells.
Table 7 effect of chrysophanol on proliferation of 3 cells (survival rate,
n＝6)
note: p <0.01, P <0.05 compared to Control (blank).
Intervention MCF-7 with luteolin of different concentrations,The results of the effects of the activities of 24, 48 and 72h cells of SGC-7901 and BEL-7404 are shown in Table 8, and experimental data show that luteolin has an inhibitory effect on the in vitro proliferation of 3 tumor cells, and the survival rate of the tumor cells is increased along with the decrease of the drug concentration; the inhibitory effect increases with increasing drug concentration. IC of 3 time points by calculating the intervention of luteolin on 3 tumor cells 50 Value, finding the IC of chrysophanol on 3 tumor cells in 72 hours of intervention 50 The values are respectively: 29.47. Mu.M, 32.66. Mu.M, 36.29. Mu.M. IC comparing the Effect of luteolin on 3 cells for 72h 50 Values, the degree of sensitivity of luteolin to 3 cells was found to be: MCF-7 > SGC-7901 > BEL-7404. Thus, luteolin was found to be sensitive to MCF-7 cells.
Table 8 effect of luteolin on proliferation of 3 cells (survival,
n＝6)
note: p <0.01, P <0.05 compared to Control (blank).
The experimental data show that chrysophanol and luteolin all have inhibition effect on in-vitro proliferation of 3 tumor cells, and have obvious anti-tumor activity, the cell survival rate is reduced along with the increase of the drug concentration, and the inhibition effect is dependent on the drug concentration. Wherein, chrysophanol is sensitive to human hepatoma carcinoma cell BEL-7404; luteolin is sensitive to human breast cancer cell MCF-7. IC of 2 monomeric Compounds at 3 time points on 3 cell lines 50 The values are shown in Table 9.
TABLE 9 IC of 3 time points of 3 monomeric compounds on 3 cell lines 50 Value (Unit:. Mu.M)
2. Apoptosis-promoting effect of monomeric compound on tumor cells
(1) Culture of tumor cells
The culture conditions of the cell strains of human breast cancer MCF-7, human gastric cancer SGC-7901 and human liver cancer BEL-7404 are respectively as follows: MCF-7 and BEL-7404 cell strains: DMEM +10% FBS +1% P/S; SGC-7901 cell line: RPMI-1640+10% FBS +1% P/S, put at 5% CO 2 And culturing in a 37 ℃ saturated humidity incubator. See the above tumor cell culture for details.
(2) Preparation of mother liquor of medicine
Luteolin (molecular weight: 286.24), mother liquor: DMSO 57mg/mL (199.13 mM).
(3) Tumor cell plating and drug intervention concentration
Taking 3 tumor cell strains which are normally cultured and are in logarithmic growth phase, digesting the 3 tumor cell strains by 0.8-1mL of 0.25% trypsin, centrifuging the cells at 800rpm for 3min, collecting the cells, counting the cells by a counter, paving a 6-hole plate on each cell, and adding 3.0-4.0 multiplied by 10 in each hole 5 Placing the individual cells (with corresponding cell plating concentration adjustable according to different cell strains) into an incubator respectively, and standing for 24h [12] . Selecting proper drug concentration for carrying out apoptosis detection experiments according to the previous MTT result, wherein the concentration of the drug groups is as follows: the concentration of the medicine chrysophanol intervening in the 3 cells is 19.67 mu M; the concentrations of the drug luteolin for intervening 3 cells are respectively 20, 40 and 61 mu M; the concentrations of the drug abrine in the 3 cells were: 1mM; the concentration of the drug protocatechuic acid to interfere with 3 cells was: 194.65. Mu.M. The next day, 2mL of the prepared culture medium containing luteolin is added to the cells with good growth state and 70% -80% fusion degree, and the cells are placed into an incubator for culturing for 48h.
(4) Detecting apoptosis by flow cytometry (Annexin V/PI double staining detection apoptosis)
After 48h drug treatment the cell status was observed and the flow analysis treatment was started by first pipetting the supernatant of the cells in the 6-well plate into a 5mL EP tube (since there were some cells in the cell supernatant that had floated apoptotic), washing the cells twice with PBS pre-chilled on ice, then treating the cells for digestion with trypsin without EDTA for 3-5min (stopping digestion depending on the cell status observed in the microscope, depending on the time of cell digestion), collecting the cells after stopping digestion into the previous 5mL EP tube, removing the supernatant after centrifugation at 800rpm for 3min and rinsing the cells with pre-chilled PBS and transferring to a 1.5mL EP tube, removing the supernatant after centrifugation at 1000rpm for 5min, and then repeating the step of washing the cells once more.
With sterile dd H beforehand 2 And O, diluting the 10 xBinding buffer into 1 xBinding buffer, standing at room temperature, re-suspending 100 mu L of the 1 xBinding buffer into cells, respectively adding 5 mu L of FITC-Annexin V and 5 mu L of PI into each tube of cells in the dark condition, after flicking and uniformly mixing, standing for 15min in the dark at room temperature, then adding 400 mu L of the 1 xBinding buffer, and filtering the mixture into clumps of cells by using 400-mesh filter cloth after uniformly blowing (preventing the flow cytometry analyzer from being blocked). And (5) placing the processed cells into an ice box to be stored in a dark place, and performing machine analysis within 1 h. Data were analyzed using FlowJo software and plotted against control cell analysis.
(5) Results of the experiment
The apoptosis of the cells is detected by Annexin V-FITC/PI flow cytometry.
The detailed results of the apoptosis promoting effect of the monomeric compound luteolin on three tumor cells of MCF-7, SGC-7901 and BEL-7404 are shown in figure 17, figure 18 and figure 19. The apoptosis rate of luteolin in human breast cancer cell MCF-7 after 48 hours intervention is 15.71%, and the apoptosis rate of a blank control group is 2.49%. The apoptosis rate of luteolin in human gastric cancer cell SGC-7901 after intervention for 48h is 17.00%, and the apoptosis rate of blank control group is 1.07%. The apoptosis rate of luteolin in human liver cancer cell BEL-7404 for 48h is 30.54%, and the apoptosis rate of blank control group is 2.1%.
Therefore, the luteolin has an advantage in apoptosis promoting effect on BEL-7404 cells, and the apoptosis rate of the luteolin is 30.54%. Experimental results show that luteolin has the effect of inducing 3 tumor cells to undergo apoptosis.
Example 9: DNA Topo I inhibition assay
(1) Preparation of mother liquor of medicine and dilution of medicine
Camptothecin (molecular weight: 348.34), mother liquor: DMSO 3.4834mg/mL (10 mM). Diluting with organic solvent DMSO, adding 10 μ L10 mM camptothecin mother liquor into 15 μ L DMSO to obtain 4mM camptothecin solution, and diluting at a multiple ratio to obtain solutions of camptothecin positive control drug with a series of concentrations of 2.5, 5, 10, 25, 50, 100, 200 μ M, etc.
Luteolin (molecular weight: 286.24), mother liquor: DMSO 57mg/mL (199.13 mM). Diluting with organic solvent DMSO, adding 4.8 μ L199.13 mM luteolin mother liquor into 194.33 μ L DMSO to obtain 4.8mM luteolin solution, and diluting at multiple ratio to obtain 15, 30, 60, 120, 240 μ M solutions.
(2) Preparation of electrophoresis-related reagents
Reaction system (50 × TAE solution formulation) Tris-Base 242g; na (Na) 2 EDTA·2H 2 O37.2 g; 57.1 mL of glacial acetic acid; dd H 2 O up to 1L (use dd H) 2 Dilute O to 1 ×).
Preparation of agarose gel: and adding a proper amount of buffer solution (the amount required by a balance weighing experiment) with proper concentration into the conical flask, heating, boiling and shaking uniformly until the buffer solution is completely melted, and obtaining the product when the liquid has no bubbles.
Preparing a rubber plate: cleaning and airing the used instruments and moulds for later use, putting the comb into a gel making groove, fixing the comb in a corresponding clamping groove, pouring agarose gel liquid with the temperature reduced to 65 ℃ into a tray, uniformly spreading the gel liquid on the surface, standing at room temperature until the gel liquid is cooled and solidified, gently vertically and upwards pulling out the comb, slowly putting the gel and the gel making groove into an electrophoresis groove, and adding electrophoresis buffer solution into the electrophoresis groove until the buffer solution is over the gel plate.
(3) Preparation of enzyme reaction mix
Reaction system 1: pBR322 DNA 0.5. Mu.g/1. Mu.L; 10 XDNA Topo I Buffer 2 u L;0.1% BSA 2. Mu.L; drug/DMSO 1 μ L; DNA Topo I1. Mu.L (U); dd H 2 O up to 20. Mu.L. If it is usedAdding 0.1% BSA directly into 10X DNA Topo I Buffer will produce a large amount of white precipitate. Therefore, the reagents are added in the following order when preparing the reaction mixture: dd H 2 O → 10 XDNA Topo I Buffer → 0.1% BSA → substrate DNA → drug/DMSO → DNA Topo I.
Reaction system 2: lambda-Hind III digest 1. Mu.L; 6 × loading Buffer 1 μ L; TE Buffer up to 6. Mu.L. The original ends of lambda DNA digest DNA Markers are often combined together by COS ends, heat treatment is carried out before electrophoresis (60 ℃,5 min), the electrophoretic image of the Marker can be clearer, and TE buffer dilution is used before the heat treatment to prevent the fragments from being degraded. Therefore, the reagents are added in the following order when preparing the reaction mixture: TE Buffer → lambda-Hind III digest → thermal treatment (60 ℃,5 min) → 6 × loading Buffer.
(4) Inhibition of DNA Topo I by drugs
Adding luteolin according to the reaction system, finally adding 1 mu L of DNA Topo I, placing in a water bath at 37 ℃ for reaction for 30min after uniformly mixing, then quickly adding 10 × loading buffer (2.22 mu L) containing SDS, uniformly mixing and stopping reaction, taking lambda-Hind III digest as a marker,1% agarose gel (1 × TAE buffer configuration), carrying out 85V electrophoresis for 90min, staining for 30min by Ethidium Bromide (EB), decoloring for 30min by pure water, and carrying out imaging analysis by a gel imager.
The plasmid DNA and ethidium bromide react in the process of running the gel to ensure that the plasmid DNA is supercoiled again, so EB cannot be added in the process of agarose gel configuration, and the staining can be carried out only by a subsequent gel soaking mode.
(5) Results of the experiment
The effectiveness of the DNA Topo I activity screening system was verified by using the positive control drug camptothecin, and the experimental results are shown in FIG. 20. Lane 2 of the figure shows the control DNA Topo I (pBR 322 DNA and DNA Topo I added) which effectively uncoils supercoiled DNA and which, when camptothecin is added, effectively inhibits the activity of DNA Topo I, resulting in an increase in supercoiled DNA. As is evident from the figure, the unwinding of DNA Topo I by camptothecin resulted in an inhibitory effect from a concentration of 200. Mu.M to 25. Mu.M (i.e., lanes 3, 4, 5, and 6) as compared to the Topo I control, which is proportional to the concentration, i.e., the inhibitory effect increases with the increase of the concentration of the drug intervention.
The experimental results of the inhibition effect of luteolin on DNA Topo I are shown in FIG. 21, wherein Lane 3 is a DNA Topo I control (pBR 322 DNA and DNA Topo I are added), and it is evident from the figure that the unwinding effect of luteolin on DNA Topo I is inhibited from drug intervention concentrations of 240 μ M to 60 μ M (i.e., lane 4, 5, and 6) compared with Topo I control and camptothecin (200 μ M) in Lane 2, and the inhibition effect is proportional to the concentration, i.e., the inhibition effect is enhanced with the increase of the drug intervention concentration; the inhibitory effect of luteolin on DNA Topo I at an intervention concentration of 240. Mu.M (lane 4) is slightly weaker than the inhibitory effect of camptothecin at an intervention concentration of 200. Mu.M (lane 2) on DNA Topo I in lane 2; however, the inhibitory effect of camptothecin on DNA Topo I was close to that of the positive control, 100. Mu.M (FIG. 20, lane 4). The results show that luteolin has a certain inhibiting effect on the activity of DNA Topo I, but the inhibiting effect is slightly weaker than that of camptothecin.
The experimental result of the inhibition effect on DNA Topo I shows that luteolin has a certain inhibition effect on the activity of DNA Topo I, but the effect of luteolin is weaker than that of camptothecin.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modifications and equivalent variations of the above embodiment according to the technical spirit of the present invention are within the scope of the present invention.
Claims (7)
1. A method for separating and extracting chrysophanol, luteolin, abrine and protocatechuic acid from abrus cantoniensis hance is characterized by comprising the following steps: the method comprises the following steps:
s1, drying and crushing the whole herb of the abrus cantoniensis hance, soaking the herb in 8-10 times of 95% ethanol, performing reflux extraction at 70-80 ℃ for 1h, repeating the extraction for 2-3 times, combining the extracting solutions, filtering, and removing the ethanol to obtain an extracted concentrated solution;
s2, diluting the extraction concentrated solution with water, extracting with ethyl acetate for 2-3 times at an extraction ratio of 1-2: 2-4, combining an extraction phase and an extraction liquid respectively, and performing reduced pressure concentration on the extraction phase and the extraction liquid respectively to obtain an ethyl acetate part extract and a water part extract;
s3, dissolving the ethyl acetate part extract by using methanol, separating by using a silica gel column chromatography to obtain a Fr.1-3 component, a Fr.4-7 component and a Fr.8-10 component,
s4, dissolving the Fr.1-3 component with ethyl acetate, filtering, concentrating, freezing, crystallizing and drying to obtain chrysophanol;
s5, preparing and separating the Fr.4-7 component by using a C18 column, and then concentrating and drying to obtain luteolin;
s6, preparing and separating the Fr.8-10 components by using a C18 column, and concentrating and drying to obtain abrine;
s7, dissolving the water-part extract, preparing and separating by using a C18 column, and concentrating and drying to obtain the protocatechuic acid.
2. The method of claim 1, wherein the chrysophanol, luteolin, abrine and protocatechuic acid are isolated and extracted from abrus cantoniensis hance, wherein the method comprises the following steps: in the step S3, the silica gel column chromatography satisfies the following conditions:
sample mixing silica gel: 200-300 meshes;
removing the lotion: dichloromethane-methanol;
gradient elution: 40: 1 → 30: 1 → 20: 1 → 10: 1.
3. The method for isolating and extracting chrysophanol, luteolin, abrin and protocatechuic acid from Abrus cantoniensis Hance according to claim 1, wherein: in the step S5, the preparation and separation of the C18 column meet the following conditions:
mobile phase: acetonitrile-0.1% formic acid water with the volume ratio of 30: 70;
wavelength: 350 And (5) nm.
4. The method of claim 1, wherein the chrysophanol, luteolin, abrine and protocatechuic acid are isolated and extracted from abrus cantoniensis hance, wherein the method comprises the following steps: in the step S6, the preparation and separation of the C18 column meet the following conditions:
mobile phase: 0.4 percent of triethylamine-0.2 percent of phosphoric acid, and the volume ratio is 18: 82;
wavelength: 280 And (5) nm.
5. The method for isolating and extracting chrysophanol, luteolin, abrin and protocatechuic acid from Abrus cantoniensis Hance according to claim 1, wherein: in the step S7, the preparation and separation of the C18 column meet the following conditions:
mobile phase: methanol-0.1% phosphoric acid, the volume ratio is 15: 85;
wavelength: 260 And (5) nm.
6. Use of the method according to any one of claims 1 to 5 for the preparation of an antitumor medicament.
7. Use of a method according to any one of claims 1 to 5 for the preparation of a DNA Topo I inhibitor medicament.
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