CN115433068B - 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
- CN115433068B CN115433068B CN202210678255.8A CN202210678255A CN115433068B CN 115433068 B CN115433068 B CN 115433068B CN 202210678255 A CN202210678255 A CN 202210678255A CN 115433068 B CN115433068 B CN 115433068B
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- Prior art keywords
- luteolin
- compound
- separating
- extracting
- ethyl acetate
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- LQGUBLBATBMXHT-UHFFFAOYSA-N chrysophanol Chemical compound C1=CC=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O LQGUBLBATBMXHT-UHFFFAOYSA-N 0.000 title claims abstract description 62
- 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 50
- 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 50
- 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 50
- 235000009498 luteolin Nutrition 0.000 title claims abstract description 50
- NZPQWZZXRKZCDU-UHFFFAOYSA-N chrysophanol Natural products Cc1cc(O)c2C(=O)c3c(O)cccc3Oc2c1 NZPQWZZXRKZCDU-UHFFFAOYSA-N 0.000 title claims abstract description 31
- 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 30
- 238000000034 method Methods 0.000 title claims abstract description 27
- CZCIKBSVHDNIDH-NSHDSACASA-N N(alpha)-methyl-L-tryptophan Chemical compound C1=CC=C2C(C[C@H]([NH2+]C)C([O-])=O)=CNC2=C1 CZCIKBSVHDNIDH-NSHDSACASA-N 0.000 title claims abstract description 18
- 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 18
- 241000220436 Abrus Species 0.000 claims abstract description 26
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
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- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 5
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C46/00—Preparation of quinones
- C07C46/10—Separation; Purification; Stabilisation; Use of additives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
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Abstract
The invention discloses a method for separating and extracting chrysophanol, luteolin, abrine and protocatechuic acid from abrus herb and application thereof.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine extraction, and in particular relates to a method for separating and extracting chrysophanol, luteolin, abrine and protocatechuic acid from abrus herb and application thereof.
Background
Herba abri (Abrus cantoensis) is a dry whole plant of Abrus genus of Leguminosae family, mainly produced in Ling nan area of China, mainly distributed in Guangdong, guangxi, etc., and belongs to one of Zhuang medicine materials. The abrus herb has a long administration history 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 whole herb. In folk, because the chicken bone herb has wide medicinal effect, is cheap and easy to cultivate, people often eat the chicken bone herb in the modes of herbal tea cooking, soup cooking and the like, and the chicken bone herb is used for relieving summer heat and eliminating dampness, and has the effects of soothing liver and relieving pain, eliminating dampness and eliminating jaundice, clearing heat and detoxicating and the like. The herba abri is rich in various biological activities, such as resisting tumor, resisting oxidation, resisting bacteria and viruses, resisting inflammation and easing pain, reducing fat and protecting liver, enhancing immunity and promoting wound healing, is derived from various chemical components such as polysaccharides, flavonoids, alkaloids, triterpenes, anthraquinone compounds, trace elements and other types, is clinically used for treating diseases such as hepatitis, jaundice due to damp-heat, hypochondriac discomfort, epigastralgia, acute mastitis swelling and pain, traumatic injury and the like, and has good application potential in industries such as foods, health products, medicines, cosmetics and the like.
Although it has various biological activities, it is widely studied mainly with pharmacological actions of anti-tumor and anti-oxidation. For example: the invention patent with publication number of CN111150752A discloses application of herba abri extract in preparing anticancer drugs, wherein application of herba abri petroleum ether extract, herba abri ethyl acetate extract, herba abri n-butanol extract and/or herba abri water extract in preparing anticancer drugs (especially gastric cancer, liver cancer and breast cancer) can promote bax protein and inhibit bcl-2 expression to cause tumor cells to apoptosis is studied respectively. For another example, the invention patent with publication number of CN107334688A discloses abrus herb flavone with antioxidant activity, and an extraction method and application thereof, wherein the abrus herb flavone is extracted mainly by adopting an enzymatic catalysis and ethanol ultrasonic extraction mode, and can be used in various skin care products and has an antioxidant function.
The existing researches show that the abrus herb not only has various biological activities, but also contains rich chemical components and has rich pharmacological actions, but the current researches on the abrus herb only stay on the pharmacodynamic actions of a certain class of compounds, such as: a method for extracting an amidic acid component from herba abri leaves or herba abri leaves disclosed in the invention patent with publication number of CN106265865A, a method for extracting herba abri total saponins disclosed in the invention patent with publication number of CN104069154A, a method for crude extraction of free amino acids of herba abri disclosed in the invention patent with publication number of CN103989733A, and the like, and have not been developed deeply for pharmacodynamics research on specific monomer compounds. As is 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 active monomer compound and the medicinal effect, so the invention has been developed for further promoting the popularization and application of the abrus herb in foods, health care products, medicines and even clinic.
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 herb, soaking the whole herb of abrus herb in 8-10 times of 95% ethanol, carrying out reflux extraction for 1h at 70-80 ℃, repeating for 2-3 times, combining the extracting solutions, and filtering and removing the ethanol to obtain an extracting concentrated solution;
s2, diluting the concentrated extract with water, extracting with ethyl acetate in the ratio of 1-2:2-4 for 2-3 times, respectively combining an extraction phase and an extraction liquid, and concentrating the extraction phase and the extraction liquid under reduced pressure to obtain ethyl acetate part extractum and water part extractum;
s3, dissolving the ethyl acetate part extractum with methanol, separating by adopting 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 components with ethyl acetate, filtering, concentrating, freezing, crystallizing and drying to obtain the chrysophanol;
s5, preparing and separating the Fr.4-7 components by using a C18 column, concentrating and drying to obtain luteolin;
s6, preparing and separating the Fr.8-10 components by using a C18 column, concentrating and drying to obtain the abrine;
s7, dissolving the water part extractum, preparing and separating by using a C18 column, concentrating and drying to obtain 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 stripping: 40:1, 30:1, 20:1, 10:1.
Further, in the step S5, the C18 column preparative 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 preparative separation satisfies the following conditions:
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 preparative separation satisfies the following conditions:
mobile phase: methanol-0.1% PA, volume ratio 15:85;
wavelength: 260nm.
And the application of the method in preparing antitumor drugs.
And the application of the method in preparing DNA Topo I inhibitor drugs.
Compared with the prior art, the invention has the following advantages:
(1) The invention separates and extracts four active monomer compounds, namely chrysophanol, luteolin, abrine and protocatechuic acid, from the abrus herb for the first time, thereby realizing the application of the abrus herb in preparing anti-tumor and inhibitor medicines.
(2) According to the invention, the whole herb of the abrus herb is taken as a raw material, ethyl acetate is taken as an extractant, and a mode of combining silica gel column chromatography and C18 column preparation separation is combined for the ethyl acetate extraction part and the water part of the abrus herb, so that chrysophanol, luteolin, abrine and protocatechuic acid are extracted and separated, pharmacological research of specific chemical components in the abrus herb can be realized, and a foundation is laid for clinical application of the abrus herb.
Drawings
FIG. 1 is a diagram of Compound 1 1 H-NMR spectrum.
FIG. 2 is a diagram of Compound 1 13 C-NMR spectrum.
FIG. 3 is an infrared spectrum of compound 1.
FIG. 4 is an ultraviolet spectrum of Compound 1.
Fig. 5 is a mass spectrum of compound 1.
FIG. 6 is a diagram of Compound 2 1 H-NMR spectrum.
FIG. 7 is a diagram of Compound 2 13 C-NMR spectrum.
Fig. 8 is an infrared spectrum of compound 2.
FIG. 9 is an ultraviolet spectrum of Compound 2.
Fig. 10 is a mass spectrum of compound 2.
Fig. 11 is a mass spectrum of compound 3.
FIG. 12 is a diagram of Compound 3 1 H-NMR spectrum.
FIG. 13 is a diagram of Compound 4 1 H-NMR spectrum.
FIG. 14 is a diagram 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 luteolin 48h on MCF-7 cells (Comp-FITC-A: annexin-V-FITC-A).
FIG. 18 shows the flow-through results of luteolin effect on SGC-7901 cells for 48h (Comp-FITC-A: annexin-V-FITC-A).
FIG. 19 shows the flow results of luteolin 48h on BEL-7404 cells (Comp-FITC-A: annexin-V-FITC-A).
FIG. 20 shows the result of the inhibition of DNA Topo I by camptothecin.
FIG. 21 shows the result of the inhibition of DNA Topo I by luteolin
In fig. 17 to 19, control refers to the flow results of the blank group without drug; 0.1% dmso refers to the flow through result of 0.1% dimethyl sulfoxide; reagent refers to the streaming results of 50. Mu.L of 0.1M NaCl.
In fig. 20, the order from left to right is: m: lambda-Hind III marker, lane 1: pBR322 was added, no DNA Topo I was added, lane 2: pBR322 and DNA Topo I were added, lanes 3, 4, 5, 6, 7, 8, 9: the concentrations of the drugs added with camptothecins are respectively 200, 100, 50, 25, 10, 5 and 2.5 mu M.
In fig. 21, the order from left to right is: m: lambda-Hind III marker, lane 1: pBR322 was added, no DNA Topo I was added, lane 2: adding positive control camptothecine 200 μm; lane 3: pBR322 and DNA Topo I were added, lanes 4, 5, 6, 7, 8: the concentrations of the medicines added with luteolin are 240 mu M, 120 mu M, 60 mu M, 30 mu M and 15 mu M respectively.
Detailed Description
The present invention will be described in further detail with reference to examples, but 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 herb, which comprises the following steps:
step one, about 44Kg of dried and crushed herba abri whole grass is soaked in 10 times of 95% ethanol, and then reflux-extracted for 1 time at 75 ℃, the steps are repeated for 2 times, the extracting solutions are combined, filtered by filter cloth, and then decompressed and concentrated to obtain an extracting concentrated solution.
And secondly, diluting the concentrated extract with pure water, extracting with ethyl acetate in the extraction ratio of 1:2 for 3 times, respectively combining an extraction phase and an extraction liquid, concentrating the extraction phase under reduced pressure to obtain ethyl acetate part extractum, and preparing the water part extractum by the extraction liquid by adopting the same method.
Dissolving the ethyl acetate extract with methanol, stirring the ethyl acetate extract with 200-mesh silica gel by a dry method, loading the ethyl acetate extract on a column by a wet method, taking methylene dichloride-methanol as a washing agent, carrying out gradient washing according to the ratio of 40:1 to 30:1 to 20:1 to 10:1, collecting components, tracking and detecting the compounds by TLC, and combining components with the same Rf value to obtain the Fr.1-component, the Fr.4-7 component and the Fr.8-10 component.
Further, the Fr.1-3 component is dissolved by ethyl acetate and filtered, and then concentrated, frozen for crystallization and dried to obtain the compound 1 (500 mg); the Fr.4-7 component takes acetonitrile-0.1% formic acid water (volume ratio is 30:70) as a mobile phase, and is prepared and separated by a C18 column at the wavelength of 350nm, and then concentrated and dried to obtain the compound 2 (500 mg); 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 of 18:82) as a mobile phase, and then concentrated and dried to obtain the compound 3 (300 mg).
And step four, dissolving the water part extract, using methanol-0.1% phosphoric acid (volume ratio is 15:85) as a mobile phase, preparing and separating by using a C18 column at a wavelength of 260nm, concentrating and drying to obtain the compound 4 (300 mg).
Example 2:
the embodiment is a method for separating and extracting chrysophanol, luteolin, abrine and protocatechuic acid from abrus herb, which comprises the following steps:
step one, about 44Kg of dried and crushed herba abri whole grass is soaked in 8 times of 95% ethanol, and then reflux-extracted for 1h at 70 ℃, the steps are repeated for 2 times, the extracting solutions are combined, filtered by filter cloth, and then decompressed and concentrated to obtain an extracting concentrated solution.
And secondly, diluting the concentrated extract with pure water, extracting with ethyl acetate in the extraction ratio of 1:3 for 2 times, respectively combining an extraction phase and an extraction liquid, concentrating the extraction phase under reduced pressure to obtain ethyl acetate part extractum, and preparing the water part extractum by the extraction liquid by adopting the same method.
Dissolving the ethyl acetate extract with methanol, stirring the ethyl acetate extract with 300-mesh silica gel by a dry method, loading the ethyl acetate extract on a column by a wet method, taking methylene dichloride-methanol as a washing agent, carrying out gradient washing according to the ratio of 40:1 to 30:1 to 20:1 to 10:1, collecting components, tracking and detecting the compounds by TLC, and combining components with the same Rf value to obtain the Fr.1-component, the Fr.4-7 component and the Fr.8-10 component.
Further, the Fr.1-3 component is dissolved by ethyl acetate and filtered, and then concentrated, frozen for crystallization and dried to obtain the compound 1 (500 mg); the Fr.4-7 component takes acetonitrile-0.1% formic acid water (volume ratio is 30:70) as a mobile phase, and is prepared and separated by a C18 column at the wavelength of 350nm, and then concentrated and dried to obtain the compound 2 (500 mg); 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 of 18:82) as a mobile phase, and then concentrated and dried to obtain the compound 3 (300 mg).
And step four, dissolving the water part extract, using methanol-0.1% phosphoric acid (volume ratio is 15:85) as a mobile phase, preparing and separating by using a C18 column at a wavelength of 260nm, concentrating and drying to obtain the compound 4 (300 mg).
Example 3:
the embodiment is a method for separating and extracting chrysophanol, luteolin, abrine and protocatechuic acid from abrus herb, which comprises the following steps:
step one, about 44Kg of dried and crushed herba abri whole grass is soaked in 10 times of 95% ethanol, then reflux-extracted for 1h at 75 ℃, the steps are repeated for 3 times, the extract is combined and filtered twice by filter cloth, the filtrate is decompressed and concentrated to be close to an alcohol-free semi-flowing state extract by using a rotary evaporator at 55 ℃, the extract obtained by enrichment and rotary evaporation is continued to be distilled out of residual ethanol until no alcohol taste exists in a water bath pot, and finally dark brown viscous extraction concentrated solution is obtained.
And secondly, diluting the concentrated extract with pure water, extracting with ethyl acetate in the extraction ratio of 1:1 for 3 times, respectively combining an extraction phase and an extraction liquid, concentrating the extraction phase under reduced pressure to obtain ethyl acetate part extractum, and preparing the water part extractum by the extraction liquid by adopting the same method.
Dissolving the ethyl acetate part extractum with methanol, stirring the ethyl acetate part extractum with 300 mesh silica gel in a dry method, taking 15 times of the ethyl acetate part extractum on a column in a wet method, taking methylene dichloride-methanol as a washing agent, carrying out gradient washing according to the ratio of 40:1 to 30:1 to 20:1 to 10:1, collecting components, tracking and detecting the compounds by TLC, and combining components with the same Rf value to obtain the Fr.1-component, the Fr.4-7 component and the Fr.8-10 component.
Further, the Fr.1-3 component is dissolved and filtered by ethyl acetate, concentrated, frozen and crystallized, and then dried by blowing at 50 ℃ and dried under reduced pressure at 40 ℃ to obtain the compound 1 (500 mg); the Fr.4-7 component takes acetonitrile-0.1% formic acid water (volume ratio is 30:70) as a mobile phase, the preparation and separation of the mobile phase are carried out by a C18 column under the wavelength of 350nm, the product preparation liquid is concentrated at 50 ℃ until solid is separated out, and the solid is dried by blowing at 50 ℃ and is dried under reduced pressure at 40 ℃ to obtain the compound 2 (500 mg); 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 of 18:82) as a mobile phase, the product preparation liquid is concentrated to be dry at 45 ℃, and is dried by blowing at 40 ℃ and reduced pressure at 40 ℃ to obtain the compound 3 (300 mg).
And step four, dissolving the water extract, using methanol-0.1% phosphoric acid (volume ratio is 15:85) as a mobile phase, preparing and separating the water extract by using a C18 column at a wavelength of 260nm, concentrating a product preparation solution at 50 ℃, and performing freeze drying and reduced pressure drying at 40 ℃ to obtain the compound 4 (300 mg).
Example 4: structural identification of Compound 1
Compound 1 was isolated and extracted as in example 3, compound 1 being an orange solid with a melting point of 196.0 ℃ to 198.0 ℃. Dissolved in DMSO, and poorly soluble in methanol, acetonitrile, and water. The compound 1 has clear and single spots when the compound is inspected and fluorescent under 365nm ultraviolet light by using 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) which are respectively spread on a silica gel G thin layer plate.
From compound 1 1 H-NMR(400MHz，CDCl 3 ) Delta is shown in the spectrogram (see FIG. 1) H 12.07 (1H, s) and delta H 11.96 (1H, s) is 2 active hydrogen signals conjugated with carbonyl group, 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 5 benzene ring hydrogen signals, delta H 2.44 (3H, s) is a methyl group with a chemical shift value up to delta H 2.44, illustrating its attachment to a benzene ring.
From compound 1 13 C-NMR(100MHz，CDCl 3 ) Delta is shown in the spectrogram (see FIG. 2) C 192.4，δ C 181.8 Is 2 carbonyl carbon signals, 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 the compound 1 has 2 benzene rings, delta C 22.2 is the carbon signal of the methyl group on the benzene ring. From the combination of the hydrogen spectrum and the carbon spectrum, the compound has 2 benzene rings, 2 carbonyl groups and 1 methyl group.
Furthermore, the presence of hydroxyl groups (3448 cm) in the molecular structure was shown in the infrared spectrum (see FIG. 3) -1 ) Double bond (1676 cm) -1 ) Aromatic ring (1605 cm) -1 ) An isocharacteristic peak signal; the UV absorption peaks λmax=224, 256, 287, 430nm (see fig. 4) show a quinone-like structure with a hyperchromic group in the structure. ESI-MS m/z (see FIG. 5): 253.2[ M-H ]] ─ Molecular weight m= 254.24.
Bonding of 1 H-NMR、 13 C-NMR, IR, UV, and the like, the molecular formula of the compound 1 is deduced to be: c (C) 15 H 10 O 4 The unsaturation was calculated to be Ω=11. In summary, 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 shown in the tables 1 and 2 respectively.
TABLE 1 Nuclear magnetic resonance Hydrogen Spectrometry for Compound 1 1 H-NMR) data against reference data
Note that: actual test conditions: 1 H NMR(400MHz,CDCl 3 )；
literature test conditions: 1 H NMR(400MHz,CDCl 3 )。
TABLE 2 Nuclear magnetic resonance carbon Spectrometry for Compound 1 13 C-NMR) data and reference data
Note that: actual test conditions: 13 C-NMR(100MHz,CDCl 3 )；
literature test conditions: 13 C-NMR(100MHz,CDCl 3 )。
example 5: structural identification of Compound 2
Compound 2 was extracted and isolated as in example 3, compound 2 being a yellow solid, dissolved in DMSO, methanol, insoluble in acetonitrile, water. Toluene-ethyl acetate-formic acid-water (5:4:1:0.2), chloroform-methanol-formic acid-water (5:1:0.5:0.2), petroleum ether-ethyl acetate-formic acid-water (4:7:0.3:0.2) are used as developing agents to be respectively developed on the silica gel G thin layer plate, taken out and dried. 1% AlCl 3 And (5) ethanol solution, airing, and placing under a 365nm ultraviolet lamp to inspect fluorescence. The spots of the compound 2 are clear and single, and the origin is free of marks.
From compound 2 1 H-NMR(400MHz，DMSO-d 6 ) Delta is shown in the spectrum (see FIG. 6) 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 to 7.37 (2H, m) are 6 benzene ring hydrogen signals, delta H 12.97 (1H, s) is 1 active hydrogen signal conjugated with carbonyl, is characteristic hydroxyl hydrogen signal of flavone 5 position, which indicates that the compound 2 is a flavone compound.
From compound 2 13 C-NMR(100MHz，DMSO-d 6 ) Delta is shown in the spectrogram (see FIG. 7) 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, indicating that 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 (1655 cm) -1 ) Aromatic group (1501 cm) -1 ) And an isocharacteristic signal peak. The UV absorption peak λmax=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 of 1 H-NMR、 13 C-NMR, IR, UV data, the molecular formula of this compound 2 was deduced to be: c (C) 15 H 10 O 6 The unsaturation was calculated to be Ω=11. In summary, monomer compound 2 was identified as Luteolin (Luteolin), the chemical structure of which is shown in formula (2), and the hydrogen signal and the carbon signal of compound 2 are shown in tables 3 and 4, respectively.
TABLE 3 Nuclear magnetic resonance Hydrogen Spectrometry for Compound 2 1 H-NMR) data against reference data
Note that: actual test conditions: 1 H NMR(400MHz,DMSO-d6)；
literature test conditions: 1 H NMR(600MHz,DMSO-d6)。
TABLE 4 Nuclear magnetic resonance carbon Spectrometry for Compound 2 13 C-NMR) data and reference data
Note that: actual measurement conditions: 13 C-NMR(100MHz,DMSO-d6)；
literature test conditions: 13 C-NMR(150MHz,DMSO-d6)。
example 6: structural identification of Compound 3
Compound 3 was extracted and isolated as in example 3, compound 3 being an off-white solid, ES-TOF ms+m/z (see fig. 11): 219.3[ M+H ]] + Molecular weight m= 218.26, molecular formula: c (C) 12 H 14 N 2 O 2 The unsaturation was calculated to be Ω=7.
From compound 3 1 H-NMR structural data showed: delta exists 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 ). At the position of 1 Delta is shown in the H-NMR spectrum (see FIG. 12) H 10.87 (1H, s) is an active hydrogen signal, presumably an 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 5 benzene rings or double bond hydrogen signals, delta H 3.33 to 3.50 (3H, m, H-10, H-11) is a hydrogen signal on aliphatic hydrogen or on a azinocarbon, delta H 2.36 (3H, s) is a methyl hydrogen signal.
Bonding of 1 H-NMR and MS data, the molecular formula of the compound 3 is estimated to be: c (C) 12 H 14 N 2 O 2 In summary, monomer compound 3 was identified as Abrine (Abrine) and its chemical structure is shown in formula (3).
Example 7: structural identification of Compound 4
Compound 4 was a white powder, ES-TOF ms+m/z of compound 4 (see fig. 15 and 16): [ M+Na ]] + ：m/z 177.01；[M+K] + : m/z 193; double peak [2M+Na] + : m/z 331.04; triple peak [3M+Na ]]+: m/z 485.36, minThe molecular weight m= 154.12.
From compound 4 1 H-NMR(400MHz，DMSO-d 6 ) Delta is shown in the spectrum (see FIG. 13) H 7.35(1H,d,J ＝2.0Hz)，δ H 7.30(1H,dd,J＝8.2,2.1Hz)，δ H 6.79 (1 h, d, j=8.2 Hz) are 3 benzene ring hydrogen signals, and from the peak type and coupling constant of hydrogen, it can be known that these 3 hydrogens are ABX coupling systems.
From compound 4 13 C-NMR(100M Hz，DMSO-d 6 ) The spectrogram (see FIG. 14) is shown in 13 Delta is shown in C-NMR C 169.2 Is a carboxylic acid or ester carbonyl carbon signal, delta 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 one benzene ring and one carboxyl group.
Bonding of 1 H-NMR、 13 C-NMR and MS data, the molecular formula of the compound 4 was estimated to be: c (C) 7 H 6 O 4 The unsaturation was calculated to be Ω=5. In summary, monomer compound 4 was identified as protocatechuic acid (Protocatechuic acid) having the chemical structure shown in the following formula (4), and the hydrogen signal and the carbon signal of compound 4 are shown in tables 5 and 6, respectively.
TABLE 5 Nuclear magnetic resonance Hydrogen Spectrometry for Compound 4 1 H-NMR) data against reference data
Note that: actual test conditions: 1 H NMR(400MHz,DMSO-d6)；
literature test conditions: 1 H NMR(300MHz,DMSO-d6)。
TABLE 6 Nuclear magnetic resonance carbon Spectrometry for Compound 4 13 C-NMR) data and reference data
Note that: actual test conditions: 13 C-NMR(100MHz,DMSO-d6)；
literature test conditions: 13 C-NMR(75MHz,DMSO-d6)。
example 8: antitumor Activity assay
1. Inhibition of proliferation of tumor cells 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, culture of 3 cancer cells: MCF-7, BEL-7404 cell lines: dmem+10% fbs+1% p/S; SGC-7901 cell line: RPMI-1640+10% FBS+1% P/S,3 cancer cells were placed in 5% CO 2 Culturing in a saturated humidity incubator at 37 ℃. Taking 3 cell strains which are normally cultured and are in logarithmic phase, rinsing 2-3 times by PBS buffer solution, stopping digestion by using a complete culture medium after 0.8-1 mL of 0.25% trypsin is digested, sucking cell suspension, transferring to a centrifuge tube, centrifuging at 1000rpm for 3min by using the centrifuge, discarding supernatant, collecting cells, counting cells, and respectively regulating the cell concentration to 3X 10 3 cell/well, inoculating to 96-well plate, and placing 90 μl of each well into incubator for static culture for 24 hr.
(2) Dissolution of the drug in dilution
Chrysophanol (molecular weight: 254.24), mother liquor: DMSO 5mg/mL (19.67 mM). 1mM:1.867mL of cell complete medium+10. Mu.L of mother liquor;
abrine (molecular weight: 218.25), mother liquor: after dissolution with 0.1M NaOH, it was neutralized again with 1M HCl to ph=7, 8.73mg/mL (40 mM);
(3) Pharmaceutical intervention
The experiment is set into a zeroing group (only adding culture medium), a 0-adding group is a Control group (containing cell fluid and culture fluid without medicine), a DMSO group (containing cell fluid, culture fluid and medicine solvent DMSO without medicine),experiment group (containing cell sap, culture solution and medicine) comprises chrysophanol and luteolin, and after cell wall-attached culture for 24 hr, the culture medium is replaced with DMEM/RPMI-1640 complete culture medium containing different concentration medicines and placed in 5% CO 2 Culturing in a saturated humidity incubator at 37 ℃. The time for the medicine to intervene in 3 tumor cells is 24h, 48h and 72h respectively, wherein the concentration of the medicine chrysophanol for intervening in 3 tumor cells in 3 time periods is as follows: 5. 10, 50, 100, 200, 300 μm; the concentration of luteolin which intervenes in 3 tumor cells in 24h, 48h and 72h is 5, 10, 20, 40, 60, 80 and 100 mu M. 100. Mu.L of medium containing different concentrations of drug was added to each well, with 6 multiplex wells per concentration.
(4) MTT colorimetric detection method
After 3 different time points of drug intervention, 100 μl of MTT solution was added to each well before termination of the experiment, and the wells were further incubated in an incubator at 37 ℃ in the dark for 4 hours. And (3) removing the culture solution after incubation, adding 150 mu L of DMSO into each well to terminate the reaction, and then putting into a shaking table to shake uniformly so as to fully dissolve the crystals, wherein the dissolving time is determined according to the size and dissolving condition of the crystals. The absorbance (OD) of each well at 490nm wavelength was measured using a multifunctional microplate reader, the experimental results were calculated according to the following formula A, and the measurement data were calculated as mean.+ -. Standard deviationExpression, and calculation of IC for drug intervention in 3 cell lines at 3 time points 50 The values, P <0.05, represent the differences as statistically significant.
Formula a: cell viability (%) = (experimental group-zeroing group)/(0 dosing group-zeroing group) ×100%.
(5) Experimental results
The results of the influence of the different concentrations of chrysophanol on the activities of MCF-7, SGC-7901 and BEL-7404 for 24, 48 and 72 hours are shown in Table 7, and experimental data show that chrysophanol has an inhibitory effect on the proliferation of 3 tumor cells in vitro at 3 time points, and the survival rate of the tumor cells increases with the decrease of the concentration of the drug; the inhibition increases with increasing drug concentration, and is concentration dependent. By calculating the chrysophanol to 3 kindsIC at 3 time points of tumor cell intervention 50 Value, found that chrysophanol intervenes in 3 tumor cells for 72 hours of IC 50 The values were 108.5. Mu.M, 39.21. Mu.M, 46.30. Mu.M, respectively. Comparison of IC of chrysophanol effect on 3 cells for 72h 50 The values, the sensitivity of chrysophanol to 3 cells was found to be: SGC-7901 > BEL-7404 > MCF-7. As can be seen, chrysophanol is more sensitive to SGC-7901 cells.
Table 7 effect of chrysophanol on proliferation of 3 cells (survival rate,n＝6)/>
note that: ratio to Control (blank) group, P <0.01, P <0.05.
The results of the effects of the luteolin with different concentrations on the activities of MCF-7, SGC-7901 and BEL-7404 cells for 24, 48 and 72 hours are shown in Table 8, and experimental data show that the luteolin has an inhibition effect on the proliferation of 3 tumor cells in vitro, and the survival rate of the tumor cells increases with the decrease of the drug concentration; the inhibition increases with increasing drug concentration. By calculating the IC of luteolin at 3 time points of intervention with 3 tumor cells 50 Value, found that chrysophanol on 3 tumor cells in 72 hours of intervention IC 50 The values are respectively: 29.47. Mu.M, 32.66. Mu.M, 36.29. Mu.M. Comparison of luteolin to 3 cells for 72h of IC 50 Values, luteolin was found to be sensitive to 3 cells to the following extent: MCF-7 > SGC-7901 > BEL-7404. Thus, luteolin is more sensitive to MCF-7 cells.
Table 8 effect of luteolin on proliferation of 3 cells (survival,n＝6)/>
note that: ratio to Control (blank) group, P <0.01, P <0.05.
The experimental data show that the chrysophanol and the luteolin have inhibition effects on the in vitro proliferation of 3 tumor cells, have obvious anti-tumor activity, the cell survival rate is reduced along with the increase of the drug concentration, and the inhibition effects are dependent on the drug concentration. Wherein, the chrysophanol is sensitive to human hepatoma cell BEL-7404; luteolin is more sensitive to human breast cancer cells MCF-7. IC of 2 monomer compounds to 3 time points of 3 cell lines 50 The values are shown in Table 9.
Table 95 IC of monomer compounds to 3 time points of 3 cell lines 50 Value (Unit: mu M)
2. Pro-apoptotic effects of monomeric compounds on tumor cells
(1) Culture of tumor cells
The culture conditions of the human breast cancer MCF-7, human gastric cancer SGC-7901 and human liver cancer BEL-7404 cell lines are respectively as follows: MCF-7, BEL-7404 cell lines: dmem+10% fbs+1% p/S; SGC-7901 cell line: RPMI-1640+10% FBS+1% P/S, placed in 5% CO 2 Culturing in a saturated humidity incubator at 37 ℃. See above for details the culture of tumor cells.
(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, and using 0.8-1 mL of 0.25% trypsinDigesting, centrifuging at 800rpm for 3min to collect cells, counting in a counter, spreading 6-well plates for each cell, and adding 3.0-4.0X10 s into each well 5 Individual cells (corresponding cell plating concentrations can be adjusted according to different cell strains) are respectively put into an incubator to stand for 24 hours [12] . And selecting a proper drug concentration according to the prior MTT result to carry out an apoptosis detection experiment, wherein the concentration of the drug group is as follows: the concentration of the drug chrysophanol intervening 3 cells was 19.67 μm; the concentrations of the 3 cells interfered by the luteolin medicines are respectively 20, 40 and 61 mu M; the concentration of the drug abrine to interfere with 3 cells was: 1mM; the concentrations of protocatechuic acid interfering 3 cells were: 194.65. Mu.M. The next day, the cells with good growth state and fusion degree reaching 70-80% are added with 2mL of the prepared culture medium containing luteolin medicine and placed into an incubator for culturing for 48h.
(4) Flow cytometry detection of apoptosis (Annexin V/PI double-staining detection of apoptosis)
After 48h of drug treatment, the cell state was observed, flow analysis was started, the supernatant of the cells in the 6-well plate was first pipetted into a 5mL EP tube (because there were some cells that had floated and had apoptotic in the cell supernatant), washed twice with PBS pre-chilled on ice, then treated with EDTA-free pancreatin for digestion for 3-5min (different cell digestion times, stop digestion according to the cell state observed in the microscope), the cells were collected after termination of digestion into the previous 5mL EP tube, centrifuged at 800rpm for 3min to remove the supernatant and the washed cells were blown with pre-chilled PBS and transferred into a 1.5mL EP tube, centrifuged at 1000rpm to remove the supernatant, and then the steps of washing the cells were repeated once again.
Previously using sterile dd H 2 O10 Xbinding buffer was diluted to 1 Xbinding buffer and left at room temperature, 100. Mu.L of 1 Xbinding buffer was taken to resuspend cells, then 5. Mu.L of FITC-Annexin V and 5. Mu.L of PI were added to each tube of cells under the condition of keeping out of light, after light flick mixing, the mixture was left at room temperature for 15min at light, then 400. Mu.L of 1 Xbinding buffer was added, after blowing uniformly, the agglomerated cells were filtered with 400 mesh filter cloth (anti-clogging flow cytometer). Placing the treated cells into ice box, preserving in dark for 1 hrAnd (5) finishing the machine analysis. Data were analyzed using FlowJo software and plotted against control cell analysis results.
(5) Experimental results
Annexin V-FITC/PI flow cytometry was used to detect apoptosis.
The apoptosis rate was counted as the sum of early and late apoptosis, and the detailed results of the pro-apoptotic effect of the monomeric compound luteolin on three tumor cells MCF-7, SGC-7901 and BEL-7404 are shown in FIG. 17, FIG. 18 and FIG. 19. The apoptosis rate of luteolin on human breast cancer cells MCF-7 after 48h intervention is 15.71%, and the apoptosis rate of a blank control group is 2.49%. The apoptosis rate of luteolin for 48h of human gastric cancer cell SGC-7901 intervention is 17.00%, and the apoptosis rate of blank control group is 1.07%. The apoptosis rate of luteolin for 48h of human liver cancer cell BEL-7404 intervention is 30.54%, and the apoptosis rate of blank control group is 2.1%.
From this, it is evident that luteolin has a better pro-apoptotic effect on BEL-7404 cells with an apoptosis rate of 30.54%. Experimental results show that luteolin has the effect of inducing apoptosis of 3 tumor cells.
Example 9: DNA Topo I inhibition assay
(1) Preparation of mother liquor of medicine and dilution of medicine
Camptothecins (molecular weight: 348.34), mother liquor: DMSO 3.4834mg/mL (10 mM). Diluting with DMSO, adding 10 μL 10mM camptothecine mother liquor into 15 μL DMSO to obtain 4mM camptothecine solution, and diluting according to the ratio to obtain a series of solutions with concentration of camptothecine positive control medicine 2.5, 5, 10, 25, 50, 100, 200 μM, etc.
Luteolin (molecular weight: 286.24), mother liquor: DMSO 57mg/mL (199.13 mM). Diluting with DMSO, adding 4.8 μl 199.13mM luteolin mother liquor into 194.33 μl DMSO to obtain 4.8mM luteolin solution, and diluting according to the ratio to obtain 15, 30, 60, 120, 240 μM solution.
(2) Preparation of electrophoresis-related reagents
The reaction system (50 xTAE solution formulation) was Tris-Base 242g; na (Na) 2 EDTA·2H 2 O 37.2g；Glacial acetic acid 57.1, mL; dd H 2 O up to 1L (dd H in use) 2 O was diluted 1×).
Preparation of agarose gel: adding a proper amount of buffer solution (the dosage required by a balance weighing experiment) and a proper concentration into the conical flask, heating and boiling the conical flask, shaking the conical flask until the conical flask is completely melted, and obtaining the conical flask when the liquid has no bubbles.
And (3) preparation of a rubber plate: cleaning the used devices and molds, airing for standby, putting a comb into a gel making groove, fixing the comb into 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 pulling the comb vertically and upwards, slowly putting the gel and the gel making groove into an electrophoresis groove, and adding electrophoresis buffer liquid into the electrophoresis groove until the buffer liquid is over a gel plate.
(3) Preparation of enzyme reaction Mixture
Reaction system 1: pBR322 DNA 0.5. Mu.g/1. Mu.L; 10X DNA Topo I Buffer. Mu.L; 2. Mu.L of 0.1% BSA; drug/DMSO 1 μl; DNA Topo I1. Mu.L (U); dd H 2 O up to 20. Mu.L. If 0.1% BSA was added directly to 10X DNA Topo I Buffer, a significant amount of white precipitate would be produced. Therefore, the reagents are added in the following order in the preparation of the reaction solution: dd H 2 O.fwdarw.10X DNA Topo I Buffer.fwdarw.0.1% BSA. Fwdarw.substrate DNA. Fwdarw.drug/DMSO. Fwdarw.DNA Topo I.
Reaction system 2: lambda-HindIII digest 1. Mu.L; 6×loading Buffer 1 μl; TE Buffer up to 6. Mu.L. The original ends of the lambda DNA digest DNA Markers are often combined together by COS ends, and heat treatment (60 ℃ for 5 min) is carried out before electrophoresis, so that an electrophoresis image of a Marker becomes clearer, and the fragment degradation can be prevented by using TE buffer dilution before the heat treatment. Therefore, the reagents are added in the following order in the preparation of the reaction solution: TE Buffer → lambda-HindIII digest → heat treatment (60 ℃ C., 5 min) → 6×loading Buffer.
(4) Inhibition of DNA Topo I by drugs
According to the reaction system, luteolin is added, finally 1 mu L of DNA Topo I is added, the mixture is placed in a water bath at 37 ℃ for reaction for 30min after uniform mixing, then 10 x loading buffer (2.22 mu L) containing SDS is rapidly added, the reaction is stopped after uniform mixing, lambda-HindIII digest is used as a marker,1% agarose gel (1 x TAE buffer configuration) is used for electrophoresis for 90min at 85V, ethidium Bromide (EB) is dyed for 30min, pure water is decolorized for 30min, and a gel imager is used for imaging analysis.
The plasmid DNA and ethidium bromide react in the process of gel running to cause the plasmid DNA to supercoil again, so EB can not be added in the process of agarose gel configuration, and dyeing can only be carried out by a mode of subsequent gel soaking.
(5) Experimental results
The effectiveness of the DNA Topo I activity screening system was verified with the positive control drug camptothecin, and the experimental results are shown in FIG. 20. In the figure, lane 2 shows a DNA Topo I control (pBR 322 DNA and DNA Topo I are added) which can effectively unwind supercoiled DNA and can effectively inhibit the activity of DNA Topo I after camptothecin is added, resulting in an increase in supercoiled DNA. It is evident from the figure that the unwinding effect of camptothecins on DNA Topo I resulted in an inhibition from 200 μm to 25 μm concentration (i.e. lanes 3, 4, 5, 6) compared to the Topo I control, which inhibition was proportional to the concentration, i.e. inhibition was increased with increasing concentration of drug intervention.
The result of the experiment of the inhibition of luteolin on DNA Topo I is shown in FIG. 21, wherein lane 3 is a DNA Topo I control (added with pBR322 DNA and DNA Topo I), and it is obvious from the figure that compared with the Topo I control group and camptothecine (200 mu M) in lane 2, the inhibition of luteolin on DNA Topo I is generated from 240 mu M to 60 mu M of drug intervention concentration (namely lanes 4, 5 and 6), the inhibition is proportional to the concentration, namely, the inhibition is enhanced with the increase of the concentration of the drug intervention; the inhibition of DNA Topo I by luteolin at an intervention concentration of 240. Mu.M (lane 4) was slightly weaker than that of camptothecine at an intervention concentration of 200. Mu.M (lane 2); but the inhibition of DNA Topo I by camptothecins was closer to the positive control 100 μm (fig. 20, lane 4). It is shown that luteolin has a certain inhibitory effect on the activity of DNA Topo I, but the inhibitory 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 is weaker than that of camptothecins.
The foregoing 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 modification, equivalent variation, etc. of the above embodiment according to the technical matter of the present invention fall within the scope of the present invention.
Claims (1)
1. A method for separating and extracting chrysophanol, luteolin, abrine and protocatechuic acid from abrus herb is characterized in that: the method comprises the following steps:
s1, drying and crushing whole herb of abrus herb, soaking the whole herb of abrus herb in 8-10 times of 95% ethanol, carrying out reflux extraction for 1h at 70-80 ℃, repeating for 2-3 times, combining the extracting solutions, and filtering and removing the ethanol to obtain an extracting concentrated solution;
s2, diluting the concentrated extract with water, extracting with ethyl acetate in the ratio of 1-2:2-4 for 2-3 times, respectively combining an extraction phase and an extraction liquid, and concentrating the extraction phase and the extraction liquid under reduced pressure to obtain ethyl acetate part extractum and water part extractum;
s3, dissolving the ethyl acetate part extractum with methanol, separating by adopting a silica gel column chromatography to obtain a Fr.1-3 component, a Fr.4-7 component and a Fr.8-10 component,
the silica gel column chromatography meets the following conditions:
sample mixing silica gel: 200-300 meshes of the powder with the diameter of 200-300 meshes,
removing the lotion: dichloromethane-methanol is used as a solvent,
gradient stripping: 40:1, 30:1, 20:1, 10:1;
s4, dissolving the Fr.1-3 components with ethyl acetate, filtering, concentrating, freezing, crystallizing and drying to obtain the chrysophanol;
s5, preparing and separating the Fr.4-7 components by using a C18 column, concentrating and drying to obtain luteolin,
the C18 column preparative separation meets the following conditions:
mobile phase: acetonitrile-0.1% formic acid water with the volume ratio of 30:70,
wavelength: 350 nm;
s6, preparing and separating the Fr.8-10 components by using a C18 column, concentrating and drying to obtain the abrine,
the C18 column preparative separation meets the following conditions:
mobile phase: 0.4 percent of triethylamine-0.2 percent of phosphoric acid, the volume ratio is 18:82,
wavelength: 280 nm;
s7, dissolving the water extract, preparing and separating by using a C18 column, concentrating and drying to obtain protocatechuic acid,
the C18 column preparative separation meets the following conditions:
mobile phase: methanol-0.1% phosphoric acid, the volume ratio is 15:85,
wavelength: 260nm.
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