CN115093426A - Method for extracting and separating benzophenanthridine enantiomer alkaloids from angelica Hovenia and application of benzophenanthridine enantiomer alkaloids - Google Patents
Method for extracting and separating benzophenanthridine enantiomer alkaloids from angelica Hovenia and application of benzophenanthridine enantiomer alkaloids Download PDFInfo
- Publication number
- CN115093426A CN115093426A CN202210899681.4A CN202210899681A CN115093426A CN 115093426 A CN115093426 A CN 115093426A CN 202210899681 A CN202210899681 A CN 202210899681A CN 115093426 A CN115093426 A CN 115093426A
- Authority
- CN
- China
- Prior art keywords
- benzophenanthridine
- enantiomer
- separating
- extracting
- alkaloids
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/04—Ortho-condensed systems
- C07D491/056—Ortho-condensed systems with two or more oxygen atoms as ring hetero atoms in the oxygen-containing ring
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
- C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
- C07J73/005—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by nitrogen as hetero atom
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
Abstract
The invention discloses a method for extracting and separating benzophenanthridine enantiomer alkaloid from hovenia dulcis thunb and application thereof, the method comprises the steps of carrying out reflux extraction, concentration, separation and purification on the hovenaia dulcis seven raw material to obtain 6R-carboxymethyl-dihydrochelerythrine and 6S-carboxymethyl-dihydrochelerythrine, the method successfully extracts and separates a pair of benzophenanthridine enantiomer alkaloids from the honey raisin tree root, has simple operation and high extraction rate and purity, realizes the separation of a class of compounds with large structural similarity and difficult separation, particularly the resolution of racemes, and further enriches the knowledge of the basis of isoquinoline substances in the honey raisin tree root, and the 6S-carboxymethyl-dihydrochelythrine extracted and separated by the method has a certain effect of inhibiting the proliferation of MCF-7 breast cancer cells in vitro, thereby providing experimental basis for the subsequent development of novel anti-breast cancer drugs or lead compounds.
Description
Technical Field
The invention belongs to the technical field of natural product extraction and separation, relates to a method for separating alkaloid from hovenia dulcis thunb, and particularly relates to a method for separating benzophenanthridine enantiomer alkaloid from hovenia dulcis thunb and application of the method.
Background
Hovenia acerba heptagenic plant Hylomecon japonica (Thunb.) Prantl is a plant of the genus Neurospora (Hylomecon japonica) of the Papaveraceae family (Papaveraceae). Hovenia acerba is the root of Hylomecon japonica (Thunb.) Prantl, has the effects of relaxing muscles and tendons, activating collaterals, dissipating blood stasis, relieving swelling, dispelling wind, removing dampness, relieving pain, stopping bleeding and the like, and is clinically used for treating diseases such as rheumatic arthritis, traumatic injury, limb weakness, dysentery, epigastric pain and the like. Modern researches show that the medicinal plant is rich in various isoquinoline alkaloids, and most of the isoquinoline alkaloids have remarkable pharmacological activities of resisting tumor, inhibiting bacteria, resisting inflammation, relieving pain and the like.
However, because the chemical components of the hovenia dulcis thunb seven are complex, the structural similarity of the active components is high, the separation of the current technology is difficult, the reports of separating and identifying the alkaloid content in the hovenia dulcis thunb seven are less, and particularly, the related reports of separating high-purity enantiomer alkaloid are not found.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for extracting and separating benzophenanthridine enantiomer alkaloids from angelica hovenia dulcis and application thereof, which can extract and separate a pair of benzophenanthridine enantiomer alkaloids and realize separation of compounds with large structural similarity and difficult separation, particularly resolution of racemates.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a method for extracting and separating benzophenanthridine enantiomer alkaloid from Hovenia dulcis Thunb, which is characterized by comprising the following steps:
1) taking crushed seven medicinal materials of hovenia dulcis thunb, performing reflux extraction by adopting an 80% ethanol-water system to obtain a total extract, and adding water to perform suspension treatment to obtain a suspension;
2) adjusting the pH value of the suspension to 2, extracting for several times by adopting dichloromethane, combining the extraction solutions, and evaporating to dryness to obtain a dichloromethane part with the pH value of 2 and an acid extraction solution part;
3) loading a dichloromethane part with the pH value of 2 by adopting a dry method, separating by using a normal pressure chromatographic column, performing gradient elution by using a petroleum ether-ethyl acetate system as an eluent, detecting by adopting TLC (thin-layer chromatography), merging and collecting same fractions to obtain a fraction A containing a target component, and concentrating and drying to obtain an extract A;
4) separating the extract A obtained in the step 3) by adopting a normal phase silica gel chromatographic column, performing gradient elution by taking a dichloromethane-methanol system as an eluent, detecting by TLC, combining and collecting the same fractions to obtain a fraction B containing a target component, and concentrating and drying to obtain an extract B;
5) performing gradient elution and TLC detection on the extract B obtained in the step 4) by using a petroleum ether-ethyl acetate system, combining and collecting the same fractions to obtain a fraction C containing a target component, and concentrating and drying to obtain an extract C;
6) separating the extract C obtained in the step 5) by using semi-preparative HPLC to obtain racemic benzophenanthridine alkaloid;
7) subjecting the racemic benzophenanthridine alkaloids obtained in step 6) to chiral chromatographic resolution by HPLC to obtain a pair of enantiomers, namely 6R-carboxymethyl-dihydrochelerythrine and 6S-carboxymethyl-dihydrochelerythrine.
Preferably, in step 1), the reflux extraction is performed 3 times, each for 3 hours.
Further preferably, the suspension treatment is carried out by adding water, and the added water is warm water.
Preferably, in step 2), the pH of the suspension is adjusted with a 10% hydrochloric acid solution.
Preferably, in the step 3), the dry-method sample loading is to mix the dichloromethane part with the pH value of 2 with 100-200 mesh column chromatography silica gel and load the sample; when the separation was performed by an atmospheric pressure column, petroleum ether and ethyl acetate were subjected to gradient elution in a volume ratio of (12:1) to (0:1) (PE/EtOAc: 12:1 to PE/EtOAc: 0:1, V/V).
Further preferably, TLC detection is to sample the obtained components on a thin layer silica gel plate and develop the components with a developing agent, observe the components under an ultraviolet lamp with 254nm, combine the components and collect the same fractions in combination with the color development of the potassium bismuth iodide test solution.
Preferably, in step 4), the chromatographic column is subjected to gradient elution (CH) of dichloromethane and methanol at a volume ratio of (20:1) to (0:1) 2 Cl 2 /MeOH＝20:1～CH 2 Cl 2 /MeOH＝0:1,V/V)。
Further preferably, TLC detection is to sample the obtained components on a thin layer silica gel plate and develop the components with a developing agent, observe the components under an ultraviolet lamp with 254nm, combine the components and collect the same fractions in combination with the color development of the potassium bismuth iodide test solution.
Preferably, in the step 5), the Petroleum ether and the Ethyl acetate are subjected to gradient elution according to the volume ratio of (8:1) to (0:1) during the chromatographic column separation (Petroleum ether/Ethyl acetate is 8:1 to Petroleum ether/Ethyl acetate is 0:1, and V/V).
Further preferably, TLC detection is to sample the obtained components on a thin layer silica gel plate and develop the components with a developing agent, observe the components under a 254nm ultraviolet lamp, combine and collect the same fractions in combination with the color development of the potassium bismuth iodide reagent,
preferably, in step 6), the chromatographic conditions for the semi-preparative HPLC separation are:
chromatograph: shimadzu LC-2030C 3D high performance liquid chromatograph;
a chromatographic column: welch Xtimate C18, 10X 250mm,5 μm;
eluent: the mobile phase adopts 50% of methanol-water and 0.5% of ammonia water, and the flow rate is as follows: 2mL/min, isocratic elution.
Preferably, in the step 7), the chromatographic conditions for chiral chromatographic resolution by HPLC are as follows:
a chromatograph: shimadzu LC-2030C 3D high performance liquid chromatograph;
a chromatographic column:
IC，4.6×250mm，5μm；
eluent: n-hexane: isopropanol-80: 20, flow rate: 0.8mL/min, isocratic elution.
The invention also discloses benzophenanthridine alkaloid separated by the method for extracting and separating benzophenanthridine enantiomer alkaloid from hovenaia dulcis, which is S-shaped in configuration and named as 6S-carboxymethyl-dihydrochelerythrine, and has the following structural formula:
the invention also discloses application of the benzophenanthridine alkaloid in preparation of anti-breast cancer drugs
Compared with the prior art, the invention has the following beneficial effects:
the invention firstly uses the honey raisin tree seven as a raw material to obtain a pair of benzophenanthridine enantiomer alkaloids by extraction, separation and purification: the 6R-carboxymethyl-dihydrochelerythrine and the 6S-carboxymethyl-dihydrochelerythrine realize the separation of a class of compounds with large structural similarity and difficult separation, particularly the resolution of racemate, further enrich the knowledge of the substance basis of the isoquinoline alkaloid in the seven elements of the hovenia acerba, and provide experimental basis for the subsequent development of novel anti-breast cancer drugs or lead compounds.
The isoquinoline alkaloid 6S-hydroxydihydrochelerythrine obtained by separation has certain cytotoxic activity on breast cancer MCF-7 cells, can further research the action mechanism of the alkaloid for inhibiting the proliferation of the breast cancer cells and the toxic and side effects on normal cells, and provides experimental basis for developing novel anti-breast cancer drugs or lead compounds.
Drawings
FIG. 1 shows HPLC chromatogram of 6R-carboxymethyl-dihydrochelerythrine and 6S-carboxymethyl-dihydrochelerythrine.
FIG. 2 is an HRESIMS spectrum of isolated 6-carboxymethyl-dihydrochelerythrine.
FIG. 3 shows a UV spectrum of the isolated 6-carboxymethyl-dihydrochelerythrine.
FIG. 4 shows an IR spectrum of the isolated 6-carboxymethyl-dihydrochelerythrine.
FIG. 5 shows the isolation of 6-carboxymethyl-dihydrochelerythrine 1 H-NMR (600MHz) spectrum.
FIG. 6 shows isolation of 6-carboxymethyl-dihydrochelerythrine 13 C-NMR (150MHz) spectrum.
FIG. 7 shows the measured ECD and calculated ECD maps of the isolated compounds 6R-carboxymethyl-dihydrochelerythrine (11a) and 6S-carboxymethyl-dihydrochelerythrine (11 b).
FIG. 8 is a process flow diagram of the present invention.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the accompanying drawings:
extraction and separation of benzophenanthridine enantiomer alkaloid from Hovenia acerba
Referring to fig. 8, a process flow chart of the process for extracting and separating a pair of benzophenanthridine enantiomer alkaloids from hovenia dulcis is shown, and the specific method is as follows:
extraction: pulverizing 100kg radix Hoveniae, soaking at 50 deg.C for 8 hr, reflux-extracting with 80% ethanol-water at 80% ethanol-water boiling temperature for 3 times (each time for 3 hr) to obtain total extract about 80L, and suspending with warm water. Adjusting the pH of the suspension to 2 with 10% hydrochloric acid solution, extracting with dichloromethane, mixing the extracts, and evaporating to dryness to obtain dichloromethane part (1272g) with pH of 2 and acidic extractive solution part; the acidic extract was adjusted to pH 9 with 10% NaOH solution, extracted with dichloromethane, and the combined extracts were evaporated to dryness to give a dichloromethane fraction (404.5g) with pH 9.
Separation: the dichloromethane fraction (1272g) with pH 2 was stirred with 100-200 mesh column chromatography silica gel and separated with normal phase normal pressure chromatography column. Gradient elution is carried out by taking petroleum ether-ethyl acetate system as eluent (PE/EtOAc: 12:1, 10:1, 8:1, 5:1, 3:1, 1:1, 0:1), each 1000mL is taken as one fraction, the obtained fraction is subjected to reduced pressure concentration, then the point sample is applied on a thin-layer silica gel plate and is developed by using a proper developing agent, the observation is carried out under an ultraviolet lamp of 254nm, the color development condition of a bismuth potassium iodide test solution is combined, and the same flow parts are combined to obtain A, B, C, D large components containing alkaloid.
Component D (604.7g) was subjected to gradient elution (CH) using normal phase silica gel column and dichloromethane-methanol as eluent 2 Cl 2 Each 500mL of the fractions was concentrated under reduced pressure, spotted on a thin layer silica gel plate and developed with a suitable developing agent, and the same fractions were combined together under a 254nm ultraviolet lamp in accordance with the color development of the potassium bismuth iodide reagent to give five alkaloid-containing components D1-D5.
D1(206.7g) was subjected to gradient elution through a normal phase silica gel column using a petroleum ether-ethyl acetate system as an eluent (PE/EtOAc: 8:1, 5:1, 3:1, 1:1, 0:1) as one fraction per 500mL, the obtained fraction was concentrated under reduced pressure, spotted on a thin layer silica gel plate and developed with a suitable developing agent, observed under an ultraviolet lamp of 254nm, and the same fractions were combined in accordance with the color development of a bismuth potassium iodide test solution to obtain 9 fractions containing alkaloids.
D1-4(1.7g) was subjected to gradient elution through a normal phase silica gel column using a petroleum ether-ethyl acetate system as an eluent (PE/EtOAc: 10:1, 8:1, 5:1, 3:1, 1:1, 0:1) as one fraction per 250mL, the obtained fractions were concentrated under reduced pressure, spotted on a thin layer silica gel plate and developed with an appropriate developing agent, observed under an ultraviolet lamp of 254nm, and the same fractions were combined in accordance with the color development of a bismuth potassium iodide test solution to obtain 6 fractions containing alkaloids.
D1-4-6(667.1mg) is subjected to gradient elution by a normal phase silica gel chromatographic column and by using a petroleum ether-ethyl acetate system as an eluent (PE/EtOAc is 10:1, 3:1, 1:1, 0:1), each 100mL is taken as a fraction, the obtained fraction is subjected to reduced pressure concentration, then the sample is applied to a thin layer silica gel plate and is spread by a proper developing agent, the observation is carried out under a 254nm ultraviolet lamp, the same fractions are combined according to the color development condition of a bismuth potassium iodide test solution, a component containing a target compound after impurity removal is obtained, then a Shimadzu LC-2030C 3D high performance liquid chromatograph is used, a Welch Xitimate C18(10 x 250mm,5 mu m) chromatographic column is adopted, 50% methanol-water and 0.2% ammonia water are taken as a mobile phase, the flow rate is 2mL/min isocratic, the detection wavelength is 210nm, and the 6-carboxymethyl-dihydrorythronine racemate is obtained.
And (3) identification:
by NMR 1 H-NMR and 13 C-NMR), HRESIMS, UV, IR, ECD and optical rotation measurement, the structure of the resolved pair of enantiomeric compounds is identified, and the spectroscopic data is shown in FIGS. 2-7.
The nuclear magnetic data were analyzed as follows:
1 H-NMR(Pyridin-d5,600MHz)：δ H 2.76(2H,d,J＝7.6Hz,H-13)，2.79(3H, s,-NCH 3 )，3.80(3H,s,7-OCH 3 )，4.01(3H,s,8-OCH 3 )，5.61(1H,t,H-6)，5.95, 6.00(2H,-OCH 2 O-)，7.10(1H,d,J＝8.6Hz,H-9)，7.28(1H,s,H-1)，7.67(1H,d, J＝8.6Hz,H-12)，7.79(1H,d,J＝8.6Hz,H-10)，7.97(1H,d,J＝8.6Hz,H-11)，8.01 (1H,s,H-4)。
13 C-NMR(Pyridin-d5,150MHz)：δ C 40.45(C-13)，43.05(-NCH 3 )，55.68(8- OCH 3 )，55.95(C-6)，60.79(7-OCH 3 )，101.36(C-4)，101.54(-OCH 2 o-), 104.64 (C-1), 112.29(C-9), 119.28(C-10), 120.34(C-11), 123.07(C-10b), 124.26 (C-12), 125.12(C-10a), 128.06(C-4a), 128.95(C-6a), 131.64(C-12a), 140.18 (C-4 b), 146.22(C-7), 148.08(C-2), 148.55(C-3), 152.73(C-8), 174.11 (C-14). The planar structure of the compound is identified as 6-carboxymethyl anhydrochelerythrine.
And because the compound has a chiral center at the C-6 position, the optical rotation value is
Presumably, the compound is a racemate. By using
IC chiral column analysis by HPLC (fig. 1), in n-hexane: under chromatographic conditions of 80:20 isopropanol, the compound can be resolved into a pair of enantiomers 11a (t) R 17.278min) and 11b (t) R 24.778 min). Thus, the compound is subjected to chiral resolution to obtain the compound
And
to determine the absolute configurations of compounds 11a and 11b, ECD calculations were performed for the two possible absolute configurations of compound 11, S-type and R-type, respectively, and the absolute configurations of 11a (6R) and 11b (6S) were obtained by comparing the calculated ECD with experimental data (fig. 7).
11a structural formula as follows:
11b structural formula as follows:
secondly, research on the activity of the breast cancer cells in vitro:
the invention adopts MTT method to measure the cytotoxicity of separated and identified alkaloid breast cancer MCF-7 cells, uses adriamycin (doxorubicin) as positive drug, and has the function characteristic of inhibiting the synthesis of RNA and DNA.
1. The experimental steps are as follows:
1) cell recovery
a) Putting the prepared culture medium into a 37 ℃ water bath kettle in advance for temperature return;
b) taking out the cell freezing tube which is placed in a refrigerator at-80 ℃ in advance and then marking;
c) rapidly melting the cell freezing solution, transferring the cell freezing solution into a centrifuge tube containing a complete culture medium, uniformly mixing, and centrifuging at 800rpm for 3 min;
d) discarding supernatant, adding 5mL complete culture medium, repeatedly blowing and sucking until cells are completely resuspended, transferring to 75cm 2 The culture bottle of (1);
e) the flask was carefully moved and placed horizontally in a cell incubator at 37 ℃ with 5% carbon dioxide.
2) Cell culture and passage
a) Observing the color and turbidity of the culture medium in time, observing the cell fusion density and activity under a microscope, and carrying out passage when the cell fusion in the culture bottle reaches more than 85% (the old culture medium is poured out firstly during passage and is rinsed for three times by sterile PBS);
b) adding a proper amount of preheated pancreatin containing 0.25% of EDTA into a culture bottle, flatly placing the culture bottle into a culture box, standing and digesting for a period of time, observing that cells lose polygonal shapes and tend to be spherical under a microscope, and adding a complete culture medium to neutralize the pancreatin when the cells do not adhere to the wall and begin to be suspended in liquid;
c) centrifuging the mixed liquid added with the culture medium at 800rpm for 3 min;
d) after discarding the supernatant, adding new culture medium, after resuspending the cells, diluting according to different passage ratios and respectively re-inoculating.
3) And the cells are frozen
a) Taking out the cells to be frozen from the incubator when the density of the cells grows to about 80%, pouring out the old culture medium, and rinsing the cells by using sterile PBS solution;
b) pouring out the PBS solution, adding pancreatin into the culture bottle, and horizontally placing the culture bottle into an incubator for standing and digestion;
c) adding a complete culture medium to neutralize pancreatin after digestion is finished, beating a culture bottle to enable cells in the bottle to completely fall into liquid, collecting the liquid, and centrifuging at 800rpm for 3 min;
d) removing the supernatant, adding 1-2 mL of serum-free cell cryopreservation solution, uniformly mixing, and transferring into a cryopreservation tube for marking and sealing;
e) and after the freezing tube is subjected to gradient cooling, transferring the tube into a liquid nitrogen tank or a low-temperature refrigerator for storage for later use.
4) Living cell workstation for detecting cell proliferation
a) Inoculating cells in a good state in a logarithmic growth phase in a 96-well plate, wherein the volume of a culture medium in each well is 100 mu L;
b) adding drug every other day (DMSO is used as solvent for dissolving compound, and isoquinoline alkaloid obtained by separation is dissolved in sequence to be used as test solution), and then putting the 96-well plate into a cell workstation for culturing for 48 h.
2. Calculating an inhibition rate: the inhibition rate was (1-number of living cells in sample/number of blank living cells) × 100%.
The IC of each compound was calculated by the inhibition ratio 50 All compounds were prescreened at a concentration of 100. mu. mol/L and doxorubicin as a positive control.
The IC of the positive control drug adriamycin is determined 50 The values are: IC of 1.08 μmol/L, 6R-hydroxydihydrochelerythrine 50 The value is more than 100 mu mol/L, 6S-hydroxydihydrochelerythrine IC of 50 The values are: 6.85. mu. mol/L.
The result shows that compared with the positive drug adriamycin, the isoquinoline alkaloid 6S-hydroxydihydrochelerythrine obtained by separation has certain cytotoxic activity on breast cancer MCF-7 cells, can further research the action mechanism of the alkaloid for inhibiting the proliferation of the breast cancer cells and the toxic and side effects on normal cells, and provides experimental basis for developing novel anti-breast cancer drugs or lead compounds.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (10)
1. A method for extracting and separating benzophenanthridine enantiomer alkaloids from Hovenia dulcis Thunb is characterized by comprising the following steps:
1) reflux-extracting pulverized radix Hoveniae with 80% ethanol-water system to obtain total extract, and adding water for suspension treatment to obtain suspension;
2) adjusting the pH value of the suspension to 2, extracting for several times by adopting dichloromethane, combining the extract solutions, and evaporating to dryness to obtain a dichloromethane part with the pH value of 2 and an acid extract part;
3) loading a dichloromethane part with the pH value of 2 by adopting a dry method, separating by using a normal pressure chromatographic column, performing gradient elution by using a petroleum ether-ethyl acetate system as an eluent, detecting by adopting TLC (thin-layer chromatography), merging and collecting same fractions to obtain a fraction A containing a target component, and concentrating and drying to obtain an extract A;
4) separating the extract A obtained in the step 3) by adopting a normal phase silica gel chromatographic column, carrying out gradient elution by adopting a dichloromethane-methanol system as an eluent, carrying out TLC detection, combining and collecting the same fractions to obtain a fraction B containing a target component, concentrating and drying to obtain an extract B;
5) performing gradient elution and TLC detection on the extract B obtained in the step 4) by using a petroleum ether-ethyl acetate system, combining and collecting the same fractions to obtain a fraction C containing a target component, and concentrating and drying to obtain an extract C;
6) separating the extract C obtained in the step 5) by using semi-preparative HPLC to obtain racemic benzophenanthridine alkaloid;
7) carrying out chiral chromatographic resolution on the racemic benzophenanthridine alkaloid obtained in the step 6) by adopting HPLC (high performance liquid chromatography) to obtain a pair of enantiomers, namely 6R-carboxymethyl-dihydrochelelytrine and 6S-carboxymethyl-dihydrochelelytrine.
2. The method for extracting and separating benzophenanthridine enantiomer alkaloids from hovenia dulcis thunb as claimed in claim 1, wherein in step 1), reflux extraction is performed for 3 times, each for 3 hours.
3. The method for extracting and separating benzophenanthridine enantiomer alkaloids from hovenia dulcis as claimed in claim 1, wherein in the step 2), 10% hydrochloric acid solution is adopted to adjust the pH value of the suspension.
4. The method for extracting and separating benzophenanthridine enantiomer alkaloids from hovenia dulcis as claimed in claim 1, wherein in the step 3), the dry-method sample loading is to mix the dichloromethane part with the pH value of 2 with 100-200 mesh column chromatography silica gel and load the mixture; when the separation is carried out by an atmospheric pressure chromatographic column, petroleum ether and ethyl acetate are subjected to gradient elution according to the volume ratio of (12:1) to (0: 1).
5. The method for extracting and separating benzophenanthridine enantiomer alkaloids from hovenia dulcis thunb as claimed in claim 1, wherein in the step 4), during the chromatographic column separation, dichloromethane and methanol are subjected to gradient elution according to the volume ratio of (20:1) - (0: 1).
6. The method for extracting and separating benzophenanthridine enantiomer alkaloids from hovenia dulcis thunb as claimed in claim 1, wherein in the step 5), petroleum ether and ethyl acetate are subjected to gradient elution according to the volume ratio of (8:1) - (0:1) during chromatographic column separation.
7. The method for extracting and separating benzophenanthridine enantiomer alkaloids from hovenia dulcis as claimed in claim 1, wherein the chromatographic conditions of semi-preparative HPLC separation in step 6) are as follows:
a chromatographic column: welch Xtimate C18, 10X 250mm,5 μm;
eluent: the mobile phase adopts 50% methanol-water and 0.5% ammonia water, and the flow rate is as follows: 2mL/min, isocratic elution.
8. The method for extracting and separating benzophenanthridine enantiomer alkaloids from hovenia dulcis as claimed in claim 1, wherein in step 7), the chromatographic conditions for chiral chromatographic resolution by HPLC are as follows:
a chromatographic column:
IC，4.6×250mm，5μm；
eluent: n-hexane: isopropanol 80:20, flow rate: 0.8mL/min, isocratic elution.
9. The benzophenanthridine alkaloid obtained by separation through the method for extracting and separating benzophenanthridine enantiomer alkaloid from hovenia dulcis thunb as claimed in any one of claims 1 to 8 is characterized in that the structure of the benzophenanthridine alkaloid is S type, namely 6S-carboxymethyl-dihydrochelerythrine, and the structural formula is as follows:
10. use of the benzophenanthridine alkaloid of claim 9 in the preparation of a medicament for treating breast cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210899681.4A CN115093426A (en) | 2022-07-28 | 2022-07-28 | Method for extracting and separating benzophenanthridine enantiomer alkaloids from angelica Hovenia and application of benzophenanthridine enantiomer alkaloids |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210899681.4A CN115093426A (en) | 2022-07-28 | 2022-07-28 | Method for extracting and separating benzophenanthridine enantiomer alkaloids from angelica Hovenia and application of benzophenanthridine enantiomer alkaloids |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115093426A true CN115093426A (en) | 2022-09-23 |
Family
ID=83299880
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210899681.4A Pending CN115093426A (en) | 2022-07-28 | 2022-07-28 | Method for extracting and separating benzophenanthridine enantiomer alkaloids from angelica Hovenia and application of benzophenanthridine enantiomer alkaloids |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115093426A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110437243A (en) * | 2019-07-12 | 2019-11-12 | 湖南农业大学 | A method of extracting six kinds of dihydrobenzo phenanthridine alkaloids of separation from macleaya microcarpa Fedde |
| MD4706B1 (en) * | 2019-07-16 | 2020-08-31 | Ана КАСИЯН | Process for producing bisulfates of benzophenanthridine alkaloids from macleaya leaves |
| CN113648312A (en) * | 2021-09-13 | 2021-11-16 | 西安交通大学 | Application of 6-methoxy dihydrosanguinarine in preparation of medicine for resisting breast cancer |
-
2022
- 2022-07-28 CN CN202210899681.4A patent/CN115093426A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110437243A (en) * | 2019-07-12 | 2019-11-12 | 湖南农业大学 | A method of extracting six kinds of dihydrobenzo phenanthridine alkaloids of separation from macleaya microcarpa Fedde |
| MD4706B1 (en) * | 2019-07-16 | 2020-08-31 | Ана КАСИЯН | Process for producing bisulfates of benzophenanthridine alkaloids from macleaya leaves |
| CN113648312A (en) * | 2021-09-13 | 2021-11-16 | 西安交通大学 | Application of 6-methoxy dihydrosanguinarine in preparation of medicine for resisting breast cancer |
Non-Patent Citations (3)
| Title |
|---|
| LI LIN: "Systematic identification of compounds in Macleaya microcarpa by high‐performance liquid chromatography/quadrupole time‐of‐flight tandem mass spectrometry combined with mass spectral fragmentation behavior of Macleaya alkaloids", vol. 34, no. 9, pages 311 - 312 * |
| OSCAR JAVIER PATIÑO LADINO: "Isoquinoline alkaloids of Zanthoxylum quinduense (Rutaceae)", BIOCHEMICAL SYSTEMATICS AND ECOLOGY, vol. 38, pages 853, XP027437782, DOI: 10.1016/j.bse.2010.07.016 * |
| 赛春梅;华会明;王建安;王其宝;王慧云;: "博落回中苯并菲啶类生物碱外消旋体的分离与鉴定", no. 04, pages 256 - 263 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113150048B (en) | Cyclocarya paliurus extract and application thereof in resisting rheumatoid arthritis | |
| CN113861114B (en) | Morphinans and dibenzyl isoquinoline alkaloid compounds, and preparation method and application thereof | |
| CN117003812A (en) | Rosin alkane type diterpenoid lactone compound, and preparation method and application thereof | |
| CN114957362B (en) | Method for separating anti-inflammatory active ingredients of coreopsis tinctoria | |
| CN115093426A (en) | Method for extracting and separating benzophenanthridine enantiomer alkaloids from angelica Hovenia and application of benzophenanthridine enantiomer alkaloids | |
| CN103191143B (en) | New application of cardiac glycoside compound | |
| CN115466248A (en) | Diterpenoid compound and extraction method and application thereof | |
| CN108484428A (en) | A kind of amides compound in matrimony vine and amides compound component and preparation method thereof | |
| CN114835668A (en) | Preparation method and application of cyclic ether type diaryl heptane in exocarpium Juglandis Immaturum | |
| CN114605422A (en) | A pair of enantiomer alkaloid dimer compounds, and preparation method and application thereof | |
| CN110204589B (en) | Effective component of feather cockscomb seed, extraction method and application thereof in preparing neuroprotective medicament | |
| CN116514916B (en) | Indole alkaloid compound and preparation method thereof | |
| CN108503682B (en) | Method for simultaneously extracting and separating triptolide A and triptolide B | |
| CN116003371B (en) | Terpenoid, and extraction method and application thereof | |
| CN115304611B (en) | Dihydrobenzophenanthridine alkaloid compound in radix zanthoxyli, and extraction method and application thereof | |
| CN114409557B (en) | Carbon keratin with neuroprotective activity and preparation method and application thereof | |
| CN116332765B (en) | Alkanoate compound and preparation method and application thereof | |
| CN115703753B (en) | Benzofuran derivative and preparation method and application thereof | |
| CN112390809B (en) | Method for extracting iridoid compound from patrinia scabiosaefolia fisch | |
| CN114409595B (en) | 1-Styryl isoquinoline derivative and preparation and application thereof | |
| CN116003238B (en) | Sesquiterpenoids in jasmine roots, and extraction method and application thereof | |
| CN110172065B (en) | Compound and preparation method and application thereof | |
| CN109705077B (en) | Coumarin compound and preparation method and application thereof | |
| CN117843602A (en) | New biflavanoid compound and extraction method and application thereof | |
| CN115819411A (en) | 7 lignans compounds in radix zanthoxyli and extraction method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |