CN115636855B - Dictamni glucoside R and preparation method and application thereof - Google Patents
Dictamni glucoside R and preparation method and application thereof Download PDFInfo
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Abstract
A dittany glycoside R and a preparation method and application thereof relate to a compound, a preparation method thereof and application of the compound. Dictamni glucoside R has molecular formula of C 21 H 36 O 9 The molecular weight is 432.2359. The preparation method comprises the following steps: 1. pulverizing cortex Dictamni Radicis, and extracting with 95% ethanol; 2. dispersing the ethanol-recovered extract with water, and sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol; 3. and (3) repeatedly performing silica gel column chromatography on the ethyl acetate extraction layer, and performing high performance liquid chromatography separation to obtain the monomer compound. The dittany glycoside R has in-vitro anti-tumor activity and is specific to MDA-MB-231 human breast cancer cell IC 50 The value was 60.5 (μg/ml) and the minimum effective concentration was 12.5 (μg/ml). Has strong inhibition on MDA-MB-231 human breast cancer cells. The invention belongs to the field of organic matter extraction.
Description
Technical Field
The invention relates to a compound, a preparation method thereof and application of the compound.
Background
Breast cancer is one of the most common malignant tumors of women, the incidence rate accounts for 7-10% of various malignant tumors of the whole body, and the breast cancer is secondary to uterine cancer in women and becomes a main cause of threatening the health of women. Its onset is often genetically related and is high in women before and after menopause between 40 and 60 years of age. It is one of the most common malignant tumors that usually occur in mammary gland epithelial tissue, severely affecting women's physical and mental health and even endangering life. Breast cancer men are rare, with only about 1-2% of breast patients being men.
Disclosure of Invention
The invention aims to provide a substance with in vitro anti-breast tumor activity.
The molecular formula of the dittany glycoside R is C 21 H 36 O 9 Molecular weight of 432.23The 59 molecular structural formula is as follows:
the preparation method of the dittany glucoside R comprises the following steps:
1. pulverizing cortex Dictamni Radicis, extracting with 95% ethanol at 90deg.C for 1 hr/time in an extraction tank of Soxhlet dynamic extraction concentration unit for three times, mixing the extractive solutions, and recovering ethanol to obtain ethanol extract;
2. dispersing the ethanol extract with equal volume of water, and sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol;
3. eluting the ethyl acetate extract by using a silica gel column chromatography, eluting an eluent a by using an ODS liquid column chromatography and using a mixed solution of methanol and water with the volume ratio of (100-0) to (0-100), eluting an eluent b by using a silica gel column chromatography and using a mixed solution of dichloromethane and methanol with the volume ratio of (100-1) to (6-100), eluting an eluent c in a SephadexLH-20 column chromatography and using methanol as a mobile phase, separating an eluent d by using a high performance liquid chromatography, and collecting an eluent with the elution time of 77.030 minutes and having an absorption peak, thus obtaining the ditallow R;
high performance liquid chromatography separation chromatographic conditions: dikma chromatographic column: c (C) 18 5 μm, 250X 4.6mm; detection wavelength is 210nm and 254nm; the ratio of mobile phase methanol to water is 20:80; sample injection amount is 100 mu L; the flow rate was 3mL/min.
In the first step, the pressure of the Soxhlet dynamic extraction and concentration unit is set to be normal pressure extraction, negative pressure concentration is carried out under the pressure of-0.8 MPa, and the extraction temperature is set to be 90 ℃.
The cortex Dictamni Radicis is dried root bark of Dictamni Radicis (Dictamnus dasycapus) which is perennial herb of Rutaceae. Mainly produced in Liaoning, hebei, sichuan, jiangsu and other places. Removing sediment and coarse skin from root of spring and autumn Ji Caiwa, peeling root skin, slicing, and drying. Alias name: cortex Dictamni Radicis, radix Calophylli Membranacei, radix et rhizoma Paeonia ostii, and herba Lespedezae Cuneatae. Cortex Dictamni Radicis has effects of clearing heat and detoxicating, dispelling pathogenic wind and removing dampness, and can be used for treating skin diseases such as: eczema, damp-heat sore, skin itching, jaundice, scabies and other diseases. Cortex Dictamni Radicis is used for treating tumor in inner Mongolia, and the literature reports that the chemical component phellodendrine in cortex Dictamni has in vitro anti-tumor activity.
The invention discloses a method for extracting dittany glucoside R from dittany bark, which has the chemical name: 4 a-hydroxymethyl-7 (1-methyl-ethyl) -1-methyl-1, 2,3, 4a,5,6, 7-octahydronaphthalene-1, 4 diol-7- (1-O-) - β -D-glucoside.
Named as dittany glycoside R.
The dittany glycoside R is white powder and dissolved in methanol. Dark spots are observed under ultraviolet 254nm, no fluorescence is observed under 365nm, and 10% sulfuric acid ethanol develops into pink purple.
(c 0.09,MeOH)；/>
263。。HR-ESI-MS:m/z455.2263[M+Na] + (calculated as 455.2257), molecular formula C was determined 21 H 36 O 9 The calculated unsaturation was 4.
The purity of the dittany glycoside R obtained by the method is up to more than 98.5 percent.
The dittany glycoside R has in-vitro anti-tumor activity and is specific to MDA-MB-231 human breast cancer cell IC 50 The value was 60.5 (μg/ml) and the minimum effective concentration was 12.5 (μg/ml). Has strong inhibition on MDA-MB-231 human breast cancer cells. The dittany glycoside R of the invention can be prepared into antitumor drugs such as injection, freeze-dried powder injection, infusion solution or oral preparation (including tablets, granules, soft capsules, hard capsules, oral liquid and sustained and controlled release preparation).
Detailed Description
The technical scheme of the invention is not limited to the specific embodiments listed below, and also includes any combination of the specific embodiments.
The first embodiment is as follows: the implementation isThe molecular formula of the dittany glycoside R is C 21 H 36 O 9 The molecular weight is 432.2359, and the molecular structural formula is:
the second embodiment is as follows: the preparation method of the dittany glycoside R in the specific embodiment is as follows:
1. pulverizing cortex Dictamni Radicis, extracting with 95% ethanol at 90deg.C for 1 hr/time in an extraction tank of Soxhlet dynamic extraction concentration unit for three times, mixing the extractive solutions, and recovering ethanol to obtain ethanol extract;
2. dispersing the ethanol extract with equal volume of water, and sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol;
3. eluting the ethyl acetate extract by using a silica gel column chromatography, eluting an eluent a by using an ODS liquid column chromatography and using a mixed solution of methanol and water with the volume ratio of (100-0) to (0-100), eluting an eluent b by using a silica gel column chromatography and using a mixed solution of dichloromethane and methanol with the volume ratio of (100-1) to (6-100), eluting an eluent c in a SephadexLH-20 column chromatography and using methanol as a mobile phase, separating an eluent d by using a high performance liquid chromatography, and collecting an eluent with the elution time of 77.030 minutes and having an absorption peak, thus obtaining the ditallow R;
high performance liquid chromatography separation chromatographic conditions: dikma chromatographic column: c (C) 18 5 μm, 250X 4.6mm; detection wavelength is 210nm and 254nm; the ratio of mobile phase methanol to water is 20:80; sample injection amount is 100 mu L; the flow rate was 3mL/min.
And a third specific embodiment: the second difference between the present embodiment and the specific embodiment is that the Soxhlet dynamic extraction and concentration unit pressure in the first step is set to normal pressure extraction, negative pressure concentration under-0.8 MPa, and extraction temperature is set to 90 ℃. The other is the same as in the second embodiment.
The specific embodiment IV is as follows: in the third embodiment, the ethyl acetate extract is eluted by silica gel column chromatography with a mixed solution of dichloromethane and methanol in a volume ratio of dichloromethane to methanol of 100:10, the eluate a is eluted by ODS liquid chromatography with a mixed solution of methanol and water in a volume ratio of methanol to water of 30:70 as a mobile phase, the eluate b is eluted by silica gel column chromatography with a mixed solution of dichloromethane and methanol in a volume ratio of dichloromethane to methanol of 100:10, and the eluate c is eluted by SephadexLH-20 column chromatography with methanol as a mobile phase to obtain the eluate d. The other embodiments are the same as those of the second or third embodiment.
Fifth embodiment: in the present embodiment, the difference from the second to fourth embodiments is that, when the eluent d is subjected to high performance liquid chromatography in the third step, the mobile phase is a mixture of methanol and water with a methanol-water volume ratio of 20:80, the detection wavelength is 210nm, the flow rate is 3mL/min, and the sample injection amount is 100 μl; chromatographic column: dikma C18, particle diameter of 5 μm,250×4.6mm, peak time of 77.030min, and obtaining dittany glycoside R. The others are the same as in the second to fourth embodiments.
Specific embodiment six: in the specific embodiments, the dittany glucoside R is used for preparing anti-tumor medicines.
The following experiments are adopted to verify the effect of the invention:
experiment one:
the preparation method of the dittany glycoside R comprises the following steps:
1. pulverizing 100kg of cortex Dictamni Radicis, extracting with 95% ethanol at 90deg.C for 1 hr/time, extracting for three times, mixing extractive solutions, and recovering ethanol to obtain ethanol extract;
2. dispersing the ethanol extract with equal volume of water, and sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol;
3. ethyl acetate extract (837 g) was eluted with a mixture of dichloromethane and methanol in a volume ratio of dichloromethane to methanol of 100:10 by using a silica gel column chromatography, eluent a was eluted with an ODS liquid chromatography column in a volume ratio of methanol to water of 30:70 by using a mixture of methanol and water as a mobile phase, eluent b was eluted with a mixture of dichloromethane and methanol in a volume ratio of dichloromethane to methanol of 100:10 by using a silica gel column chromatography, and eluent c was eluted with methanol as a mobile phase in a SephadexLH-20 column chromatography; performing high performance liquid chromatography separation on the eluate d, and collecting the eluate of the absorption peak occurring in 77.030min to obtain the dittany glucoside R4.2mg;
in the first step, the pressure of the Soxhlet dynamic extraction and concentration unit is set to be normal pressure extraction, negative pressure concentration is carried out under the pressure of-0.8 MPa, and the extraction temperature is set to be 90 ℃.
In the third step, the eluent d is subjected to high performance liquid chromatography, the mobile phase is a mixture of methanol and water with a methanol-water volume ratio of 20:80, the detection wavelength is 210nm, the flow rate is 3mL/min, and the sample injection amount is 100 mu L; chromatographic column: dikma C18,5 μm, 250X 4.6mm.
The experiment adopts a Waters 2545 high performance liquid chromatograph in the United states, and the detector is a Waters2489 ultraviolet detector. The packing of the chromatographic column of the silica gel column chromatography is normal-phase merck silica gel.
100kg of dittany bark is crushed and extracted for the experiment, and finally the obtained dittany glycoside R is 4.2mg, the purity reaches 98.5%, and nuclear magnetic resonance data of the dittany glycoside R are shown in table 1.
TABLE 1 Dictamni glycoside R 1 H-NMR 13 C-NMR data
The dittany glycoside R is white powder and dissolved in methanol. Dark spots are observed under ultraviolet 254nm, no fluorescence is observed under 365nm, and 10% sulfuric acid ethanol develops into pink purple.
(c 0.09,MeOH)；/>
263。HR-ESI-MS:m/z455.2263[M+Na] + (calculated as 455.2257), molecular formula C was determined 21 H 36 O 9 The calculated unsaturation was 4.
The molecular formula of the dittany glycoside R is C 21 H 36 O 9 The molecular weight is 432.2359, and the molecular structural formula is:
human breast cancer MDA-MB-231 cells in the logarithmic growth phase were inoculated into 96-well plates (the inoculation density was 7000 pieces/well), cultured at 37℃for 24 hours, the medium was aspirated off, and medium containing different concentrations of dittany glycoside R (blank group, concentration group: 6.25. Mu.g/ml, 12.5. Mu.g/ml, 25. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml) was added to the well plates, respectively, and incubation was continued at 37 ℃. After 24h, 20. Mu.L of MTT (diphenyltetrazolium bromide) was added at a concentration of 5mg/mL per well and incubated for 4h at 37 ℃. After the incubation, the supernatant was discarded, 150. Mu.L of DMSO (dimethyl sulfoxide) was added to each well and shaken for 10min to sufficiently dissolve the purple crystals in the well plate, and then absorbance was measured at 570nm using an ELISA reader, and IC was calculated 50 The value was 60.5 (μg/ml) and the minimum effective concentration was 12.5 (μg/ml).
Claims (5)
1. The dittany glycoside R is characterized in that the molecular formula of the dittany glycoside R is C 21 H 36 O 9 The molecular weight is 432.2359, and the molecular structural formula is:
2. the preparation method of the dittany glycoside R as claimed in claim 1, which is characterized in that the preparation method of the dittany glycoside R comprises the following steps:
1. pulverizing cortex Dictamni Radicis, extracting with 95% ethanol at 90deg.C for 1 hr/time in an extraction tank of Soxhlet dynamic extraction concentration unit for three times, mixing the extractive solutions, and recovering ethanol to obtain ethanol extract;
2. dispersing the ethanol extract with equal volume of water, and sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol;
3. eluting the ethyl acetate extract by adopting silica gel column chromatography with a mixed solution of dichloromethane and methanol with the volume ratio of dichloromethane to methanol of 100:10 to obtain an eluent a; eluting the eluent a by adopting ODS liquid column chromatography and taking a mixed solution of methanol and water with the volume ratio of methanol to water of 30:70 as a mobile phase to obtain an eluent b; eluting the eluate b by adopting silica gel column chromatography, and eluting with a mixed solution of dichloromethane and methanol with the volume ratio of dichloromethane to methanol of 100:10 to obtain an eluate c; eluting the eluate c in SephadexLH-20 column chromatography with methanol as mobile phase to obtain eluate d; performing high performance liquid chromatography separation on the eluate d, and collecting eluate with absorption peak at 77.030min to obtain dittany glycoside R;
high performance liquid chromatography separation chromatographic conditions: dikma chromatographic column: c (C) 18 5 μm,250×4.6mm; detection wavelength is 210nm and 254nm; the ratio of mobile phase methanol to water is 20:80; sample injection amount is 100 mu L; the flow rate was 3mL/min.
3. The method for preparing the dittany glycoside R according to claim 2, characterized in that in the step one, the Soxhlet dynamic extraction and concentration unit pressure is set to normal pressure extraction, negative pressure concentration is carried out under the pressure of-0.8 MPa, and the extraction temperature is set to 90 ℃.
4. The method for preparing the dittany glycoside R according to claim 2, characterized in that when the eluent d is subjected to high performance liquid chromatography separation in the step three, the mobile phase is a mixture of methanol and water with a methanol-water volume ratio of 20:80, the detection wavelength is 210nm, the flow rate is 3mL/min, and the sample injection amount is 100 mu L; chromatographic column: dikma C18, particle size of 5 μm,250×4.6mm, peak time 77.030min, and preparation of dittany glycoside R.
5. The use of the dictamnus glucoside R as claimed in claim 1, characterized in that the dictamnus glucoside R is used for preparing a tumor drug against human breast cancer MDA-MB-231.
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