CN107320554B - Exocarpium Citri Grandis extract and its application in preparing medicine for treating APE1 mediated diseases - Google Patents
Exocarpium Citri Grandis extract and its application in preparing medicine for treating APE1 mediated diseases Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/75—Rutaceae (Rue family)
- A61K36/752—Citrus, e.g. lime, orange or lemon
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
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- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
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- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention relates to the technical field of medicinal chemistry, and particularly discloses an exocarpium citri grandis extract and application thereof in preparing a medicament for treating APE 1-mediated diseases. The pummelo peel extract comprises the following effective components: nobiletin, quercetin and naringin. Experiments show that the extract can effectively inhibit the expression of APE1 in lung cancer cells, can be used as an APE1 inhibitor and is used for treating APE 1-mediated diseases; the experiments of the examples also show that the pummelo peel extract has very obvious in-vitro anti-lung cancer effect; in addition, the exocarpium citri grandis extract provided by the invention is clear in effective components and content, and is beneficial to quality control and medication safety of the extract.
Description
Technical Field
The invention relates to the technical field of medicinal chemistry, in particular to an exocarpium citri grandis extract and application thereof in preparing a medicament for treating APE 1-mediated diseases.
Background
Exocarpium Citri Grandis, namely exocarpium Citri Grandis, is prepared from immature or nearly mature dry outer pericarp of Citrus grandis or fructus Citri Grandis of Rutaceae. Has effects in eliminating phlegm, relieving cough, and treating cough due to wind-cold evil.
Lung cancer is one of the most rapidly growing malignancies that threaten human health and life. At present, the main treatment means of lung cancer are radiotherapy and chemotherapy, wherein the combined treatment of radiotherapy and chemotherapy is also a common treatment mode.
APE1 is apurinic/apurinic endonuclease, is a biological macromolecular functional complex, is a key rate-limiting enzyme of DNA base excision repair pathway, has endonuclease and redox functions, and is an important repair factor for cell chemotherapy drug injury. The existing research (Zhangmin. APE1 action-related protein and action research [ D ] of the protein in tumor radiotherapy, third department of military medicine university, 2011.) shows that the expression of APE1 in lung cancer cells can promote the expression of Nm23-H1, and APE1 and Nm23-H1 can synergistically repair DNA of the lung cancer cells, so that the lung cancer cells generate radiotherapy or chemotherapy resistance. Therefore, the development of a medicament which not only has the APE1 expression inhibition function, but also has the lung cancer resistance function has wide market prospect.
Disclosure of Invention
The invention aims to solve the technical problem of providing an exocarpium citri grandis extract which can inhibit the expression of APE1 and has a very good anti-lung cancer effect.
The technical problem to be solved by the invention is realized by the following technical scheme:
an exocarpium Citri Grandis extract comprises the following effective components: nobiletin, quercetin and naringin.
Preferably, the pummelo peel extract is characterized by comprising the following effective components in percentage by weight: 20-30% of nobiletin; 10-20% of quercetin; 30-40% of naringin.
The preparation method of the pummelo peel extract comprises the following steps:
(1) extracting exocarpium Citri Grandis with ethanol under reflux, and concentrating the extractive solution to obtain exocarpium Citri Grandis ethanol extract;
(2) dissolving the ethanol extract of exocarpium Citri Grandis in water to obtain suspension, extracting with petroleum ether, discarding the petroleum ether extract, extracting with ethyl acetate, and concentrating the ethyl acetate extract to obtain exocarpium Citri Grandis extract;
(3) subjecting exocarpium Citri Grandis extract to silica gel column chromatography to enrich effective components to obtain exocarpium Citri Grandis extract;
the specific method for enriching the effective components by silica gel column chromatography in the step (3) comprises the following steps: putting the pummelo peel extract into a silica gel column, and eluting and removing impurities by using a mixed solvent system consisting of a solvent A and a solvent B in a volume ratio of 85: 15-80: 20; then, eluting by using a mixed solvent system consisting of a solvent A and a solvent B in a volume ratio of 75: 25-72: 28, and collecting eluted parts to obtain the effective parts of the pummelo peel; the solvent A is petroleum ether, and the solvent B is ethyl acetate;
the dosage of a mixed solvent system consisting of the solvent A and the solvent B in a volume ratio of 85: 15-80: 20 is 1-3 times of the volume of the silica gel column; the dosage of a mixed solvent system consisting of the solvent A and the solvent B in a volume ratio of 75: 25-72: 28 is 3-5 times of the volume of the silica gel column.
Preferably, the specific method for enriching the effective components by silica gel column chromatography in the step (3) comprises the following steps: putting the pummelo peel extract on a silica gel column, and firstly, adding a solvent A: eluting a mixed solvent system consisting of the solvent B to remove impurities; then adding solvent A with volume ratio of 72: 28: eluting with a mixed solvent system consisting of the solvent B and collecting the eluted part to obtain the effective part of the pummelo peel;
the dosage of a mixed solvent system consisting of the solvent A and the solvent B with the volume ratio of 80:20 is 3 times of the volume of the silica gel column; the dosage of the mixed solvent system consisting of the solvent A and the solvent B with the volume ratio of 72:28 is 5 times of the volume of the silica gel column.
The volume of the silica gel column is the volume from the column bottom to the silica gel deposition surface after the silica gel is filled into the column.
Preferably, the weight of the silica gel used in the silica gel column in the step (3) is 20-40 times of the dry weight of the exocarpium citri grandis extract.
Preferably, the ethanol in the step (1) is an ethanol aqueous solution with a volume fraction of 80-100%; the ratio of the amount of the ethanol to the amount of the pummelo peel is 8-15 mL:1 g; the heating reflux extraction time is 1-3 h; and (3) extracting for 1-3 times by using petroleum ether with the same volume as the suspension in the step (2), discarding the petroleum ether extract, extracting for 1-3 times by using ethyl acetate with the same volume as the suspension, and concentrating the ethyl acetate extract to obtain the pummelo peel extract.
The exocarpium Citri Grandis extract can be used for preparing APE1 inhibitor.
The application of the pummelo peel extract in preparing a medicament for treating APE1 mediated diseases; the APE1 mediated disease is APE1 mediated lung cancer.
The exocarpium Citri Grandis extract is used in preparing anticancer drugs for treating lung cancer.
The invention also provides an anti-cancer pharmaceutical composition derived from pummelo peel, which comprises nobiletin, quercetin and naringin, wherein the weight ratio of the nobiletin to the quercetin to the naringin is (2-3): 1-2: 3-4; the anti-cancer drug is an anti-lung cancer drug.
Has the advantages that: the invention provides a brand-new pummelo peel extract, which can effectively inhibit the expression of APE1 in lung cancer cells, can be used as an APE1 inhibitor and is used for treating APE 1-mediated diseases; the experiments of the examples also show that the pummelo peel extract has very obvious in-vitro anti-lung cancer effect; in addition, the exocarpium citri grandis extract provided by the invention is clear in effective components and content, and is beneficial to quality control and medication safety of the extract.
Detailed Description
The present invention is further explained below with reference to specific examples, which are not intended to limit the present invention in any way.
Example 1 preparation of exocarpium Citri Grandis extract
(1) Heating and refluxing exocarpium Citri Grandis with 95% ethanol water solution by volume for 1.5 hr, and concentrating the extractive solution to obtain exocarpium Citri Grandis ethanol extract;
(2) dissolving exocarpium Citri Grandis ethanol extract in water to obtain suspension, extracting with petroleum ether of the same volume for 3 times, discarding petroleum ether extract, extracting with ethyl acetate of the same volume for 3 times, mixing the ethyl acetate extracts, and concentrating to obtain exocarpium Citri Grandis extract;
(3) subjecting exocarpium Citri Grandis extract to silica gel column chromatography to enrich effective components to obtain exocarpium Citri Grandis extract;
the method for enriching the effective components by silica gel column chromatography comprises the following steps: passing the exocarpium Citri Grandis extract through silica gel column (the weight of silica gel in silica gel column is 30 times of the dry weight of exocarpium Citri Grandis extract, and the specification of silica gel is 200 meshes), dissolving in solvent A: eluting and removing impurities in a mixed solvent system (the dosage is 3 times of the column volume) consisting of the solvent B; then adding solvent A with volume ratio of 72: 28: eluting with a mixed solvent system (8 times of column volume) composed of solvent B, and collecting the eluted part to obtain exocarpium Citri Grandis effective part; the solvent A is petroleum ether, and the solvent B is ethyl acetate.
Through detection, the pummelo peel extract contains 28% of nobiletin, 19% of quercetin and 37% of naringin by weight percentage.
The content determination method of the components comprises the following steps: detecting with liquid chromatography, and measuring with nobiletin, quercetin and naringin as standard substances by external standard method.
The liquid chromatogram conditions are as follows: with Agilent Zorbax SB C18(column length 250mm, inner diameter 4.6mm, particle size 5 μm) as chromatographic column; acetonitrile is used as a mobile phase A, 0.05% acetic acid solution is used as a mobile phase B, and the detection wavelength is 330 nm; the column temperature is 25 ℃, and the flow rate is 1.0 mL/min; mobile phase A: mobile phase B15: 85. Wherein the retention time of naringin is 5.1min, the retention time of quercetin is 9.3min, and the retention time of nobiletin is 14.4 min.
Example 2 preparation of exocarpium Citri Grandis extract
(1) Heating and refluxing exocarpium Citri Grandis with 95% ethanol water solution by volume for 1.5 hr, and concentrating the extractive solution to obtain exocarpium Citri Grandis ethanol extract;
(2) dissolving exocarpium Citri Grandis ethanol extract in water to obtain suspension, extracting with petroleum ether of the same volume for 3 times, discarding petroleum ether extract, extracting with ethyl acetate of the same volume for 3 times, mixing the ethyl acetate extracts, and concentrating to obtain exocarpium Citri Grandis extract;
(3) subjecting exocarpium Citri Grandis extract to silica gel column chromatography to enrich effective components to obtain exocarpium Citri Grandis extract;
the method for enriching the effective components by silica gel column chromatography comprises the following steps: passing the exocarpium Citri Grandis extract through silica gel column (the weight of silica gel in silica gel column is 30 times of the dry weight of exocarpium Citri Grandis extract, and the specification of silica gel is 200 meshes), dissolving in solvent A with volume ratio of 85: 15: eluting and removing impurities in a mixed solvent system (the dosage is 3 times of the column volume) consisting of the solvent B; then adding solvent A with a volume ratio of 75: 25: eluting with a mixed solvent system (8 times of column volume) composed of solvent B, and collecting the eluted part to obtain exocarpium Citri Grandis effective part; the solvent A is petroleum ether, and the solvent B is ethyl acetate.
Through detection, the pummelo peel extract contains 26% of nobiletin, 16% of quercetin and 34% of naringin by weight percentage.
Example 3 preparation of exocarpium Citri Grandis extract
(1) Heating and refluxing exocarpium Citri Grandis with 95% ethanol water solution by volume for 1.5 hr, and concentrating the extractive solution to obtain exocarpium Citri Grandis ethanol extract;
(2) dissolving exocarpium Citri Grandis ethanol extract in water to obtain suspension, extracting with petroleum ether of the same volume for 3 times, discarding petroleum ether extract, extracting with ethyl acetate of the same volume for 3 times, mixing the ethyl acetate extracts, and concentrating to obtain exocarpium Citri Grandis extract;
(3) subjecting exocarpium Citri Grandis extract to silica gel column chromatography to enrich effective components to obtain exocarpium Citri Grandis extract;
the method for enriching the effective components by silica gel column chromatography comprises the following steps: passing the exocarpium Citri Grandis extract through silica gel column (the weight of silica gel in silica gel column is 30 times of the dry weight of exocarpium Citri Grandis extract, and the specification of silica gel is 200 meshes), dissolving in solvent A with volume ratio of 85: 15: eluting and removing impurities in a mixed solvent system (the dosage is 3 times of the column volume) consisting of the solvent B; then adding solvent A with volume ratio of 72: 28: eluting with a mixed solvent system (8 times of column volume) composed of solvent B, and collecting the eluted part to obtain exocarpium Citri Grandis effective part; the solvent A is petroleum ether, and the solvent B is ethyl acetate.
Through detection, the pummelo peel extract contains 23% of nobiletin, 12% of quercetin and 31% of naringin by weight percentage.
EXAMPLE 4 extract inhibition of APE1 expression assay
Plasmid pcDNA3.0-APE1 was constructed and transfected into human lung cancer cell A549, then the human lung cancer cell A549 was cultured in cell culture medium containing the exocarpium citri grandis extract described in example 1, the exocarpium citri grandis extract described in example 2, the exocarpium citri grandis extract described in example 3, the pharmaceutical composition (the composition consists of nobiletin, quercetin and naringin in a weight ratio of 3:2: 3) and no drug for 48h, and then Western blotting was used to detect the expression of APE1 protein in the cells. The specific method is referred to as 'Zhangmin. APE1 action-related protein and action research thereof in tumor radiotherapy [ D ]. third university of military medicine, 2011'.
The experimental result shows that the APE1 protein content of the human lung cancer cell A549 cultured in the cell culture solution containing the pummelo peel extract and the pharmaceutical composition is obviously lower than that of the human lung cancer cell A549 cultured in the cell culture solution without any medicine. This indicates that: the pummelo peel extract and the pharmaceutical composition consisting of nobiletin, quercetin and naringin can obviously inhibit the expression of APE1 in lung cancer cells, can be used as an APE1 inhibitor, and can be used as a medicine for treating APE 1-mediated diseases.
Example 5 anti-Lung cancer Activity assay of extracts
Culturing human lung cancer cell A549 in culture medium (RPMI 1640 culture medium) at a ratio of 10 × 103Each well was placed in a 96 well plate containing 5% CO at 37 deg.C2The original culture medium is sucked off after the wall is attached after the culture for 24 hours in the incubator. Then adding RPMI 1640 culture medium containing 10% fetal bovine serum into the blank group; the experimental groups are respectively added into RPMI 1640 culture media containing 0.01-100 mug/mL of drugs to be detected; after further culturing for 48 hours, MTT was added at a concentration of 5mg/mL, further culturing was carried out for 4 hours, the supernatant was aspirated, 100. mu.L of DMSO was added, the mixture was left in the dark for 10 minutes, the absorbance was measured at 570nm using a microplate reader (Sunrise), and the survival of the cells was calculated from the absorbance, with 6 duplicate wells for each treatment. Cell survival rate (%). DELTA.ODDrug treatment group/ΔODBlank groupX 100. The semi-Inhibitory Concentration (IC) of the cells was then calculated from the dose-response curve50) The results are shown in Table 1. The drugs in the experimental groups were as follows: experimental group 1 used nobiletin alone; experimental group 2 quercetin alone; experiment group 3 used naringin alone; experiment group 4 used composition 1 consisting of nobiletin, quercetin and naringin in a weight ratio of 3:2: 4; experiment group 5 used composition 2 consisting of hesperetin, quercetin and naringin in a weight ratio of 3:1: 3; experimental group 6 uses hesperetin, quercetin and naringin at a weight ratio of 2:1:3Composition 2 as prepared; experimental group 7 the pummelo peel extract prepared in example 1 was used; experimental group 8 the pummelo peel extract prepared in example 2 was used; experimental group 9 the pummelo peel extract prepared in example 3 was used.
TABLE 1 IC of drugs on human lung carcinoma cells A54950Test results
As can be seen from the experimental groups 1-6 in Table 1, the nobiletin, quercetin and naringin have certain anti-lung cancer effects when used independently, and the anti-lung cancer effects are more remarkable when the nobiletin, the quercetin and the naringin are mixed according to the proportion of the invention, which shows that the nobiletin, the quercetin and the naringin can generate a synergistic anti-lung cancer effect after being mixed. Therefore, the combination of nobiletin, quercetin and naringin can be used as anti-lung cancer medicine.
As can be seen from the experimental groups 7-9 in the table 1, the pummelo peel extract has a very significant anti-lung cancer effect, and the anti-lung cancer effect of the pummelo peel extract is stronger than that of any monomer in the pummelo peel extract, because the effective components of the pummelo peel extract, quercetin and naringin generate a synergistic anti-lung cancer effect.
Claims (8)
1. The pummelo peel extract is characterized by comprising the following effective components in percentage by weight: 20-30% of nobiletin; 10-20% of quercetin; 30-40% of naringin.
2. The method of producing an exocarpium Citri Grandis extract according to claim 1, comprising the steps of:
(1) extracting exocarpium Citri Grandis with ethanol under reflux, and concentrating the extractive solution to obtain exocarpium Citri Grandis ethanol extract;
(2) dissolving the ethanol extract of exocarpium Citri Grandis in water to obtain suspension, extracting with petroleum ether, discarding the petroleum ether extract, extracting with ethyl acetate, and concentrating the ethyl acetate extract to obtain exocarpium Citri Grandis extract;
(3) subjecting exocarpium Citri Grandis extract to silica gel column chromatography to enrich effective components to obtain exocarpium Citri Grandis extract;
the specific method for enriching the effective components by silica gel column chromatography in the step (3) comprises the following steps: putting the pummelo peel extract into a silica gel column, and eluting and removing impurities by using a mixed solvent system consisting of a solvent A and a solvent B in a volume ratio of 85: 15-80: 20; then, eluting by using a mixed solvent system consisting of a solvent A and a solvent B in a volume ratio of 75: 25-72: 28, and collecting eluted parts to obtain the effective parts of the pummelo peel; the solvent A is petroleum ether, and the solvent B is ethyl acetate;
the dosage of a mixed solvent system consisting of the solvent A and the solvent B in a volume ratio of 85: 15-80: 20 is 1-3 times of the volume of the silica gel column; the dosage of a mixed solvent system consisting of the solvent A and the solvent B in a volume ratio of 75: 25-72: 28 is 3-5 times of the volume of the silica gel column.
3. The preparation method according to claim 2, wherein the specific method for enriching the effective components by silica gel column chromatography in the step (3) comprises the following steps: putting the pummelo peel extract on a silica gel column, and firstly, adding a solvent A: eluting a mixed solvent system consisting of the solvent B to remove impurities; then adding solvent A with volume ratio of 72: 28: eluting with a mixed solvent system consisting of the solvent B and collecting the eluted part to obtain the effective part of the pummelo peel;
the dosage of a mixed solvent system consisting of the solvent A and the solvent B with the volume ratio of 80:20 is 3 times of the volume of the silica gel column; the dosage of the mixed solvent system consisting of the solvent A and the solvent B with the volume ratio of 72:28 is 5 times of the volume of the silica gel column.
4. The preparation method according to claim 2, wherein the weight of the silica gel used in the silica gel column in the step (3) is 20 to 40 times of the dry weight of the exocarpium Citri Grandis extract.
5. The preparation method according to claim 2, wherein the ethanol in the step (1) is an aqueous ethanol solution with a volume fraction of 80-100%; the ratio of the amount of the ethanol to the amount of the pummelo peel is 8-15 mL:1 g; the heating reflux extraction time is 1-3 h; and (3) extracting for 1-3 times by using petroleum ether with the same volume as the suspension in the step (2), discarding the petroleum ether extract, extracting for 1-3 times by using ethyl acetate with the same volume as the suspension, and concentrating the ethyl acetate extract to obtain the pummelo peel extract.
6. Use of an exocarpium Citri Grandis extract of claim 1 in the preparation of a medicament for the treatment of APE 1-mediated diseases; the APE1 mediated disease is APE1 mediated lung cancer.
7. The use of the exocarpium Citri Grandis extract of claim 1 in the preparation of anticancer drug, wherein the anticancer drug is anti-lung cancer drug.
8. An anticancer pharmaceutical composition derived from pummelo peel is characterized by comprising nobiletin, quercetin and naringin, wherein the weight ratio of the nobiletin to the quercetin to the naringin is (2-3): 1-2: 3-4; the anti-cancer drug is an anti-lung cancer drug.
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| CN106728205A (en) * | 2016-12-29 | 2017-05-31 | 中山大学 | Application of the Exocarpium Citri Grandis extract in preventing and treating PM2.5 particle inducing chronic obstructive disease of lung acute attack medicines are prepared |
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| CN106728205A (en) * | 2016-12-29 | 2017-05-31 | 中山大学 | Application of the Exocarpium Citri Grandis extract in preventing and treating PM2.5 particle inducing chronic obstructive disease of lung acute attack medicines are prepared |
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