CN108771671A - A kind of composition and the preparation method and application thereof with anti-cutaneum carcinoma effect - Google Patents

A kind of composition and the preparation method and application thereof with anti-cutaneum carcinoma effect Download PDF

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CN108771671A
CN108771671A CN201810469779.XA CN201810469779A CN108771671A CN 108771671 A CN108771671 A CN 108771671A CN 201810469779 A CN201810469779 A CN 201810469779A CN 108771671 A CN108771671 A CN 108771671A
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composition
licochalcone
neoglycyrol
preparation
licoisoflavanone
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CN108771671B (en
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郑云枫
陆睿寅
彭国平
杨敏
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Nanjing University of Chinese Medicine
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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
    • A61K31/33—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 
    • 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
    • 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
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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Abstract

The invention discloses a kind of compositions and preparation method thereof with anti-cutaneum carcinoma effect, and the composition is by 1~2 parts by weight of licoisoflavanone B, 1~2 parts by weight of neoglycyrol, 2~4 parts of compositions of Licochalcone A.Wherein licoisoflavanone B can promote the differentiation of skin cancer cell with neoglycyrol by P38MAPKs accesses, PI3K/AKT accesses respectively, inhibit the proliferation of skin cancer cell, and Licochalcone A can be by the apoptosis of PARP access induced skin cancer cells, to reach the growth for inhibiting skin cancer cell.The present invention by many experiments screen to obtain optimum amount than 3 monomer isopentene group flavonoid compounds composition, the composition can be cooperateed with by multiple target point, it is obviously improved and inhibits skin cancer cell growth, have the function of good prevention and treatment cutaneum carcinoma, there is good development prospect.

Description

A kind of composition and the preparation method and application thereof with anti-cutaneum carcinoma effect
Technical field
The invention belongs to Chinese medicine and natural medicine technical fields, and in particular to a kind of composition with anti-cutaneum carcinoma effect And preparation method and purposes.
Background technology
Cutaneum carcinoma is a kind of common malignant tumour, and incidence is in ascendant trend year by year, at present still without effective treatment Drug.Cutaneum carcinoma is broadly divided into squamous cell carcinoma, malignant mela noma, syringocarcinoma, angiosarcoma etc., and wherein melanoma is most Dangerous cutaneum carcinoma, it can spread with blood, lymph, if melanoma has been spread when treatment, mortality risk is high, most of skin The lethal case of skin cancer is caused by melanoma.Some researches show that inhibit the proliferation of skin cancer cell in addition to that can lead in the recent period The common mechanisms such as promotion cancer cell-apoptosis, autophagy are crossed to play a role outside, it can also be by promoting cancer cell to break up to normal cell And play and inhibit its proliferation, it is final to play prevention or therapeutic effect.
Radix Glycyrrhizae has been widely used in medicine, food, makeup as a kind of excellent natural plant resource and Chinese medicine The fields such as product, herding.Licorice medicinal materials are mainly carried using water at present or buck extraction method produces extract of licorice root, glycyrrhizic acid crude extract We have discovered that, in Radix Glycyrrhizae other than moderately polar reactive compound, there is also abundant low pole Huangs Deng, early period Ketone active material has antioxidant activity (Fang Shiqi, cold health, section gold storehouse for grain, etc., Li Cunyu, Wei Juanhua, Zheng Yunfeng, Peng Guoping.It is sweet Straw colour ketones component and its Study on Its Antioxidant Activity in Vitro [J] Chinese patent drugs, 2015,37 (11):2443-2448.).But about The activity of the anti-cutaneum carcinoma of licoflavone constituents is less.
Invention content
Goal of the invention：In order to preferably develop and use Licorice, extraction of the present invention to low pole general flavone in Radix Glycyrrhizae Process for refining optimizes, and is found in screening active ingredients research process, and Radix Glycyrrhizae low pole total flavone part has good suppression The effect of black cancer cell (B16-F10, A375, A2058) growth processed.Further screening bioactive compounds the study found that its In 3 tool prenyl different type flavone component-licoisoflavanone B (isoflavones), neoglycyrol (cumarin) and Licochalcone A (chalcone) is its main active substances, and study on mechanism shows that this 3 main actives can be distinguished It is played a role by different approach (Carcinoma cell differentiation Huo zhou is promoted to die).The research of medical instrument multicomponent multiple target point is thought according in Road, the present invention carry out a large amount of screening weight Experiment of Compatibility according to different weight ratio with this 3 main actives, filter out most Good dosage, and have effects that significantly to cooperate with the composition for inhibiting skin cancer cell.
Technical solution：In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is as follows：
A kind of composition with anti-cutaneum carcinoma effect, the composition includes licoisoflavanone B, and neoglycyrol and Radix Glycyrrhizae are looked into Ear ketone A；
Preferably, a kind of above-described composition with anti-cutaneum carcinoma effect, the composition are different by Radix Glycyrrhizae Flavones B1 parts by weight, 1~3 parts by weight of neoglycyrol and 1~3 parts by weight of Licochalcone A composition.
Preferably, above-described a kind of composition with anti-cutaneum carcinoma effect, the composition is by Radix Glycyrrhizae Isoflavones B1 parts by weight, 2 parts by weight of neoglycyrol, 2 parts by weight of Licochalcone A composition.
A kind of composition with anti-cutaneum carcinoma effect of the present invention, the licoisoflavanone B, neoglycyrol are sweet The preparation method of careless chalcone A is：
(1) extracting liquorice is extracted using ethyl alcohol, and extracting solution is recovered under reduced pressure, and it is 1.20~1.35 thick pastes to be concentrated into relative density；
(2) take thick paste, adsorbent be added, mixes thoroughly, dry, pulverize, be fitted into supercritical extract axe, with extracting pressure 10~ 30MPa, 45~55 DEG C of extraction temperature, entrainer are ethyl alcohol, and entrainer dosage is 5%~10%, 4~10MPa of separating pressure, is divided 30~40 DEG C from axe temperature, extraction time is 1~3 hour, collects extract liquor from separation axe, recycles ethyl alcohol, obtain overcritical essence Position processed；
(3) the super critical refined position of gained is adsorbed through ODS reverse phase silica gel chromatographic columns, with 60%~80% ethyl alcohol of volumetric concentration Aqueous solution elutes, and separation obtains licoisoflavanone B, neoglycyrol and Licochalcone A.
Preferably, above-described a kind of composition with anti-cutaneum carcinoma effect, the licoisoflavanone B, The preparation method of neoglycyrol and Licochalcone A is：
(1) extracting liquorice adds 95% ethyl alcohol of volumetric concentration to extract, extracting solution is recovered under reduced pressure, and it is 1.28 to be concentrated into relative density Thick paste；
(2) thick paste is taken, the silica gel of 3 times of weight is added, mixes thoroughly, dry, pulverize, be fitted into supercritical extract axe, with extraction Pressure 20MPa, 55 DEG C of extraction temperature, entrainer are ethyl alcohol, and entrainer dosage is 15%, separating pressure 6MPa, detaches axe temperature 30 DEG C, extraction time 2 hours collects extract liquor from separation axe, recycles ethyl alcohol, dry, obtains super critical refined position；
(3) the super critical refined position ODS reverse phase silica gel chromatographic columns absorption of gained, with volumetric concentration for 60%~80% ethyl alcohol Aqueous solution elutes, and separation obtains licoisoflavanone B, neoglycyrol and Licochalcone A.
A kind of pharmaceutical preparation, including claims 1 to 3 any one of them have the composition of anti-cutaneum carcinoma effect, institute The preparation stated is external preparation or oral preparation.
Above-described pharmaceutical preparation, the external preparation are one kind in outer ointment, gelling agent or liniment；It is described Oral preparation be tablet, one kind in capsule or pill.
Advantageous effect：The present invention has the following advantages that compared with prior art：
(1) present invention is carried out by many experiments according to having the different compounds for inhibiting skin cancer cell mechanism of action Combined sorting, illustrated and proved and obtained with a variety of skin cancer cell strain models.Mtt assay is used first from Radix Glycyrrhizae The ingredient for having notable growth inhibitory activity to skin cancer cell (B16-F10 cell strains) has been filtered out in general flavone, and has been found that These reactive compounds can be played a role by different approaches, i.e., licoisoflavanone B passes through respectively with neoglycyrol P38MAPKs accesses, PI3K/AKT accesses promote the differentiation of skin cancer cell, it is suppressed that the proliferation of skin cancer cell, and Radix Glycyrrhizae Chalcone A can be by PARP accesses induction of the apoptosis of skin cancer cell, to reach the growth for inhibiting skin cancer cell. Compatibility combined sorting further is carried out with these three reactive compounds, is finally obtained the composition of optimization, the combination filtered out Object shows activity advantage more apparent than one-component, plays the unexpected technique effect of good synergy！
(2) " the alcohol extracting-supercritical CO that preparation method of the invention uses2Filling extraction combination " technique prepares sweet Careless low pole general flavone, then with reversed phase chromatography separation, obtain licoisoflavanone B, neoglycyrol and Licochalcone A monomer at Point, efficiency obtains great promotion, and toxic organic solvents are not used in technique, with conventional solvent extraction, resin spirit Preparation method, which is compared, has apparent advantage.
Advantageous effect for a better understanding of the present invention, below using corresponding experiment as further instruction.
A. SFE-CO2 technologic study is studied
Purpose：Using general flavone content as index, the supercritical CO of Radix Glycyrrhizae ethanol extract is investigated using orthogonal experiment2Extraction Take process for refining.
1. instrument and material：
Supercritical CO2Extraction equipment and extraction gas：(Nantong flies FY230-40-11 types supercritical fluid extraction equipment Space petroleum science and technology development corporation, Ltd.)；CO2Gas is food-grade (Nanjing Tong Guang special gas factory)；Ultraviolet specrophotometer.
Take dry Radix Glycyrrhizae (i.e. with water boiling and extraction twice after medicinal material, drying) 10kg, add ethyl alcohol extract 2 times (8+6 times Amount), merge, filtering is recovered under reduced pressure ethyl alcohol, is concentrated into thick paste (relative density 1.28), obtains 967g, and 100-200 mesh silicon is added Glue 3000g, mixing, 60 DEG C of drying crush to get the refined raw material of supercritical extract.
2. method and result
Sample is taken, supercritical CO is packed into2Extractor (volume 1L), the extraction of according to the form below conditional, remaining extraction time are 2h, Separating pressure is 6Mpa, and separation temperature is 40 DEG C.When in view of means of supercritical extraction, extracting pressure, temperature these three factors are the most Key, therefore select L9(34) orthogonal arrage carried out 9 experiments using general flavone as inspection target, preferred optimum extraction condition, respectively Factor level see the table below 1.
1 supercritical CO of table2Extraction factor water-glass
According to table 1, if experimental factor level is A1B1C1, that is, indicate the tested number with extracting pressure 10Mpa, extraction temperature 35 DEG C, entrainer dosage is 0, extraction time 2h, and parsing pressure and temperature is respectively 6Mpa and 40 DEG C of collection extract, freezing It is dry, detect general flavone content.The rest may be inferred for remaining.
2 supercritical CO of table2Extract orthogonal arrage
The selection of 2.2 indexs
This orthogonal test selects general flavone content as its performance assessment criteria.Method is as follows：
The preparation of reference substance solution：It is appropriate to weigh control substance of Rutin, adds 60% ethanol solution, prepares reed containing 0.08mg/mL Fourth reference substance solution, then it is diluted to 0.04 successively, 0.02,0.01mg/mL control series product solution.
The preparation of sample liquid：Sample about 5mg is weighed, it is accurately weighed, it sets in 50mL measuring bottles, adds the dissolving of 60% ethanol solution simultaneously Be settled to scale to get.
It takes rutin control series product solution, each 5mL of sample solution to set in 10mL measuring bottles respectively, adds 5% sodium nitrite solution 0.3mL shakes up, and places 6min, adds 10% pin acid aluminum solutions 0.3mL, shake up, and stands 6min, and it is molten that 1mol/L sodium hydroxides are added Liquid 4mL, shakes up, and with 30% ethyl alcohol constant volume, stands 15min, suction degree degree is measured at wavelength 510nm, is vertical sit with absorbance A Mark, concentration C is abscissa, draws standard curve, and calculate general flavone content in sample.
2.3 results and analysis
According to orthogonal array, 9 experiments are carried out altogether, and correlation test data and its calculating see the table below 3.
3 supercritical CO of table2Extract orthogonal experiments table
4 supercritical CO of table2Extract orthogonal analysis of variance table
Soruces of variation Quadratic sum Degree of freedom It is square F Conspicuousness
Extracting pressure 1589.29 2 794.64 33.52 Significantly
Extraction temperature 124.46 2 62.23 2.63 Not significantly
Entrainer 845.01 2 422.50 17.82 Not significantly
Error 47.41 2 23.70
Extraction temperature influences effect of extracting with entrainer not notable it can be seen from the variance analysis of table 4, and extracts Pressure has a significant impact to effect of extracting.
The K it can be seen from the simple computation of table 32a>K3a>K1a, but K2aClose to K3a, therefore factor A selects level 2 for most Good, i.e., best extracting pressure is 20Mpa；Although K3b>K2b>K1b, but three is not much different, therefore extraction temperature is selected as in 35- Between 45 DEG C；K2c>K3c>K1c, but K2aClose to K3a, factor C selection levels 2, i.e. entrainer Optimum is 15%.
From the point of view of orthogonal experiments, supercritical extraction process may be selected to be：With 10~30MPa of extracting pressure, extraction temperature 35~45 DEG C of degree, entrainer dosage are 5%~30%, 4~10MPa of separating pressure, 30~40 DEG C of separation axe temperature, extraction time 1~3 hour, extract liquor is collected from separation axe, recycling design, freeze-drying to get.
Best extraction process is：With extracting pressure 20MPa, 55 DEG C of extraction temperature, entrainer dosage is 15%, separating pressure 6MPa, 30 DEG C of separation axe temperature, extraction time 2 hours collect extract liquor from separation axe, recycling design, freeze-drying to get Radix Glycyrrhizae supercritical extract.
B. chemical constitution study in Radix Glycyrrhizae supercritical extract
(1) experiment material and instrument
Ultraviolet spectra is measured with Japanese Shimadzu UV-2401 type UV-vis spectroscopy degree analyzer.Nuclear magnetic resoance spectrum is used BrukerACF-300 types, BrukerASR-500 type nmr determinations.Mass spectrum is by Agilent1100HPLC/TOF-MS matter Spectrometer measures.
Liquid phase analysis Agilent1100, VWD detector, AgilentLC chromatographic work stations.Chromatographic column Chinese nation science and technology Co., Ltd's ThermoC18 columns (5 μm, C18,250 × 4.6mm).C18 medium pressure column chromatographies are used BUCHIChromatographyPumpB-688 is pumped, and filler is C18 (25-50 μm), and acetonitrile dispenses chromatographically pure (MERCK) with import Reagent, ethyl alcohol are chromatographically pure (Huaiyin fine chemistry industry institute), and water is sub-boiling distillation water, remaining reagent is that analysis is pure.
Licorice medicinal materials are purchased from Gansu.
(2) high-efficient liquid phase chromatogram condition：
With ThermoC18Column (4.6 × 50mm, 5 μm) is chromatographic column, and Agilent1100 is efficient liquid phase chromatographic analysis Instrument, UV UV detector, flow velocity 1.0mL/min, sampling volume are 10 μ L, Detection wavelength 254nm, mobile phase actual conditions Such as the following table 5：
5 condition of gradient elution of table
Time (min) Acetonitrile (%) 0.2% formic acid (%)
0 18 82
10 25 75
30 45 55
60 70 30
(3) extraction separation
Licorice medicinal materials are concentrated under reduced pressure into thick paste with 95% ethyl alcohol heating and refluxing extraction, with silica gel mixed sample (mass ratio 1: 1) the super critical refined object of Radix Glycyrrhizae, is prepared by above overcritical optimization rear method, then through compression leg chromatography in C18, with 50% The ethanol water of (4BV), 60% (4BV), 70% (4BV) carry out gradient elution, are detected through HPLC, merge the stream containing similar component Part, tetra- fractions of Fra.A, Fra.B, Fra.C, Fra.D are respectively obtained, each fraction distinguishes again upper C18 columns：Fra.A fractions with 50%~60% ethanol water carries out gradient elution, and separation obtains GF-5, GF-7；Fra.B fractions with 55%~65% ethanol water into Row gradient elution, separation obtain compound GF-3, GF-8, GF-9；Fra.C fractions carry out gradient with 60%~65% ethanol water and wash De-, separation obtains compound GF-6, GF-10；Fra.D fractions carry out gradient elution with 65%~70% ethanol water, and separation obtains Compound GF-1, GF-2, GF-4.
(4) Structural Identification
LC-HRTOF-MS testing results：GF-1：m/z353.1024[M+H]+, GF-2：m/z337.1071[M+H]+, chemical combination Object 3：m/z353.1012[M+H]+, GF-4：m/z367.1187[M+H]+, GF-5：m/z269.0808[M+H]+, GF-6：m/ z339.1590[M+H]+, GF-7：m/z257.0809[M+H]+, GF-8：m/z257.0805[M+H]+, GF-9：m/z323.1278 [M+H]+, GF-10：m/z315.0866[M+H]+；13CNMR measurement results (see the table below 6).
6 GF 1-10's of table13C NMR(DMSO-d6)
NO. 1 2 3 4 5 6 7 8 9 10
C-1 113.5 125.7
C-2 155.7 155.1 162.5 157.9 153.0 158.2 78.9 131.1 162.2 155.8
C-3 120.5 122.1 104.4 102.7 124.2 99.9 43.1 115.8 104.4 137.7
C-4 180.6 176.0 176.2 158.6 174.5 159.6 190.0 160.3 176.2 178.0
C-4a 104.6 116.2 16.1 100.2 127.2 113.5 115.8 105.1
C-5 161.8 127.2 126.4 154.3 116.6 126.5 128.3 115.8 124.5 160.9
C-6 98.8 115.3 114.7 120.1 115.0 127.7 110.5 131.1 127.2 97.6
C-7 164.1 162.8 160.6 160.0 162.5 164.6 160.3 165.0
C-8 93.6 102.0 102.4 99.7 102.0 102.5 101.7 92.2
C-8a 157.7 157.5 157.3 153.4 158.9 163.1 155.6 160.2
C-α 117.8 144.2
C-β 138.7 117.4
C-γ 187.3 191.5
C-1' 111.2 112.7 121.7 114.7 123.1 129.6 129.3 112.9 121.9 120.4
C-2' 151.2 151.3 128.0 156.6 130.0 130.6 128.2 165.8 127.9 130.1
C-3' 109.7 110.1 115.8 99.0 113.5 115.2 115.1 102.5 115.8 115.6
C-4' 153.6 153.5 162.4 157.4 157.4 161.6 157.6 165.4 160.5 156.2
C-5' 107.4 107.6 115.8 114.5 113.5 115.2 115.1 108.0 115.8 115.6
C-6' 131.3 130.9 128.0 120.9 130.0 130.6 128.2 132.7 127.9 130.1
C-1” 22.5 39.5 27.5
C-2” 75.4 75.4 122.9 147.5 121.8
C-3” 128.8 128.8 131.4 109.9 132.3
C-4” 116.9 117.0 18.2 26.9 25.5
C-5” 27.4 27.5 25.9 26.9 17.6
C-6” 27.4 27.5
2-OCH3 55.4
3-OCH3 56.0
4'-OCH3 55.1
5-OCH3 62.8
7-OCH3 59.6
It is compareed according to the above measurement result, and with data in literature, authenticating compound 1-10 is respectively：Licoisoflavanone B, light Licoricon, 7,4'- dihydroxyflavones, neoglycyrol, onocerin, Licochalcone A, glycyrrhizin, isoliquiritigenin, Radix Glycyrrhizae are yellow Ketone, kumatakenin A.
C. anti-cutaneum carcinoma screening active ingredients
1. experiment material
(1) by test product：Compound the 1-10 isolated above b, i.e.,：Licoisoflavanone B, glabrone, 7,4'- dihydroxies Base flavones, neoglycyrol, onocerin, Licochalcone A, glycyrrhizin, isoliquiritigenin, licoflavone, kumatakenin A.
(2) experimental cell strain and source：Skin cancer cell B16-F10 (is purchased from ATCC)
(3) experiment reagent DMEM culture mediums, fetal calf serum：Gibco；Methyl- azoles salt (MTT), dimethyl sulfoxide (DMSO) (DMSO), NaHCO3：Shanghai Aladdin biochemical technology limited liability company；Microplate reader：U.S. Thermo；96 hole cell culture Plate.
2. method and result
Medicine preparation：Above each compound 1~10 is weighed, is dissolved with DMSO, is made into the mother liquor of a concentration of 80mM, -20 DEG C It preserves.80 μM, 60 μM, 40 μM, 20 μM, 10 μM are diluted to corresponding culture solution before use to be tested.
Administration is handled：The cell of logarithmic growth phase adjusts cell density appropriate, is inoculated in 96 orifice plates, 7.5 × 103Cells/well is incubated at 37 DEG C, 5%CO2Incubator in, culture for 24 hours after dosing, act on 48h.Set up separately blank group, to Medicine group and positive controls, every group sets 3 multiple holes, repeats experiment 3 times.
MTT is measured：Cell survival rate, which measures, uses MTT analytic approach, and being metabolized reduction tetramethyl azo azoles salt with living cells is Principle measures the OD values that go out of 492nm using microplate reader, the size of OD values and reflects influence of the drug to cell survival rate.Carefully Born of the same parents' inhibiting rate %=(1- dosing groups OD values/control group OD values) × 100%
Cell differentiation rate：Inverted microscope (100 times ×), observation administration treated each group cell, counts 150/group, The cell number of cell body dendron length >=3 cell length is counted, and calculates cell differentiation rate (%).
IC50Computational methods：The parameters such as each dosage and inhibiting rate nonlinear regression and fitting is calculated into IC50.Such as the following table 7.
Table 7GF1-10 acts on (n=3) to B16-F10 skin cancer cell Inhibit proliferatons
Compound IC50(μM) Compound IC50(μM)
Licoisoflavanone B (1) 16.0±3.2 Licochalcone A (6) 18.1±2.9
Glabrone (2) 45.8±3.3 Glycyrrhizin (7) >80
7,4'- dihydroxyflavones (3) >80 Isoliquiritigenin (8) >80
Neoglycyrol (4) 17.5±3.7 Licoflavone (9) 42.4±3.4
Onocerin (5) >80 Kumatakenin A (10) >80
The results show that compound licoisoflavanone B (GF-1), neoglycyrol (GF-4) and Licochalcone A (GF-6) tool There is stronger activity, has the function of that it is significantly inhibited to be proliferated to B16-F10 skin cancer cells, IC50<20μM；Chemical combination object light Licoricon and licoflavone activity are relatively weak, and other ingredients then fail to show corresponding activity.
D. licoisoflavanone B, neoglycyrol and Licochalcone A inhibit skin cancer cell proliferation function Mechanism Study
1. experiment material
(1) by test product：Licoisoflavanone B (GF-1), neoglycyrol (GF-4) and Licochalcone A (GF-6), self-control, HPLC purity>98%.
(2) experimental cell strain and source：Skin cancer cell B16-F10 (is purchased from ATCC)
(3) experiment reagent DMEM culture mediums, fetal calf serum：Gibco；Methyl- azoles salt (MTT), dimethyl sulfoxide (DMSO) (DMSO), NaHCO3：Shanghai Aladdin biochemical technology limited liability company；Microplate reader：U.S. Thermo；100mm cell culture Ware.Antibody anti-GAPDH (1:5000),anti-p38(1:2500),anti-AKT(1:2500),anti-P-CREB(1: 1000),anti-MITF(1:200),anti-PI3K(P85)(1:1000),anti-Cl-PARP(1:1000)、anti-Cl- Casp9(1:1000),anti-Cl-Casp3(1:1000) it (purchases in CellSignalingTechnology, Danvers, MA)
2. method and result
Medicine preparation：Each compound is weighed, is dissolved with DMSO, the mother liquor of a concentration of 80mM, -20 DEG C of preservations are made into.Face use It is preceding to be diluted to 40 μM, 20 μM with corresponding culture solution and tested.
Administration is handled：The cell of logarithmic growth phase adjusts cell density appropriate, is inoculated in 100mm culture dishes, 1 ×106Cells/well is incubated at 37 DEG C, 5%CO2Incubator in, culture for 24 hours after dosing, act on 48h.Set up separately blank group, Administration group repeats experiment 3 times.After processing, twice by cells rinsed with PBS, RIPA lysis buffers (150mMNaCl, 1mMEDTA, 1mMEGTA, 50mMTris-Cl, 0.35%w/v, 1- NaTDC, 1mM phenylmethylsulfonyl fluorides, 1%v/ VNP-40,1mMNaF, 1mMNa3VO4, pH7.4) in cracked 30 minutes in 4 DEG C, collect lysate, 30 points centrifuged under 15,000g Clock.Take supernatant, -20 DEG C of preservations.
Westernblot is measured in each group cell pyrolysis liquid with cell differentiation and the relevant antibody of proliferation, and is used Imagelab softwares (ThermoScientific) make reaction band visualize, and then pass through Chemidoc imaging systems (Bio- Rad；Hercules, CA) calibration.
Such as Fig. 1 the results show that licoisoflavanone B (GF-1), neoglycyrol (GF-4) and Licochalcone A (GF-6) although It can inhibit the proliferation of B6-F10 cancer cells, but its mechanism of action and differ：Licoisoflavanone B and neoglycyrol can be distinguished By raising p-p38 and p-CREB albumen and lowering P-AKT, PI3K albumen, downstream differentiation GAP-associated protein GAP MITF is finally promoted Expression, prompt the two compounds can by promote skin cancer cell differentiation by play inhibit tumour cell growth.And it is sweet Careless chalcone A shows the mechanism of action different from other two compounds, mainly can be by inducing Cl-PARP/Cl- The apoptosis-related proteins such as Casp3/Cl-Casp9 make cancer cell that apoptosis occur and generate the effect for inhibiting growth.
E. licoisoflavanone B, neoglycyrol and the research of Licochalcone A composition optimization blends
1. experiment material
(1) by test product：Licoisoflavanone B (GF-1), neoglycyrol (GF-4) and Licochalcone A (GF-6) composition.
(2) experimental cell strain and source：Skin cancer cell B16-F10, A375, A2058 (are purchased from ATCC)
(3) experiment reagent DMEM culture mediums, fetal calf serum：Gibco；Methyl- azoles salt (MTT), dimethyl sulfoxide (DMSO) (DMSO), NaHCO3：Shanghai Aladdin biochemical technology limited liability company；Microplate reader：U.S. Thermo；96 hole cell culture Plate.
2. method and result
Medicine preparation：The sample for weighing the combination of according to the form below weight ratio, is dissolved with DMSO, is made into the mother of a concentration of 50mM Liquid, -20 DEG C of preservations.50 μM, 25 μM, 10 μM, 5 μM, 1 μM are diluted to corresponding culture solution before use to be tested.
Administration is handled：The cell of logarithmic growth phase adjusts cell density appropriate, is inoculated in 96 orifice plates, 7.5 × 103Cells/well is incubated at 37 DEG C, 5%CO2Incubator in, culture for 24 hours after dosing, act on 48h.Set up separately blank group, to Medicine group and positive controls, every group sets 3 multiple holes, repeats experiment 3 times.
MTT is measured：Cell survival rate, which measures, uses MTT analytic approach, and being metabolized reduction tetramethyl azo azoles salt with living cells is Principle measures the OD values that go out of 492nm using microplate reader, the size of OD values and reflects influence of the drug to cell survival rate.Carefully Born of the same parents' inhibiting rate %=(1- dosing groups OD values/control group OD values) × 100%.
IC50Computational methods：The parameters such as each dosage and inhibiting rate nonlinear regression and fitting is calculated into IC50.Such as 8 institute of table Show.
Three kinds of skin cancer cell Inhibit proliferaton effects (n=3) of composition pair of 8 different weight ratio of table
The results show that in B16-F10, on tri- kinds of skin cancer cell models of A375, A2058, the above different proportion Radix Glycyrrhizae is different The composition of flavones B (GF-1), neoglycyrol (GF-4) and Licochalcone A (GF-6), all show more excellent than one-component Inhibition growth of tumour cell effect.Especially looked into licoisoflavanone B1 parts by weight, neoglycyrol 1-3 parts by weight and Radix Glycyrrhizae The composition formed when ear ketone A1-3 parts by weight, compatibility synergistic effect is stronger, to the IC of three kinds of skin cancer cells50<10μM；? In various compositions, particularly preferred licoisoflavanone B1 parts by weight, 2 parts by weight of 2 parts by weight of neoglycyrol and Licochalcone A When, the activity of the composition is most strong, to B16-F10, A375, the IC of A2058 skin cancer cell models50Respectively 3.3 μM, 3.7 μ M and 4.2 μM.Show apparent unexpected technique effect！
Description of the drawings
Fig. 1 is that licoisoflavanone B (GF-1), neoglycyrol (GF-4) and Licochalcone A (GF-6) pass through unlike signal Regulatory pathway inhibits B6-F10 skin cancer cell proliferation function result figures.
Specific implementation mode
Form by the following examples is described in further detail the above of the present invention again, but should not be by this The range for being interpreted as the above-mentioned theme of the present invention is only limitted to example below, and all technologies realized based on the above of the present invention are equal Belong to the scope of the present invention.
It is prepared by the separation of 1 anti-skin chemical compound for treating cancer of embodiment
(1) extracting liquorice 2kg is extracted with ethyl alcohol, is extracted 2 times (8+6 times is measured), each 2h, is merged extracting solution, is recovered under reduced pressure, And it is 1.28 thick paste 145g to be concentrated into relative density；
(2) thick paste is taken, 450g silica gel absorbers (100-200 mesh) are added, mixes thoroughly, dry, pulverize, is packed into supercritical extract In axe, with extracting pressure 20MPa, 55 DEG C of extraction temperature, entrainer ethanol consumption is 15%, separating pressure 6MPa, detaches axe temperature 40 DEG C of degree, extraction time are 2 hours, and extract liquor is collected from separation axe, recycle ethyl alcohol, freeze-drying, i.e., Radix Glycyrrhizae is overcritical carries Take object, total 28.4g.
(3) extracting liquorice supercritical extract adds a small amount of methanol to dissolve, upper C18Middle compression leg chromatography (1000g, 25-50 μm, 6.0 × 80cm), with EtOH-H2O(50:50；60:40,70:30) gradient elution, each concentration elute 6L, and 1 is collected as per 0.5L Fraction, HPLC-UV tracing detections merge according to testing result：A is fraction 16-20 (containing Licochalcone A) 3.2g, and B is stream Part 24-26 (containing licoisoflavanone B) 2.9g, C are fraction 28-30 (containing neoglycyrol) 3.5g；
(4) after fraction A recycling concentration, then through 60%~65% ethanol water progress gradient elution, 1 is collected as per 200mL Fraction, HPLC-UV tracing detections collect purity>90% fraction merges, and recycles, recrystallization, obtains compound Radix Glycyrrhizae and looks into ear Ketone A0.43g；After fraction B recycling concentrations, then through 65%~70% ethanol water progress gradient elution, 1 stream is collected as per 200mL Part, HPLC-UV tracing detections collect purity>90% fraction merges, and recycles, and recrystallization obtains compound licoisoflavanone B0.26g；After fraction C recycling concentrations, then through 65%~70% ethanol water progress gradient elution, it is collected as 1 fraction per 200mL, HPLC-UV tracing detections collect purity>90% fraction merges, and recycles, and recrystallization obtains compound neoglycyrol 0.38g.
It is prepared by 2 anti-skin chemical compound for treating cancer of embodiment
(1) extracting liquorice 5kg is extracted with ethyl alcohol, is extracted 2 times (10+8 times is measured), each 2h, is merged extracting solution, is recovered under reduced pressure, And it is 120 thick paste 145g to be concentrated into relative density；
(2) thick paste is taken, 850g diatomite adsorbants are added, mixes thoroughly, dry, pulverize, be fitted into supercritical extract axe, with extraction Pressure power 10MPa, 45 DEG C of extraction temperature, entrainer dosage are 30%, separating pressure 8MPa, 30 DEG C of separation axe temperature, when extraction Between be 3 hours, collect extract liquor from separation axe, recycle ethyl alcohol, freeze-drying, i.e. Radix Glycyrrhizae supercritical extract, total 67.4g.
(3) extracting liquorice supercritical extract adds a small amount of methanol to dissolve, upper C18Middle compression leg chromatography (1500g, 25-50 μm, 8.0 × 80cm), with EtOH-H2O(65:35) 20L is eluted, 1 fraction, HPLC-UV tracing detections, according to detection are collected as per 0.5L As a result merge：I is fraction 12-15 (containing Licochalcone A) 10.2g, and II is fraction 21-24 (containing licoisoflavanone B) 12.9g, III is fraction 29-31 (containing neoglycyrol) 13.2g；
(4) it after the recycling of fraction I concentration, then is eluted through 60% ethanol water, 1 fraction, HPLC- is collected as per 500mL UV tracing detections collect purity>90% fraction merges, and recycles, and recrystallization obtains compound Licochalcone A 0.91g；Stream It after part II recycling concentration, then is eluted through 65% ethanol water, 1 fraction is collected as per 200mL, HPLC-UV tracing detections are received Collect purity>90% fraction merges, and recycles, and recrystallization obtains compound licoisoflavanone B0.64g；The recycling concentration of fraction III Afterwards, then through 70% ethanol water it is eluted, 1 fraction is collected as per 200mL, HPLC-UV tracing detections collect purity>90% Fraction, merge, recycle, recrystallization obtains compound neoglycyrol 0.87g
The preparation of 3 anti-cutaneum carcinoma ointment of embodiment
Prescription：
Preparation process：Above-mentioned weight is weighed into licoisoflavanone B, neoglycyrol, Licochalcone A with a small amount of 1,2- the third two Alcohol dissolves, and adds in the stearic acid of heating and melting, then adds atoleine, mixing, is heated to 70 DEG C, standby as oil phase With；It by remaining 1,2-PD and deionized water mixing, then adds in Tween 80, is sufficiently stirred, and be heated to 75 DEG C, make It is spare for water phase；Finally oil phase is slowly added into water phase, it is stirring while adding, until condensation is to get anti-cutaneum carcinoma ointment.
The preparation of 4 anti-cutaneum carcinoma gelling agent of embodiment
Prescription：
Above-mentioned weight is weighed into licoisoflavanone B, neoglycyrol, Licochalcone A and glycerine mixed dissolution, adds card Wave nurse, hyaluronic acid and methyl p-hydroxybenzoate, mixing are add to deionized water under homogeneous, are uniformly mixed, are cooled to 40-50 DEG C to get to anti-cutaneum carcinoma gelling agent.
The preparation of 5 anti-cutaneum carcinoma capsule of embodiment
Prescription：
Above-mentioned weight is weighed into licoisoflavanone B, neoglycyrol, Licochalcone A and starch, dextrin mixing, is pelletized, then Jia Ru Austria crystalline celluloses, mixing, filling capsule to get.

Claims (8)

1. a kind of composition with anti-cutaneum carcinoma effect, which is characterized in that the composition includes licoisoflavanone B, new Radix Glycyrrhizae Phenol and Licochalcone A；
2. a kind of composition with anti-cutaneum carcinoma effect according to claim 1, which is characterized in that the composition is by sweet Careless 1 parts by weight of isoflavones B, 1~3 parts by weight of neoglycyrol and 1~3 parts by weight of Licochalcone A composition.
3. a kind of composition with anti-cutaneum carcinoma effect according to claim 2, which is characterized in that the composition by 1 parts by weight of licoisoflavanone B, 2 parts by weight of neoglycyrol, 2 parts by weight of Licochalcone A composition.
4. a kind of composition with anti-cutaneum carcinoma effect according to any one of claims 1 to 3, which is characterized in that institute Licoisoflavanone B is stated, the preparation method of neoglycyrol, Licochalcone A is：
(1) extracting liquorice is extracted using ethyl alcohol, and extracting solution is recovered under reduced pressure, and it is 1.20~1.35 thick pastes to be concentrated into relative density；
(2) take thick paste, adsorbent be added, mixes thoroughly, dry, pulverize, be fitted into supercritical extract axe, with extracting pressure 10~ 30MPa, 45~55 DEG C of extraction temperature, entrainer are ethyl alcohol, and entrainer dosage is 5%~10%, 4~10MPa of separating pressure, is divided 30~40 DEG C from axe temperature, extraction time is 1~3 hour, collects extract liquor from separation axe, recycles ethyl alcohol, obtain overcritical essence Position processed；
(3) the super critical refined position of gained is adsorbed through ODS reverse phase silica gel chromatographic columns, water-soluble with 60%~80% ethyl alcohol of volumetric concentration Liquid elutes, and separation obtains licoisoflavanone B, neoglycyrol and Licochalcone A.
5. a kind of composition with anti-cutaneum carcinoma effect according to claim 4, which is characterized in that the different Huang of Radix Glycyrrhizae The preparation method of ketone B, neoglycyrol and Licochalcone A is：
(1) extracting liquorice adds 95% ethyl alcohol of volumetric concentration to extract, extracting solution is recovered under reduced pressure, and it is 1.28 thick pastes to be concentrated into relative density；
(2) thick paste is taken, the silica gel of 3 times of weight is added, mixes thoroughly, dry, pulverize, be fitted into supercritical extract axe, with extracting pressure 20MPa, 55 DEG C of extraction temperature, entrainer are ethyl alcohol, and entrainer dosage is 15%, separating pressure 6MPa, detaches 30 DEG C of axe temperature, Extraction time 2 hours collects extract liquor from separation axe, recycles ethyl alcohol, dry, obtains super critical refined position；
(3) the super critical refined position ODS reverse phase silica gel chromatographic columns absorption of gained is that 60%~80% ethyl alcohol is water-soluble with volumetric concentration Liquid elutes, and separation obtains licoisoflavanone B, neoglycyrol and Licochalcone A.
6. a kind of pharmaceutical preparation, which is characterized in that there is anti-cutaneum carcinoma effect including claims 1 to 3 any one of them Composition, the preparation are external preparation or oral preparation.
7. pharmaceutical preparation according to claim 6, which is characterized in that the external preparation is outer ointment, gelling agent Or one kind in liniment；The oral preparation is tablet, one kind in capsule or pill.
8. claims 1 to 3 any one of them Chinese medicine composition is preparing the application in preventing or treating skin cancer drug.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113975253A (en) * 2021-12-10 2022-01-28 上海运佳黄浦制药有限公司 Application of glycyrrhizic acid phenol in antitumor drugs and pharmaceutical composition

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113975253A (en) * 2021-12-10 2022-01-28 上海运佳黄浦制药有限公司 Application of glycyrrhizic acid phenol in antitumor drugs and pharmaceutical composition

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