CN105669796B - A kind of flavone compound TA34a and preparation method thereof and purposes - Google Patents
A kind of flavone compound TA34a and preparation method thereof and purposes Download PDFInfo
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Abstract
The invention belongs to field of medicaments; more particularly to a kind of flavone compound TA34a and preparation method thereof and purposes; the structural formula of compound such as formula 1; it is named as 6; 8; 4 ＇ trihydroxyflavones 7C (2 " E mustard acyls) glucopyranosyl, 4 ＇ O (4 " ＇ glucopyranosyls) glucopyranoside, chemical formula C powdered in glassy yellow44H50O24.The preparation method of the compound, it is characterised in that using Semen thlaspis as raw material, purified through medicinal extract extraction, organic solvent extraction, reversed-phase silica gel column chromatography, high performance liquid chromatography separation.The compounds of this invention has specific antioxidant activity, antitumor activity, immune-enhancing activity and PC12 cytoprotections, and a kind of new selection is provided for clinical application.
Description
Technical field
The invention belongs to field of medicaments, and in particular to a kind of flavone compound TA34a and preparation method thereof and purposes.
Background technology
Semen thlaspis is common a kind of drug in Tibetan medicine, is under the jurisdiction of Cruciferae (Brassicaceae), herba thlaspis genus
The dry seed of (Thlaspi arvense L.) plant Thlaspi.According to traditional medicine ancient books and records《Sheng Nong's herbal classic》And《China is originally
Grass》Record that , Semen thlaspis is pungent, tepor.It is swollen to cure mainly hot eyes for main improving eyesight, mesh pain lacrimation, strengthening the essence light, tonifying five zang organs, eliminating impediment, wind-damp dispelling
Bitterly, tearing against wind, arthralgia pain due to rheumatism etc..Tibetan medicine ancient books and records《Jingzhubencao》Recording Semen thlaspis has clear kidney heat, lung heat, treats various kidneys
The effect of sick, is widely used in Tibetan medicine, has very important effect.The research of Semen thlaspis is not many at present, only few
It has the function of antitumor, antidepression and liver protection to amount document report, there is no the active research report of other biological both at home and abroad
Road.
Less, the research predominantly in terms of Thlaspi seed oil is reported to the research of the chemical composition of Semen thlaspis both at home and abroad simultaneously.
Have on a small quantity about Semen thlaspis seed oil extraction process, the report of seed oil component analysis both at home and abroad.
Such as：
Apply outstanding person et al. (it is apply outstanding, the .GC-MS analysis Thlaspi seeds such as Zhang Xinshen, Li Xiang stir-fry front and back volatile oil chemical composition and
Its West China variation [J] pharmaceutical journal, 2007, (01):1-4.；The GC/MS of the Thlaspi seed volatile oils such as Tu Jie, Zhang Xinshen, Luo Xia
It analyzes [J] chemical research and applies, 2006, (11):1340-1342.) carried out using the volatile oil component of GC/MS Dui Semen thlaspis
Analysis shows and contains a large amount of unsaturated fatty acid in Semen thlaspis volatile oil, and main chemical composition is 4- isothiocyanic acids
Base -1- butylene and allyl group isosulfocyanate.
Roque L.Evangelista(R.L.Evangelista,T.A.Isbell,S.C.Cermak.Extraction
of pennycress (Thlaspi arvense L.)seed oil by full pressing[J].Industrial
Crops and Products,2012,37(1):The shadow of seed moisture content and frying Dui Semen thlaspis squeezing property 76-81.) is inquired into
It rings, its seed oil quality is evaluated by solid content, free fatty, color, Ca, P, Mg, S equal size, is shown
Moisture is low and what the Semen thlaspis of frying was higher than moisture has higher seed oil recovery rate, and solid content
Higher, free aminoacid content is slightly higher, and color is slightly good, and the content of phosphatide increases.And the content difference of sulphur is larger, depends primarily on
The moisture of seed and the degree of frying.
Bryan R.Moser(B.R.Moser,S.N.Shah,J.K.Winkler-Moser,et al.Composition
and physical properties of cress(Lepidium sativum L.)and field pennycress
(Thlaspi arvense L.)oils[J].Industrial Crops and Products,2009,30(2):199-
205.) fatty acid profile in Thlaspi seed oil, vitamin E and content of phytosterol are had studied, the physical property of oil is also measured, wherein wrapping
Include oxidation stability, kinematic viscosity, viscosity index (VI), low temperature flow, proportion, acid value, lubricity and iodine number.The results show that Xi
Mi seed oil contents are 29.0%.Aliphatic acid in Semen thlaspis is mainly made of erucic acid (32.8%) and linoleic acid (22.4%)；It is raw
Phenol content is educated for 851ppm, and predominantly alpha-tocopherol (714ppm).Content of phytosterol in Semen thlaspis is 8.55mg/g,
Predominantly sitosterol and campesterol.
Sinigrin is one of significant ingredient of Semen thlaspis, and (Chen Yu, all Min, the .HPLC such as 5 beautiful duckweeds measure Xi to Chen Yu etc.
The West China sinigrin [J] pharmaceutical journal in Mi, 2012, (01):94-95.) establish a kind of easy, reproducible survey
Ding the HPLC methods of Semen thlaspis sinigrin.Chromatographic condition is, using C18 as chromatographic column, acetonitrile -20mmol 4-butyl ammonium hydrogen sulfates
Aqueous solution=20:80 be mobile phase, Detection wavelength 227nm, flow velocity 1mL/min.
Wang Leilei etc. (Wang Leilei, Chen Cong, the near-infrareds such as all Min diffuse spectrometry measure Sichuan-Tibet genunie medicinal materials Semen thlaspis
Middle sinigrin content [J] spectroscopy and spectrum analysis, 2009, (10):2673-2676.) establish near infrared spectrum detection
The method of Semen thlaspis sinigrin.Author determines the absorption bands of Semen thlaspis using Fourier Transform Near Infrared instrument first,
Then go out optimal testing conditions by software screening method, the method can prepare to determine black mustard in the case where sample size is sufficiently large
The content of sub- glycosides.
There is document report that , Thlaspis is claimed to be mainly the flavone compound based on Isoorientin.Cheng Qing etc. (Cheng Qing,
Measurement [J] the China traditional Chinese medicine science and technology of the Semen thlaspis general flavone contents such as Qian Yu, Guo Huiqing, 2012, (05):It is 430-431.) sharp
It is measured with the general flavone in colorimetric method Dui Semen thlaspis.70% ethyl alcohol extracts, and passes through the selection of assay method, investigation side
Linear relationship, precision, stability and the repeatability of method are established using rutin as reference substance, and 503nm is to measure wavelength,
The development process of NaNO2-Al (NO3) 3-NaOH is used for the measurement of Semen thlaspis general flavone content.
There is author to detect the content of rubimaillin in 13 Wei Thlaspis capsules and 13 Wei Thlaspi balls by HPLC.Zhang Ning
Deng (content [J] Pharmaceutical Analysis of rubimaillin in Zhang Ning, Yang Xiaohong, Ma Xiaofan .HPLC methods 13 taste Thlaspi capsules of measurement
Magazine, 2007, (09):1475-1477.) establish a kind of using C18 as chromatographic column, methanol：0.2% phosphoric acid solution=85:15 are
Mobile phase, 250nm are the detection method of Detection wavelength.Author [12] finds best Detection wavelength by full wavelength scanner
270nm, and compare distinct methods, it is methanol to eventually find most suitable mobile phase：Water=84:16, the method is reproducible, exclusive
Property is strong.
Apply it is outstanding equal (apply outstanding, the Thlaspi seed Chemical forms of trace element such as Zhang Xinshen, Luo Xia studies [J] analytical instrument,
2006,(04):It 28-33.) uses atomic absorption spectrophotometry and analyzes 13 kinds of micro- contents in Semen thlaspis.Xi
Micro- content characteristics are in Mi:K>Ca>P>Mg>Fe>Na>Zn>Mn>Cu>As>Cd>Pb>C.Ca in Qie Semen thlaspis,
The micronutrient levels such as Mg, K, P, Fe, Zn are all relatively abundanter, and wherein Fe is suitable with fleece-flower root iron content.
Hojilla-Evangelista(M.P.Hojilla-Evangelista,R.L.Evangelista,
T.A.Isbell,et al.Effects of cold-pressing and seed cooking on functional
properties of protein in pennycress(Thlaspi arvense L.)seed and press cakes
[J].Industrial Crops and Products,2013,45(0):223-229.) and Selling (G.W.Selling,
M.P.Hojilla-Evangelista,R.L.Evangelista,et al.Extraction of proteins from
pennycress seeds and press cake[J].Industrial Crops and Products,2013,41(0):
113-119.) etc. researchs report the extraction of Semen thlaspis albumen, the influence of protein function in cold pressing and boiling Dui Semen thlaspis.Pass through
The comparison of various extracting conditions, finds , Semen thlaspis protein extracting ratios highest 35% when 77 DEG C of Extracting temperature, Er the general egg of Semen thlaspis
Bai Hanliang is 25% or so, other most of ingredients are all carbohydrate and oil.Analysis finds the amino acid of , Semen thlaspis albumen
Composition is typical vegetable protein, including glycine, glutamic acid and alanine.In addition, comparing different squeezing process Dui Thlaspis
The influence of sub- albumen, to determine the potential use of its diet.It takes cold pressing and is heated to the extracting method of 82 DEG C of squeezings, compare egg
White composition and functional character (dissolubility, blistering, emulsification and moisture holding capacity).Semen thlaspis albumen is mainly by albumin and ball egg
White composition, a small amount of glutelin, no prolamin.Boiling can be such that albumin and globulin content significantly reduces.In pH4, egg
White solubility minimum 10%, as pH value raising also can only achieve moderate solubility 35-45%.Semen thlaspis albumen has good
Foaminess and emulsifiability, moisture holding capacity it is general.Experiment shows that boiling meeting Dui Semen thlaspis albumen has an adverse effect, still
It still has useful functional characteristic.In addition Hojilla-Evangelista [16] also reported heavy by brine extraction or acid
Shallow lake Cong Thlaspi seed protein isolate matter evaluates the emulsibility of protein, the function of stability and foaminess, it is found that two methods carry
The protein functional taken has difference.
A kind of flavone compound (6,8,4 ＇-in yellow powder are isolated in present inventor's Cong Semen thlaspis
Trihydroxyflavone 7C- (2 ＇ ＇-E- mustard acyls) glucopyranosyl -4 ＇-O- (4 ＇ ＇ ＇-glucopyranosyl) glucopyranose
Glycosides), and it is found that its medical usage.
Invention content
First technical problem solved by the invention is to provide a kind of flavone compound.The compound is Cong Thlaspis
Isolated in son, structural formula such as formula 1 is named as 6,8,4 ＇-trihydroxyflavones 7C- (2 ＇ ＇-E- mustard acyls) glucopyra
- 4 ＇-O- of glycosyl (4 ＇ ＇ ＇-glucopyranosyl) glucopyranoside.The compounds of this invention is powdered in glassy yellow, ESI-MSm/
Z shows its molecular weight 960, comprehensive1H-NMR and13C-NMR data, chemical formula C44H50O24, structural formula is as follows：
Second technical problem solved by the invention is to provide the flavone compound (6,8,4 ＇-trihydroxyflavones 7C-
(2 ＇ ＇-E- mustard acyls) glucopyranosyl -4 ＇-O- (4 ＇ ＇ ＇-glucopyranosyl) glucopyranoside) preparation method.
The preparation method of the flavone compound is using Semen thlaspis as raw material, through medicinal extract extraction, organic solvent extraction, reverse phase silica gel column layer
Analysis, high performance liquid chromatography separation step prepare gained, specially：
A, medicinal extract extracts：Qu Semen thlaspis is crushed to 20~40 mesh, with 90%~100% alcohol reflux extract 2~5 times, often
Secondary 40~80min merges extracting solution, filtering, is concentrated under reduced pressure into medicinal extract；
B, organic solvent extracts：Step A medicinal extract is suspended with distilled water, and (60 DEG C -90 DEG C) extractions 5 of isometric petroleum ether are added
~8 times, aqueous phase solution is collected, isometric ethyl acetate is added and extracts 5~8 times, collects aqueous phase solution, isometric n-butanol is added
Extraction 5~8 times collects n-butanol phase solution, is concentrated under reduced pressure into medicinal extract；
C, reversed-phase silica gel column chromatography：Step B medicinal extract is dissolved with ethyl alcohol, carries out reversed-phase silica gel column chromatography；It is with volume proportion
1:9~6:4 ethanol-water solution carries out gradient elution, merges identical part, collects each section eluent and concentrates；
D, high performance liquid chromatography separation：The 4 of step C eluent:6 parts are further purified with high performance liquid chromatography separation,
Up to the flavone compound.
In above-mentioned technical proposal, the concentration of alcohol in preferred steps A is 90%~100%.
In above-mentioned technical proposal, after medicinal extract is suspended with distilled water in preferred steps B, petroleum ether, ethyl acetate is used to extract respectively
It removes miscellaneous, takes the aqueous phase solution after extraction, with extracting n-butyl alcohol, collect n-butanol phase solution.
In above-mentioned technical proposal, ethanol-water solution volume proportion is 1 in preferred steps C:9、2:8、3:7、4:6、5:5、6:
4。
In above-mentioned technical proposal, high performance liquid chromatography separation purifying is to use 20mm × 250mm in preferred steps D, 5 μm
C18Chromatographic column, flow velocity are 5~20ml/min, and the methanol that mobile phase is 70~100%, UV detector Detection wavelength is 254nm,
190~210 μ l of each sample introduction collect the chromatographic peak of 8.22~11.6min, dry after repeatedly adding up.
Third technical problem solved by the invention is to provide the flavone compound (6,8,4 ＇-trihydroxyflavones 7C-
(2 ＇ ＇-E- mustard acyls) glucopyranosyl -4 ＇-O- (4 ＇ ＇ ＇-glucopyranosyl) glucopyranoside) have in preparation
The drug of antioxidant activity or the application in health food.
Specifically, it is by the flavone compound with clearly to embody flavone compound of the present invention with antioxidant activity
Except the ability of DPPH free radicals, the ability of scavenging hydroxyl, with remove ABTS free radicals ability, with reducing power body
It is existing.
4th technical problem solved by the invention is to provide the flavone compound (6,8,4 ＇-trihydroxyflavones 7C-
(2 ＇ ＇-E- mustard acyls) glucopyranosyl -4 ＇-O- (4 ＇ ＇ ＇-glucopyranosyl) glucopyranoside) preparing anti-swell
The drug of tumor activity or the application in health food.
Specifically, embody flavone compound of the present invention with antitumor activity be by the flavone compound for
The inhibiting effect of HGC-27 tumor cell proliferations is relatively strong and S180 growth of tumour cell in KM Mice Bodies is inhibited to embody,
5th technical problem solved by the invention is to provide the flavone compound (6,8,4 ＇-trihydroxyflavones 7C-
(2 ＇ ＇-E- mustard acyls) glucopyranosyl -4 ＇-O- (4 ＇ ＇ ＇-glucopyranosyl) glucopyranoside) have in preparation
The drug of immune-enhancing activity or the application in health food.
Specifically, embodiment flavone compound of the present invention is can by the flavone compound with immune-enhancing activity
Promote macrophage to generate NO, the phagocytic function of macrophage can be promoted, embodiment is had a significant impact to the proliferation of macrophage.
6th technical problem solved by the invention is to provide the flavone compound (6,8,4 ＇-trihydroxyflavones 7C-
(2 ＇ ＇-E- mustard acyls) glucopyranosyl -4 ＇-O- (4 ＇ ＇ ＇-glucopyranosyl) glucopyranoside) have in preparation
The drug of PC12 cytoprotections or the application in health food.
Specifically, it is by the flavone compound to embody flavone compound of the present invention to have PC12 cytoprotections
H can be mitigated2O2Damage to PC12 cells can reduce in PC12 cells by H2O2MDA contents caused by damage increase, and reduce
The LDH burst sizes of PC12 cells, can improve the vigor of the intracellular SOD of PC12, can improve the vigor of the intracellular CAT enzymes of PC12, energy
The vigor for improving the intracellular GSH-Px of PC12, can inhibit by H2O2Intracellular ROS contents caused by damage increase, and can stablize line
Mitochondrial membrane potential embodies,
To sum up, flavone compound (6,8,4 ＇-trihydroxyflavones 7C- (2 ＇ ＇-E- mustard acyls) glucopyra of the present invention
- 4 ＇-O- of glycosyl (4 ＇ ＇ ＇-glucopyranosyl) glucopyranoside) there is specific antioxidant activity, antitumor activity, tool
There are immune-enhancing activity and PC12 cytoprotections, a kind of new selection is provided for clinical application.
Description of the drawings
Fig. 1 flavone compound hydrogen spectrograms
Fig. 2 flavone compound carbon spectrograms
Fig. 3 flavone compound high resolution mass spectrum figures
Fig. 4 flavone compounds HBMC
Fig. 5 flavone compounds HSQC
Fig. 6 flavone compound HMBC correlation figures
The Scavenging activity of Fig. 7 flavone compound DPPH free radicals.
The Scavenging activity of Fig. 8 flavone compound hydroxy radicals.
The Scavenging activity of Fig. 9 flavone compound ABTS free radicals.
Figure 10 flavone compound reducing powers.
Figure 11 NO standard curves.
Figure 12 flavone compounds generate RAW264.7 macrophages the influence of NO.
Wherein：*, indicate to compare P with blank group<0.05；*, indicate to compare P with blank group<0.01；*, indicate and blank
Group comparison P<0.01.
Figure 13 flavone compounds generate RAW264.7 macrophages the influence of phagocytic function.
Wherein：*, indicate to compare P with blank group<0.05；*, indicate to compare P with blank group<0.01.
Figure 14 flavone compounds generate RAW264.7 macrophages the influence of proliferation.
Wherein：*, indicate to compare P with blank group<0.05；*, indicate to compare P with blank group<0.01.
Figure 15 flavone compound HGC-27 inhibition rate of tumor cell.
Influence of Figure 16 flavone compounds to PC12 cell survival rates.
Wherein：*, indicate to compare P with model group<0.05；#, indicate to compare P with blank group<0.05.
Influence of Figure 17 flavone compounds to intracellular MDA.
Wherein：*, indicate to compare P with model group<0.05；#, indicate to compare P with blank group<0.05.
Influence of Figure 18 flavone compounds to LDH contents.
Wherein：*, indicate to compare P with model group<0.05；##, indicate to compare P with blank group<0.01.
Influence of Figure 19 flavone compounds to SOD enzyme activities.
Wherein：*, indicate to compare P with model group<0.05；#, indicate to compare P with blank group<0.05.
Influence of Figure 20 flavone compounds to CAT enzyme activities.
Wherein：*, indicate to compare P with model group<0.05；##, indicate to compare P with blank group<0.01.
Influence of Figure 21 flavone compounds to GSH-Px enzyme activities.
Wherein：*, indicate to compare P with model group<0.05；##, indicate to compare P with blank group<0.01.
Figure 22 flavone compounds influence intracellular ROS contents.
Wherein：*, indicate to compare P with model group<0.05；##, indicate to compare P with blank group<0.01.
Influence of Figure 23 flavone compounds to mitochondrial membrane potential.
Wherein：A indicates normal group；B indicates H2O2Model group；E indicates TA34a experimental groups.
Specific implementation mode
A kind of flavone compound of the present invention be with the following method in Cong Semen thlaspis extraction separation and purification and obtain：
Qu Semen thlaspis 10kg, are crushed to 20~40 mesh, and 10 times of 95% alcohol refluxs of volume are added and extract 3 times, every time
60min merges extracting solution, filtering, is concentrated under reduced pressure, obtains medicinal extract 1.57kg, medicinal extract is suspended with distilled water, is added in separatory funnel
(60 DEG C -90 DEG C) extractions of isometric petroleum ether fully after oscillation, after liquid layering to be mixed, collect aqueous phase solution, operate 5 repeatedly
After secondary, isometric ethyl acetate extraction is added in aqueous phase solution, after fully vibrating mixing, after liquid clarification layering to be mixed, collects water
Phase solution, after operating 5 times repeatedly, isometric extracting n-butyl alcohol is added in aqueous phase solution, after fully vibrating mixing, waits for clarification layering
Afterwards, n-butanol phase solution is collected, is concentrated under reduced pressure, obtains medicinal extract 182g.After the 20% ethyl alcohol dissolving of 3 times of weight ratio of medicinal extract, carry out
Reverse phase silica gel column (5cm × 25cm) chromatographs, and multiple loading elution, each loading 20ml is 1 with volume proportion:9、2:8、3:7、
4:6、5:5、6:4 ethanol-water solution gradient elution, TLC monitorings merge identical part, obtain 6 parts；The 4 of eluent:
6 parts are further isolated and purified with half preparative high-performance liquid chromatographic, using 40% methanol as mobile phase, using 20mm × 250mm, 5 μ
The C of m18It is stationary phase to prepare column, and flow velocity 10ml/min, UV detector Detection wavelength is 254nm, 200 μ l of each sample introduction, is collected
The chromatographic peak of 9.5min, it is dry after repeatedly adding up, you can to obtain the flavone compound.
ESI-MS mass spectral analyses, high resolution mass spec analysis and 1D, 2D nuclear magnetic resonance point are carried out to the flavone compound
Analysis (13C NMR、1H NMR, DEPT135, HMBC and HSQC).
Flavone compound of the present invention shows its molecular weight 962 by ESI-MS m/z, comprehensive1H-NMR and13C-NMR numbers
According to chemical formula C44H50O24。1H-NMR and13C-NMR data are shown in Table 1.
The hydrocarbon ownership of 1 flavone compound of table
In conjunction with one-dimensional nuclear magnetic data, two-dimentional nuclear magnetic data and high-resolution data analysis (see 1~attached drawing of attached drawing 5) can be true
The structure for determining flavone compound is shown in formula 1.Attached drawing 6 is the HMBC correlation figures of the flavone compound.The flavonoid
Object is accredited as -4 ＇-O- (4 ＇ ＇ ＇-glucopyra of 6,8,4 ＇-trihydroxyflavones 7C- (2 ＇ ＇-E- mustard acyls) glucopyranosyl
Glycosyl) glucopyranoside.
It is the chemism research experiment of flavone compound of the present invention (hereinafter referred to as TA34a) below.
1, antioxidant activity
To remove DPPH, ABTS, hydroxy radical and reducing power as evaluation index, TA31a, TA31b and TA34a are carried out
Determination oxidative.
1) DPPH free radical scavenging abilities measure：The sample of 100 μ L, room temperature reaction is added in the DPPH of 100 μ L 0.1mM
30min, 517nm measure absorbance value.It is not added with the blank control of sample, and only sample Background control with sample simultaneously.
DPPH clearance rates %=1- (A1-A2)/A0
A1：The absorbance value of sample+DPPH；A2:The absorbance value of sample+solvent；A0：The absorbance value of DPPH
Flavone compound of the present invention has the ability for removing DPPH free radicals, and has dose-dependence, but whole
Scavenging activity it is weak compared with positive controls Vc.In concentration<Under the conditions of 80 μ g/mL, identical dose is better than to the Scavenging activity of DPPH
The Vc of amount, in concentration>After 80 μ g/mL, Scavenging activity will be less than Vc.It is learnt by calculating, flavone compound of the present invention is removed
The IC of DPPH50Value is 15.72 μ g/mL, as a result as Fig. 7 is shown.
2) Hydroxyl radical-scavenging ability measures：25 μ L 1.5mM FeSO are added in the sample of 150 μ L various concentrations4With 10 μ
L20mM salicylic acids add 15 μ L 6mM H2O2.Absorbance value is measured at 37 DEG C of reactions 1h, 520nm.
Clearance rate %=1- (A1-A2)/A0
Wherein：A1：Sample absorbance value；A2:The absorbance value of sample Background control；A0：It is not added with sample and H2O2Absorbance
Value
As a result such as Fig. 8 is shown, flavone compound of the present invention has the ability of certain scavenging hydroxyl, and has agent
Measure dependence.By calculating the IC it is found that flavone compound scavenging hydroxyl of the present invention50Value is respectively 771.02 μ g/
mL。
3) ABTS free radical scavenging abilities measure：The potassium peroxydisulfate of the ABTS and 2.45mM of 7mM mix in equal volume, in dark place
16h is reacted at room temperature, 50 times are diluted with the phosphate buffer of pH7.2, until absorbance value 0.7 or so (734nm).This dilution is drawn again
The sample containing 100 μ L various concentrations is added in 100 μ L of liquid, reacts at room temperature 5min, and 734nm measures absorbance value.
Clearance rate %=1- (A1-A2)/A0
A1：The absorbance value of sample+ABTS；A2:The absorbance value of sample+solvent；A0：The absorbance value of ABTS
As a result show such as Fig. 9, flavone compound of the present invention has the ability for removing ABTS free radicals, and have dosage according to
The relationship of relying, in 30 μ g/mL of concentration, to the clearance rates of ABTS free radicals close to 100%.By calculating, flavonoids of the present invention
Compound removes the IC of ABTS50Value is respectively 4.18 μ g/mL.
4) reducing power measures：1% potassium ferricyanide of 50 μ L 0.2M pH6.0PBS and 25 μ L is added in 25 μ L samples, in 45
DEG C reaction 30min.0.1% ferric trichloride of 40 μ L, 10% trichloroacetic acids and 60 μ L is added, absorbance value is measured at 700nm.
As a result such as Figure 10 is shown, flavone compound of the present invention has certain reducing power, and is closed with dose-dependant
System.
2, antitumor activity and immune-enhancing activity
To promote RAW264.7 macrophage proliferations, phagocytosis and produce NO, in anti-HGC-27 tumor cell proliferations and Mice Body
Inhibition test is test platform, evaluates the antitumor activity and immune-enhancing activity of flavone compound of the present invention.
(1) influence of NO is produced to macrophage
RAW264.7 cell culture：
264.7 cells of RAW are incubated at 37 DEG C of 5%CO with the RPMI-1640 culture solutions of 10% fetal calf serum2Saturated humidity
It is primary every 1d passages in the cell incubator of air.When passage, adherent cell 60s is digested with 0.25% trypsin solution
Left and right, is poured out enzyme solution, the fresh RPMI-1640 culture solutions containing 10% fetal calf serum is added, suction pipe pressure-vaccum is used in combination for several times,
With 1:4 ratio secondary cultures.
NO's induces：
By 1 × 106The RAW264.7 macrophages of exponential phase are added in 24 orifice plates in a/hole.It, will after overnight incubation
Sample empirically designs, and 24 well culture plates is added, and complement to final volume with PBS as 1000 μ L；Each concentration set 3 it is parallel
Hole, totally 5 concentration, final concentration are respectively 0,1,10,20,100,200 μ g/mL.Meanwhile it doing Positive control wells and (final concentration being added
For the LPS of 10 μ g/mL).In 37 DEG C of 5%CO22d is cultivated under saturated humidity air conditions.After culture, culture is drawn respectively
Supernatant carries out NO detections immediately.
NO is detected：
Using the content of NO in Griess method measurement systems.NO is unstable, and Griess methods are made of measurement is decomposed by NO
NO-2/NO-3Ratio react its content.
The drafting of standard curve：
Accurately weigh NaNO26.9g, with ddH2O is settled to 100mL, and further gradient dilution is a concentration of 100 μM
Working solution.Utilize ddH2Working solution is diluted to 50 μM, 40 μM, 30 μM, 20 μM, 10 μM, 5 μM, 2 μM, 0 μM of final concentration by O.In every
100 μ L of isometric mixed Griess reagent As liquid and B liquid are added in hole.After reacting at room temperature 20min, in being measured in microplate reader
Absorbance at 540nm.Using standard concentration and absorbance value as transverse and longitudinal coordinate, standard curve is drawn.
Sample detection：
The 100 μ L of culture supernatant after sample effect are accurately added in ELISA Plate.Isometric mixing is added in every hole
100 μ L of Griess reagent As liquid and B liquid afterwards.React at room temperature 20min after, in microplate reader measure 540nm at absorbance.It utilizes
Calibration curve equation finds out the concentration of NO in supernatant.
Using standard concentration as abscissa, absorbance is ordinate, draws standard curve (Figure 11), derives standard curve
Equation is Y=0.0068X+0.0428 (R2=0.9985).Bring the absorbance data of each concentration of sample into calibration curve equation,
Acquire the concentration of NO in each concentrations of cells liquid supernatant of sample.
Experimental result such as Figure 12 shows that the NO amounts that RAW264.7 macrophages itself generate are less, but are stimulated by LPS
Afterwards, NO largely generates (P<0.01).Flavone compound of the present invention can promote macrophage to generate NO (P in 100 μ g/mL<
0.01)。
(2) to the influence of macrophages phagocytic capacity
By 1 × 105The RAW264.7 macrophages of exponential phase are added in 96 orifice plates in a/hole.It, will after overnight incubation
Three compounds empirically design, and are added in 96 orifice plates, and complement to final volume with PBS as 200 μ L；Each concentration sets 3 and puts down
Row hole, totally 5 concentration, final concentration are respectively 0,1,10,20,100,200 μ g/mL.Meanwhile it doing Positive control wells and (being added dense eventually
Degree is the LPS of 10 μ g/mL).
Cell is in 37 DEG C of 5%CO2It is cultivated for 24 hours under saturated humidity air conditions.After culture, supernatant is gently sucked,
100 μ L of 1mg/mL neutral red solutions are added.After 30min, dimethyl diaminophenazine chloride dye liquor is sucked, and for several times with 37 DEG C of PBS board-washings, until thin
Extracellular dyestuff is all cleaned.After centrifuging and carefully sucking PBS, cell pyrolysis liquid (ethyl alcohol is added：Acetic acid=24：1) 100 μ L, 4 DEG C
1h is placed, until cell all cracks.After shaking mixing, OD values at wavelength 540nm are measured in microplate reader.
Phagocytosis is the important way that macrophage exercises its function.By detecting the dimethyl diaminophenazine chloride in macrophage
Dyestuff can react the phagocytic activity of macrophage.It is huge that experimental result such as Figure 13 shows that flavone compound of the present invention can promote
Phagocytic function (the P of phagocyte<0.05).
(3) to the influence of macrophage proliferation
The RAW264.7 macrophages of exponential phase are pressed 2 × 104A/hole concentration is added in 96 orifice plates.Overnight incubation
Afterwards, sample is empirically designed, 96 well culture plates is added, and final volume is complemented to as 200 μ L to contain PBS；Each concentration sets 3
Parallel hole, totally 5 concentration, final concentration are respectively 0,1,10,20,100,200 μ g/mL.It is blank control wells (refinement born of the same parents simultaneously
Culture solution).
In 37 DEG C of 5%CO after cell sample-adding248h is cultivated under saturated humidity air conditions, and cell is observed under inverted microscope
Upgrowth situation.Terminate preceding 4h in culture, abandons supernatant.The PBS of 100 μ L is added per hole, is then added 5 a concentration of 5mg/mL's of μ L
MTT continues to cultivate, and after culture, 100 μ L of 10%SDS (being prepared with the HCl of 0.01M) is added per hole, are stood overnight at 37 DEG C.It shakes
Mixing is swung, crystal is made fully to dissolve.It is measured per hole OD values with microplate reader at 570nm wavelength.
Find that flavone compound of the present invention has obviously mononuclear macrophage strain RAW264.7 by experiment such as Figure 14
Promotion or Inhibit proliferaton effect (P<0.05), illustrate that it has a significant impact the proliferation of macrophage.
(4) measurement of anti-HGC-27 cell Proliferations
Cell culture：
HGC-27 cells are incubated at 37 DEG C of 5%CO with the DMEM culture solutions containing 10% fetal calf serum2Saturated humidity air
It is primary every 1d passages in cell incubator.When passage, adherent cell 30s or so is digested with 0.25% trypsin solution, is inclined
Enzyme solution is poured out, the fresh DMEM culture solutions containing 10% fetal calf serum are added, suction pipe pressure-vaccum are used in combination for several times, with 1：4 ratios pass
It is commissioned to train foster.
Cell proliferation experiment：
The HGC-27 macrophages of exponential phase are added by 3000/hole concentration in 96 orifice plates.In 37 DEG C of incubators
Middle overnight incubation empirically designs sample, 96 well culture plates is added, and to be cultivated without phenol red DMEM containing 10% fetal calf serum
It is 200 μ L that liquid, which complements to final volume,；Each concentration sets 3 parallel holes, totally 5 concentration, and final concentration is respectively 0,1,10,20,
100,200 μ g/mL.Blank control wells (only plus cell culture fluid) are done simultaneously.
In 37 DEG C of 5%CO after cell sample-adding248h is cultivated under saturated humidity air conditions, and cell is observed under inverted microscope
Upgrowth situation.Terminate preceding 4h in culture, abandon supernatant, 100 μ L PBS are added per hole, the MTT of 5 a concentration of 5mg/mL of μ L is then added
Continue to cultivate, after culture, 100 μ L of 10%SDS (being prepared with the HCl of 0.01M) is added per hole, are stood overnight at 37 DEG C.Oscillation
Mixing makes crystal fully dissolve.It is measured per hole OD values with microplate reader at 570nm wavelength.
As a result as shown in figure 15, flavone compound of the present invention for HGC-27 tumor cell proliferations inhibiting effect all compared with
By force, there is antitumor action.
(5) inhibition test in Mice Body
The tumor-bearing mice after intraperitoneal inoculation S180 7d is taken, ascites is aseptically extracted with syringe, ascites should be breast
White extremely faint yellow, no color.It is diluted to 4 × 10 with sterile saline7A/ml is placed in spare in ice-water bath.
After KM mouse (18-22g) are bought back, it is subcutaneously accurately inoculated with tumor liquid 0.2ml in every right side of mice armpit, raising is overnight
Afterwards, it is grouped.It is inoculated with next day, mouse is randomly divided into 5 groups, every group 10, half male and half female.Wherein physiology is injected intraperitoneally in blank group daily
Flavone compound 0.1ml/10g (2mg/10g) of the present invention, successive administration 7 days is injected intraperitoneally in brine 0.1ml/10g, administration group.
After last dose 4h, cervical dislocation puts to death mouse, and separation oxter tumour, which is existed side by side, claims knurl weight.
The results are shown in Table 2, and flavone compound of the present invention is 69.6% for the internal tumour inhibiting rate of S180 tumour cells,
With antineoplastic action.
Inhibition test in 2 KM Mice Bodies of table
3, PC12 cytoprotections
(1) cell viability measures (MTT colorimetric methods)
Cell survival rate is measured with cell growth inhibition assay (MTT colorimetric methods).The PC12 cells of logarithmic growth phase, are pressed
40000/hole is added in 96 orifice plates, 37 DEG C of 5%CO2It is incubated overnight in incubator.Given the test agent is empirically designed to addition 96
In orifice plate, and 200 μ L of final volume are complemented to culture medium；3 parallel holes are arranged in each concentration, totally 5 concentration gradients, final concentration
Respectively 0,1,10,20,100,200 μ g/mL.After given the test agent is added, it is incubated 6h jointly with cell, final concentration is then added
200 μM of H2O2Cellular damage is carried out, while being not added with given the test agent and H2O2Control wells, and only plus H2O2Model group.
Cell plates are placed in 37 DEG C of 5%CO2Continue to be incubated 18h in incubator, discard supernatant liquid, the buffer solution and 5 μ L of 100 μ L is added
The MTT of 5mg/mL continues after cultivating 4h, 100 μ L SDS is added per hole, are incubated overnight.Absorbance value is measured at 570nm.It inhales
Shading value has positive correlation with cell survival rate, and therefore, absorbance value can react the survival rate of PC12 cells.Each group cell is deposited
Motility rate is calculated by following equation.
Cell viability (%)=experimental group absorbance value/control group absorbance value × 100
As can be seen from Figure 16, compared with control group (regulation cellular control unit vigor is 100%), model group cell viability
Significantly reduce (P<0.05), cell viability 52% shows the H of 200 μM of PC12 cells pair2O2It is very sensitive, modeling success.With mould
Type group is compared, and within the scope of 100 μ of μ g/mL~200 g/mL of flavone compound concentration of the present invention, has the significance difference opposite sex (P<
0.05), illustrate that it can mitigate H2O2Damage to PC12 cells, effect have dose dependent.When 200 μ g/mL, the present invention is yellow
The PC12 cell survival rates of ketone compounds are 73.14%.
(2) intracellular MDA, LDH content and the measurement of SOD, CAT, GSH-Px enzyme activity
Preparation of samples：
The PC12 cells of logarithmic growth phase, by 5.0 × 105/ hole is added in 24 orifice plates, 37 DEG C of 5%CO2It is incubated in incubator
It educates overnight.After the sample of 100 μ g/mL is added, it is incubated 6h jointly with cell, the H of 200 μM of final concentration is then added2O2Carry out cell
Damage, while being not added with sample and H2O2Control wells, and only plus H2O2Model group.Cell plates are placed in 37 DEG C of 5%CO2Training
It supports and continues to be incubated 18h in case, collect supernatant and be used for LDH assays, it is molten that 100 μ L 1%Triton are added in cell precipitation
Liquid, under the conditions of cell plates are placed in -80 DEG C, lytic cell 1h is to get detection sample, for malonaldehyde (MDA), lactic dehydrogenase
(LDH), the survey of superoxide dismutase (SOD), glutathione peroxidase (GSH-px), catalase (CAT) content
It is fixed.
The assay of MDA, LDH, SOD, GSH-px, CAT：
Reference reagent box specification carries out MDA, LDH content to sample and SOD, GSH-Px, CAT enzyme activity are measured.
As a result as shown in figure 17, compared with the control group (regulation control group MDA levels are 100%), MDA contents in model group
Apparent increase (P<0.05)；And compared with model group, the content of MDA is substantially reduced (P in flavone compound administration group of the present invention
<0.05), illustrate that it can be reduced in PC12 cells by H2O2MDA contents caused by damage increase.
Influence of the sample to LDH burst sizes is as shown in figure 18, and (regulation control group LDH burst sizes are compared with the control group
100%), LDH contents apparent increase (P in model group<0.01)；And compared with model group, flavone compound administration of the present invention
The content of LDH is substantially reduced (P in group<0.05).The result shows that the LDH that flavone compound of the present invention can reduce PC12 cells is released
High-volume.
As a result as shown in figure 19, compared with the control group (regulation control group SOD enzyme activities are 100%), SOD enzymes in model group
Content is substantially reduced (P<0.05)；And compared with model group, the apparent liter of the activity of SOD in flavone compound administration group of the present invention
Height (P<0.05), illustrate that it can improve the vigor of the intracellular SOD of PC12.
Sample is to shown in CAT enzyme activities influence diagram 20, compared with the control group (regulation control group CAT enzyme activities are 100%),
CAT enzymes content is substantially reduced (P in model group<0.01)；And compared with model group, in flavone compound administration group of the present invention
Active apparent increase (the P of CAT enzymes<0.05).Illustrate that it can improve the vigor of the intracellular CAT enzymes of PC12.
Flavone compound of the present invention influences GSH-Px enzyme activities as shown in figure 21, (regulation control compared with the control group
100%) group GSH-Px enzyme activities are that GSH-Px enzymes content is substantially reduced (P in model group<0.01)；And compared with model group, this
Active apparent increase (the P of GSH-Px enzymes in invention flavone compound administration group<0.05).It is intracellular to illustrate that it can improve PC12
The vigor of GSH-Px.
(3) measurement of reactive oxygen species (ROS)
The PC12 cells of logarithmic growth phase, by 1.0 × 106/ hole is added in 6 orifice plates, 37 DEG C of overnight incubations.Random point
Group：Normal group, 300 μM of H2O2Group, 300 μM of H2O2+ sample sets.For 24 hours with the sample pretreatment cell of 100 μ g/mL, rear to be added
300μM H2O2Induction 3 hours.Reactive oxygen species detection is carried out according to active oxygen kit.Suspension PC12 cells, with concentration 10
μM active oxygen fluorescence probe, reacted under the conditions of being protected from light, cell dyed, 37 DEG C be incubated 30 minutes after, 3000 turns/
Point, it centrifuges 5 minutes, abandons supernatant.Cell is washed using PBS liquid 3 times, extracellular remaining fluorescent reagent wash clean, removes the back of the body
The interference of scape fluorescence.On multi-function microplate reader, using excitation wavelength 488nm, launch wavelength 525nm, the glimmering of cell ROS is measured
Luminous intensity, intracellular ROS contents are directly proportional to fluorescence intensity, can pass through the content of ROS in the fluorescent value reacting cells that measure.
Influence of the sample to intracellular ROS contents is as shown in figure 22, and compared with the control group, ROS contents are apparent in model group
Increase (ROS contents are 100% in regulation model group) (P<0.01)；And compared with model group, flavone compound of the present invention is given
ROS contents are substantially reduced (P in medicine group<0.05), illustrate that it can inhibit by H2O2Intracellular ROS contents caused by damage increase.
(4) measurement of PC12 mitochondrial membrane potential in anoxic
After JC-1 dyeing cells, mitochondrial membrane potential completely loses, and is in green fluorescence after dyeing.Normal cell is in red
Color fluorescence.
The PC12 cells for taking logarithmic logarithm growth period, by 5 × 105/ hole is added in 24 orifice plates, 37 DEG C of overnight incubations.At random
Grouping：Normal group, 300 μM of H2O2Group, 300 μM of H2O2+ sample sets.The sample of 100 μ g/mL is incubated 6h jointly with cell, then
The H of 300 μM of final concentration is added2O2Carry out cellular damage.Cell plates are placed in 37 DEG C of 5%CO2Continue to be incubated 18h in incubator,
At the end of culture, cell is collected by centrifugation, with 0.5ml cell culture fluids again suspension cell, the JC-1 dyes of 5 μ g/mL are then added
Material, reacts 20min in 37 DEG C, cell is washed 3 times with PBS.Fluorescence is observed using inverted fluorescence microscope.
As a result such as Figure 23 is shown, most cells are in reddish yellow fluorescence in normal group.And H2O2Model group cell is in almost green
The faintly visible yellow fluorescence of fluorescence, only a few cell shows that mitochondrial membrane potential in anoxic declines and even loses.With model group phase
Than flavone compound of the present invention can be such that reddish yellow Fluorescence Ratio in cell increases, and show that it can stablize mitochondrial membrane potential.
Claims (8)
1. flavone compound is named as 6,8,4 ＇-trihydroxyflavones -7-C- (2 ＇ ＇-E- mustard acyls) glucopyranosyl -
4 ＇-O- (4 ＇ ＇ ＇-glucopyranosyl) glucopyranoside, molecular formula C44H50O24, structural formula such as formula 1：
2. flavone compound according to claim 1 is being used to prepare with antioxidant activity, antitumor activity, is being immunized
The drug or the application in health food of enhancing activity or PC12 cytoprotections.
3. application according to claim 2, it is characterised in that：Meet following any one：
It is described with antioxidant activity refer to remove DPPH free radicals ability；
It is described with antioxidant activity refer to scavenging hydroxyl ability；
It is described with antioxidant activity refer to remove ABTS free radicals ability；
It is described have antioxidant activity refer to reducing power.
4. application according to claim 2, it is characterised in that：
The antitumor activity refers to the inhibiting effect for HGC-27 tumor cell proliferations.
5. application according to claim 2, it is characterised in that：Meet following any one：
It is described have immune-enhancing activity be refer to promote macrophage generate NO；
It is described have immune-enhancing activity refer to the phagocytic function that can promote macrophage；
It is described have immune-enhancing activity refer to being had a significant impact to the proliferation of macrophage.
6. application according to claim 2, it is characterised in that：Meet following any one：
It is described have PC12 cytoprotections refer to that can mitigate H2O2Damage to PC12 cells；
It is described have PC12 cytoprotections be refer to reduce PC12 cells in by H2O2MDA contents caused by damage increase；
It is described with PC12 cytoprotections refer to reduce PC12 cells LDH burst sizes；
It is described have PC12 cytoprotections refer to the vigor that can improve the intracellular SOD of PC12；
It is described have PC12 cytoprotections refer to the vigor that can improve the intracellular CAT enzymes of PC12；
It is described have PC12 cytoprotections refer to the vigor that can improve the intracellular GSH-Px of PC12；
It is described have PC12 cytoprotections refer to that can inhibit by H2O2Intracellular ROS contents caused by damage increase；
It is described have PC12 cytoprotections be to refer to stablize mitochondrial membrane potential.
7. the preparation method of flavone compound described in claim 1, including using Semen thlaspis as raw material, extracted through step A medicinal extract,
The extraction of step B organic solvents, step C reversed-phase silica gel column chromatographies, step D high performance liquid chromatography separation steps are prepared, special
Sign is：
A, medicinal extract extracts：Qu Semen thlaspis is crushed to 20~40 mesh, and 2~5 times are extracted with 90%~100% alcohol reflux, and every time 40
~80min merges extracting solution, filtering, is concentrated under reduced pressure into medicinal extract；
B, organic solvent extracts：Step A medicinal extract is suspended with distilled water, isometric petroleum ether extraction is added 5~8 times, collects water phase
Solution is added isometric ethyl acetate and extracts 5~8 times, collect aqueous phase solution, isometric extracting n-butyl alcohol is added 5~8 times, receives
Collect n-butanol phase solution, is concentrated under reduced pressure into medicinal extract；
C, reversed-phase silica gel column chromatography：Step B medicinal extract is dissolved with ethyl alcohol, carries out reversed-phase silica gel column chromatography；It is carried out with ethanol-water solution
The initial volume proportioning of gradient elution, alcohol-water is 1:9, it is 6 to terminate volume proportion:4, merge identical part, collects each portion
Divide eluent and concentrates；
D, high performance liquid chromatography separation：The 4 of step C eluent:6 parts further with high performance liquid chromatography separation purify to get
The flavone compound.
8. the preparation method of flavone compound according to claim 7, it is characterised in that：Meet following any one：
Concentration of alcohol in the step A is 90%~100%；Or
After medicinal extract is suspended with distilled water in the step B, petroleum ether, ethyl acetate abstraction impurity removal are used respectively, take the water after extraction
Phase solution collects n-butanol phase solution with extracting n-butyl alcohol；Or
Gradient elution is carried out with ethanol-water solution in the step C, the initial volume proportioning of alcohol-water is 1:9, terminate volume
Proportioning is 6:4, during gradient elution, ethanol-water solution concentration is at least 1:9、2:8、3:7、4:6、5:5 or 6:The one of 4
Kind；Or
High performance liquid chromatography separation purifying is to use 20mm × 250mm, 5 μm of C in the step D18Chromatographic column, flow velocity be 5~
20ml/min, the methanol that mobile phase is 70~100%, UV detector Detection wavelength are 254nm, 190~210 μ of each sample introduction
L collects the chromatographic peak of 8.22~11.6min, dry after repeatedly adding up.
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| US20080268024A1 (en) * | 2004-05-25 | 2008-10-30 | Santiago Rull Prous | Compositions for Oral and/or Topical Administration |
| US20080220101A1 (en) * | 2007-03-09 | 2008-09-11 | Sybille Buchwald-Werner | Compositions of extracts of aloe for oral administration |
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