CN105153255A - Sansevieria trifasciata Prain flavones, and preparation method and application thereof - Google Patents
Sansevieria trifasciata Prain flavones, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses Sansevieria trifasciata Prain flavones which are prepared by the following steps: by using fresh Sansevieria trifasciata Prain as the raw material, pretreating to obtain dry Sansevieria trifasciata Prain powder; carrying out ultrasonic ethanol extraction; extracting with petroleum ether and ethyl acetate; and purifying by silica gel chromatography to obtain the four components TFS-a, TFS-b, TFS-c and TFS-d of the Sansevieria trifasciata Prain flavones. The Sansevieria trifasciata Prain flavones have obvious inhibiting actions on Salmonella and Escherichia coli, and the MIC (minimal inhibitory concentration) is respectively 3.75 mg/mL. The Sansevieria trifasciata Prain flavone TFS-b has obvious removal actions on hydroxy free radicals, DPPH free radicals and superoxide anions (respectively 59.8%, 78.6% and 84.2%). The Sansevieria trifasciata Prain flavones TFS-c and TFS-d have obvious inhibiting actions on growth of human cervical cancer cell line Hela and mouse myeloma cell line SP2/0, and do not have significant difference for growth of human lung cancer cell line A549, laryngocarcinoma cell line Hep-2 and normal cell line DK.
Description
Technical field
The invention belongs to biological technical field, be specifically related to blue flavones of tiger fur and its preparation method and application.
Background technology
Tiger fur is blue, Liliaceae, and Folium Sansevieriae Trifasclatae belongs to, and originates in Africa western, and accommodative ability of environment is strong, and leaf fibres is tough, can be used for braiding.All there is plantation in China various places at present, can be used as Ornamental Foliage House Plants and carry out potted plant, because it can be good at absorbing indoor formaldehyde, can purify air, be called " natural street cleaner " by people.Research shows, containing major element calcium 172.85mg/g, magnesium 42.87mg/g, potassium 0.1212mg/g, sodium 0.0687mg/g in tiger fur orchid, and trace elements iron 0.1066mg/g, manganese 0.0770mg/g, copper 0.0073mg/g, zinc 0.0040mg/g, the average mass fraction containing essential amino acid is 2.384%; The method utilizing gas chromatography-mass spectrum, nucleus magnetic resonance to combine has isolated 12 kinds of steroidal saponins from Folium Sansevieriae Trifasclatae.
Show the pharmacological research of tiger fur orchid, tiger fur orchid has certain latent effect in anti-inflammatory, analgesia, antidiarrheal, antianaphylaxis, diabetes-alleviating etc.But tiger fur is blue as natural phant, has various active composition, specify structure and the function thereof of a certain or certain several activeconstituents, for the pharmacological action of deep excavation tiger fur orchid and new drug (or protective foods) exploitation thereof, there is vital role.
Flavonoid compound proportion in the activeconstituents of plant is comparatively large, containing higher Flavonoid substances in the medicinal plant of 1/5th, pharmaceutical use and nutritive value remarkable.In the last few years, people were more and more darker to probing into of flavones, found that its pharmacological action is extensive.Some flavonoid compounds are applied to actual production, cholesterol is reduced significantly as soybean isoflavones composition isolated from leguminous plant has, reduce effect of cardiovascular disease incidence rate, U.S. FDA has been recommended in heath food in 1999 and has been used.
In view of the report or the patent application document that there is not yet flavonoid component and efficacy study thereof in tiger fur orchid.Patent of the present invention have developed the extraction of Flavonoid substances in tiger fur orchid, refining and detection method, and confirms that the blue flavones of tiger fur has antibacterial, anti-oxidant, inhibition tumor cell growth isoreactivity.
Summary of the invention
An object of the present invention is to provide the blue flavones of tiger fur.
The blue flavones of tiger fur, it is prepared by following method: by blue for tiger fur drying and crushing, extraction using alcohol; Petroleum ether extraction, abandons petroleum ether layer, extraction into ethyl acetate, volatilizes and obtains the blue flavones of tiger fur;
Described extraction using alcohol be using volume fraction be the ethanol of 60% as extraction agent, preparation liquid ratio is 25:1, soaked overnight, at 80 DEG C, ultrasonic frequency 20-35Hz, power 90-100W, supersound extraction 34min, suction filtration;
Be dissolved in methanol solution by blue for above-mentioned tiger fur flavones, add 15g silica gel mixed sample, after fully stirring, 48 DEG C-52 DEG C volatilize methyl alcohol in an oven; Silica gel containing sample being added to silicagel column capital, treating that silica-gel powder precipitation surface is smooth, take chloroform-methanol as elutriant, and flow velocity is that 1mL/min carries out gradient elution, obtains elution peak and is labeled as elution peak I, elution peak II, elution peak III, elution peak IV;
Merge and collect elution peak II elutriant, through concentrated, lyophilize, obtain the blue flavones TFS-b of tiger fur;
Merge and collect elution peak III elutriant, through concentrated, lyophilize, obtain the blue flavones TFS-c of tiger fur;
Merge and collect elution peak IV elutriant, through concentrated, lyophilize, obtain the blue flavones TFS-d of tiger fur.
Another object of the present invention is to provide the application of the blue flavones of tiger fur.
The application in anti-salmonella and intestinal bacteria medicine prepared by the blue flavones of described tiger fur.
The blue flavones TFS-b of described tiger fur applies the removing of hydroxy radical qiao, DPPH free radical and superoxide anion.
The application of the blue flavones of described tiger fur in the medicine of preparation treatment Human cervical cancer cell lines Hela or mouse myeloma cell line SP2/0.
The blue flavones TFS-c of described tiger fur or the application of the blue flavones TFS-d of tiger fur in preparation treatment Human cervical cancer cell lines Hela or mouse myeloma cell line SP2/0 medicine.
The invention provides the blue flavones of tiger fur, it is blue for raw material with fresh tiger fur, obtains the blue powder of dry tiger fur through pre-treatment; Ultrasonic extraction using alcohol; Obtain with sherwood oil, extraction into ethyl acetate again; Liquid ratio 25:1 is dissolved in 60% ethanol, ultrasonic time 34min, ultrasonic temperature 80 DEG C, ultrasonic power 90-100W, and the extracted amount of the blue flavones of tiger fur reaches maximum value under this condition, is 8.501mg/g; Through purified on silica, obtain four component TFS-a of the blue flavones of tiger fur, TFS-b, TFS-c and TFS-d; The blue flavones raw product of tiger fur, significant restraining effect is all had to intestinal bacteria, Salmonellas, streptococcus aureus, subtilis, to Salmonellas and colibacillary restraining effect particularly evident, being 3.75mg/mL to the MIC of intestinal bacteria, Salmonellas, subtilis, is 7.5mg/mL to the MIC of streptococcus aureus; Four component TFS-a of the blue flavones of tiger fur, TFS-b, TFS-c and TFS-d, all have significant scavenging(action) to hydroxy radical qiao, DPPH free radical, superoxide anion, and the scavenging(action) of TFS-b is the strongest, and clearance rate can reach 59.8%, 78.6%, 84.2% respectively; Crude flavonoid powder and each flavonoid component all effective to Hela cell, especially TFS-c and TFS-d; TFS-a is to murine myeloma cell SP2/0 without action effect, but crude flavonoid powder and other components all can suppress SP2/0 Growth of Cells, especially TFS-c and TFS-d; In addition, the blue flavones of tiger fur and four kinds of components thereof to human lung cancer cell line A549, Human Laryngeal Cancer Cell Hep-2 without positive effect, to normal Madin-Darby canine kidney(cell line) (MDCK) DK almost fanout free region.
Accompanying drawing explanation
Fig. 1 rutin standard curve;
The silica gel column chromatography elution curve of the blue flavones of Fig. 2 tiger fur;
Blue flavones qualitative detection tomographic map (ethyl acetate: formic acid: water=8:3:1) of Fig. 3 tiger fur;
Blue flavones qualitative detection tomographic map (benzene: ethyl acetate: formic acid=8:4:2) of Fig. 4 tiger fur;
The blue flavones of Fig. 5 tiger fur to press down subtilis active;
The blue flavones of Fig. 6 tiger fur press down E. coli Activity;
The blue flavones of Fig. 7 tiger fur to press down streptococcus aureus active;
The blue flavones of Fig. 8 tiger fur to press down Salmonellas active;
The blue flavones of Fig. 9 tiger fur four component scavenging hydroxyl are active;
The blue flavones of Figure 10 tiger fur four component free radical scavengings;
It is active that the blue flavones of Figure 11 tiger fur four components remove superoxide anion;
The cytotoxic effect (contrast 10 × 4) of Figure 12 HeLa;
The blue flavones TFS-a of Figure 13 tiger fur is to the cytotoxic effect (10 × 4) of HeLa;
The blue flavones TFS-b of Figure 14 tiger fur is to the cytotoxic effect (10 × 4) of HeLa;
The blue flavones TFS-c of Figure 15 tiger fur is to the cytotoxic effect (10 × 4) of HeLa;
The blue flavones TFS-d of Figure 16 tiger fur is to the cytotoxic effect (10 × 4) of HeLa
The blue flavones of Figure 17 tiger fur is to the cytotoxic effect (10 × 4) of HeLa;
The cytotoxic effect (contrast 10 × 4) of Figure 18 SP2/0;
The blue flavones TFS-b of Figure 19 tiger fur is to the cytotoxic effect (10 × 4) of SP2/0;
The blue flavones TFS-c of Figure 20 tiger fur is to the cytotoxic effect (10 × 4) of SP2/0;
The blue flavones TFS-d of Figure 21 tiger fur is to the cytotoxic effect (10 × 4) of SP2/0;
The blue flavones of Figure 22 tiger fur is to the cytotoxic effect (10 × 4) of SP2/0;
The blue flavones of Figure 23 tiger fur is on the impact (contrast 10 × 4) of A549;
The blue flavones of Figure 24 tiger fur is on the impact (10 × 4) of A549;
The blue flavones of Figure 25 tiger fur is on the impact (contrast 10 × 4) of Hep-2;
The blue flavones of Figure 26 tiger fur is on the impact (10 × 4) of Hep-2;
The blue flavones of Figure 27 tiger fur is on the impact (contrast 10 × 4) of DK;
The blue flavones of Figure 28 tiger fur is on the impact (10 × 4) of DK.
Embodiment
the optimal extraction technology of the blue flavones of embodiment 1 tiger fur
(1) typical curve preparation
Take the rutin standard substance 0.0050g of dry fully also constant weight, be the dissolve with ethanol of 60% by concentration, and be settled to 50mL, obtain the rutin standardized solution that concentration is 0.lmg/mL.Get standardized solution 0.0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL in 6 10mL scale test tubes, mend to 5.0mL with 60% ethanol, add 0.3mL5% (W/V) sodium nitrite solution, shake up, 0.3mL10% (W/V) aluminum nitrate solution is added after placing 6min, place 6min, add 4.0mL1mol/L sodium hydroxide solution again, mixing, with 60% alcohol dilution to scale (adding the ethanol of 0.4mL60% again), after normal temperature puts 15min, measure its absorbancy, the ethanol that blank gets 60% adds above-mentioned solvent, and drawing standard curve, as Fig. 1.
(2) the blue flavones content method of calculation of tiger fur
Get fresh tiger fur blue, shred after cleaning, be cooled to room temperature 5min, under normal pressure, steam 6-8min again, be placed in drying, dry in the shade in ventilation, after pulverizing with disintegrating machine, cross 40 mesh sieves, obtain the blue powder of dry tiger fur.Take the blue powder 2.0g of dry tiger fur in 50mL triangular flask, add ethanolic soln by certain liquid ratio (V/m), extract by certain hour, suction filtration.Filter residue extracts with aforesaid method, suction filtration, and merging filtrate, then uses appropriate petroleum ether degreasing, obtain sample liquid.Get each 1.0mL of gained sample solution under 60% ethanol and various extracting conditions, in 10mL scale test tube, adopt above-mentioned Sodium Nitrite-aluminum nitrate solution treatment process, at its light absorption value of 510nm wavelength place's colorimetric estimation, blank solution is done with the ethanol of above-mentioned 60%, utilize the standardized solution of preparation to measure the content of the blue flavones of tiger fur, be yield.
Total flavones yield (mg/g)=(C*V
1* V
2* 10
-3)/(W*V
0)
In formula: C: the concentration (mg/mL) measuring sample liquid;
V
0: the volume (mL) measuring absorbancy sample liquid used;
V
1: dilute volume (mL) during mensuration;
V
2: volume (mL) after sample liquid constant volume;
W: sample quality (g).
(3) the blue extracting flavonoids processing parameter of tiger fur is selected and optimizes
The blue flavones of ultrasonic wave added extraction using alcohol tiger fur, respectively with alcohol concn, solid-liquid ratio, ultrasonic time and ultrasonic temperature are that principal element carries out single factor experiment.Wherein, volume fraction of ethanol is respectively 40%, 50%, 60%, 70%, 80%, and solid-liquid ratio is respectively 10:1,15:1,20:1,25:1,30:1; Ultrasonic time is respectively 10min, 20min, 30min, 40min, 50min; Ultrasonic temperature is respectively 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C; Ultrasonic power is respectively 50W, 60W, 70W, 80W, 90W, 100W, 110W, 120W.The blue flavones of the tiger fur obtained after each single factor test process adopts above-mentioned formula to calculate.
The extraction process of the blue flavones of final optimization pass tiger fur is, alcohol concn 60%, liquid ratio 25:1, ultrasonic time 34min, ultrasonic temperature 80 DEG C, ultrasonic power 90-100W, the blue flavones content of tiger fur is 8.501mg/g under this condition, relatively with predictor is reduced to 0.097mg/g.
the purifying of the blue flavones of embodiment 2 tiger fur
(1) preparation of the blue flavones of tiger fur
2 times of volume sherwood oils are added in the blue ultrasonic extract of tiger fur described in embodiment 1, extracted several times, layer to be extracted is close to discarding petroleum ether layer solution time colourless, flavone extractive after rotary evaporation decolouring, volatilization ethanol, add the extraction into ethyl acetate collection ethyl acetate layer of 1 times of volume, repeated multiple times extraction in surplus solution after, combined ethyl acetate evaporates on water-bath, is the blue flavones raw product of tiger fur after volatilizing.
(2) the blue flavones silica column purification of tiger fur
Be loaded in beaker by 400g silica gel, the baking oven being placed in 120 DEG C activates 5h, is saved backup by the silica gel sealing after activation.The silica gel activated is dissolved in chloroform, constantly prolong same direction to stir, remove bubble, pour in chromatography column (3.5cm × 50cm) with glass stick drainage is disposable, open chromatography column bottom piston, silica gel is slowly sunk under gravity, silica gel will be kept during the course to be in all the time below chloroform liquid level.The flavones raw product getting the above-mentioned preparation of 0.5g is dissolved in methanol solution, adds 15g silica gel mixed sample, and after fully stirring, (50 DEG C) volatilize methyl alcohol in an oven, make the abundant adsorption sample of silica gel.Silica gel containing sample is added to silicagel column capital, treats that silica-gel powder precipitation surface is smooth, add a cover a circular filter paper sheet above in order to avoid break up silica gel when moving phase is dripped.Selection speed is 1mL/min, and eluent system is chloroform-methanol, adopts 18:1,15:1,12:1,9:1,6:1,3:1,1:1,1:3,1:6,1:9,1:12,1:18 to carry out gradient elution successively, each gradient elution 4h.Collect elutriant respectively according to I, II, III, IV 4 elution peaks in Fig. 2, obtain the blue flavonoid component of four kinds of tiger furs through concentrated, lyophilize and be followed successively by TFS-a, TFS-b, TFS-c, TFS-d, yield is respectively 18.1%, 25.7%, 16.4%, 10.3%.
the foundation of embodiment 3 tiger fur blue flavones HPLC detection method
(1) the blue flavones qualitative detection of tiger fur
Measure a small amount of rutin 1, rutin 2, Quercetin, kaempferol standard substance are dissolved in 10mL methyl alcohol, solution in contrast.Get the blue flavones 0.2g of tiger fur to be dissolved in 10mL methanol solution.Get contrast liquid and each 6 μ L points of trial-product liquid in same silica gel g thin-layer plate.Be ethyl acetate at developping agent: formic acid: water=8:1:1 and developping agent are benzene: ethyl acetate: launch in formic acid=8:3:1.After expansion terminates, dry thin layer plate, evenly spray with 1% aluminum chloride, after drying colour developing in stink cupboard, observe under 254nm.Result shows, when being ethyl acetate at developping agent: formic acid: during water=8:3:1, polarity is bigger than normal, as shown in Figure 3, can run out of rutin band, but Quercetin and kaempferol all goes to forward position, has the spot corresponding with rutin in flavones raw product; When developping agent is benzene: ethyl acetate: during formic acid=8:4:2, polarity is relatively little, as shown in Figure 4, flavones raw product not with kaempferol, spot that Quercetin is corresponding, and similar to rutin standard substance speckle displacement, be present in zero position.Illustrate containing rutin flavonoid substances in the blue flavones of extracted tiger fur, not containing Quercetin, kaempferol analogue.
(2) the blue flavones detection by quantitative of tiger fur.
Precision takes rutin standard substance 0.032g, and methanol constant volume is in 100mL volumetric flask, and 0.45 μm/mL membrane filtration, is 0.32mg/mL rutin standard substance for subsequent use.Get blue flavones TFS-a, TFS-b, TFS-c, the TFS-d of above-mentioned tiger fur tetra-each 0.05g of component powders, with dissolve with methanol, constant volume in 10mL volumetric flask, 0.45 μm/mL membrane filtration, as sample solution.Adopt HPLC method to detect, through groping, testing conditions is: WondaSilC18 silicagel column, 25 DEG C of column temperatures, and moving phase is methyl alcohol: water=(40:60), and determined wavelength is 284nm, and flow velocity is 1mL/min, sample size 20 μ L.Result shows, detect precision 0.54%, the blue flavonoid component content of TFS-a, TFS-b, TFS-c, TFS-d tetra-kinds of tiger furs is respectively 0.62mg/g, 1.24mg/g, 1.04mg/g, 0.27mg/g, and the rate of recovery is respectively 100.21%, 99.06%, 99.31%, 99.71%.Measure rutin Flavonoid Content in four kinds of compositions through thin layer chromatography scanner and be respectively 91.2%, 92.3%, 90.4% and 90.1%.
the bacteriostatic activity of the blue flavones of embodiment 4 tiger fur
Tiger fur described in Example 2 blue flavones raw product 0.15g, is dissolved in 10mL distilled water, is made into the blue flavonoids solution of tiger fur that concentration is 15mg/mL, through 0.45 μm of filtering with microporous membrane slagging-off.With intestinal bacteria, Salmonellas, streptococcus aureus, subtilis for examination criteria bacterial strain.Conventional filter paper enzyme is adopted to detect inhibition zone size, criterion: it is less sensitive that antibacterial circle diameter is less than 12.0mm, inhibition zone 12.0 ~ 16.0 is medium sensitivity, and antibacterial circle diameter 16.0 ~ 20.0mm is extremely sensitive, and antibacterial circle diameter is greater than 20.0mm for extremely responsive.Result shows, as shown in figures 5-8, all has certain restraining effect when the blue flavones concentration of tiger fur is 15mg/mL to four kinds of bacteriums, wherein to Salmonellas and colibacillary restraining effect particularly evident; As shown in table 1, the blue flavones of tiger fur to streptococcus aureus less sensitive, to the medium sensitivity of subtilis, to intestinal bacteria and Salmonellas extremely sensitive.
blue flavones filter paper antibacterial circle diameter (mm) of table 1 tiger fur
Conventional tube dilution method is adopted to detect blue flavones minimum inhibitory concentration (MIC) of tiger fur, criterion: muddy shape appears in blank group test tube, and under the prerequisite of medicine (Pen .-Strep) contrast composition transparence, observe the opacity of tiger fur blue flavones group test tube.Result shows, as shown in table 2, and the MIC of the blue flavones of tiger fur to intestinal bacteria, Salmonellas, subtilis is 3.75mg/mL, is 7.5mg/mL to the MIC of streptococcus aureus.
the blue flavones of table 2. tiger fur is to the MIC of bacterium
In vitro the muddy a little colony growth having minority, represents with "+", if in test tube relatively muddiness have slightly many colony growths, represent with " ++ ", if complete muddiness in test tube, bacterium colony grows fine, and represents with " +++ "
the anti-oxidant activity of the blue flavones of embodiment 5 tiger fur
(1) hydroxy radical qiao (OH) of the blue flavones of tiger fur is removed
Getting Vc and 4 kind of tiger fur blue flavones TFS-a, TFS-b, TFS-c, TFS-d is respectively dissolved in 10mL distilled water, configuration strength of solution gradient is 2,4,6,8mg/mL.
Get 2mmol/L ferrous sulfate and each 2mL of 6mmol/L hydrogen peroxide, mix the salicylic ethanolic soln of rear 6mmol/L and be settled to 25mL, in the water bath with thermostatic control of 37 DEG C, react 15min, record absorbance at 510nm place, be A0.Adopt the distilled water solution of aforesaid method 2mL to replace above-mentioned hydrogen peroxide solution, the absorbance of acquisition is Ax0.Get 2mmol/L ferrous sulfate and each 2mL of 6mmol/L hydrogen peroxide, add 5mL different concns sample, after mixing, be settled to 25mL with the salicylic ethanolic soln of 6mmol/L, in the water bath with thermostatic control of 37 DEG C, react 15min, record absorbance at 510nm place.Clearance rate with following formulae discovery hydroxy radical qiao: OH clearance rate=1-[(Ax-Ax0)/A0] * 100%
Result shows, as shown in Figure 9, four kinds of blue flavonoid components of tiger fur all have scavenging(action) to hydroxy radical qiao.When concentration is 8mg/mL, the scavenging(action) of TFS-b is the strongest, clearance rate can reach 59.8%, TFS-a be 42.6%, TFS-c, TFS-d is 38.4%.
(2) the DPPH free radical scavenging activity of the blue flavones of tiger fur
Getting Vc and 4 kind of tiger fur blue flavones TFS-a, TFS-b, TFS-c, TFS-d is respectively dissolved in 10mL distilled water, configuration strength of solution gradient is 2,4,6,8mg/mL; Weigh 4mgDPPH free radical, adopt dehydrated alcohol to be made into the DPPH ethanolic soln of 0.02mg/mL.
In test tube, add 1.0mL dehydrated alcohol and 4.0mLDPPH ethanolic soln, mixing, measure absorbance at 517nm place, be designated as A0; Add 4.0mL0.02mg/mLDPPH ethanolic soln and l.0mL liquid to be measured, mixing, measure absorbance, be designated as A1; Add 4.0mL dehydrated alcohol and 1.0mL liquid to be measured, mixing, measure absorbance, be designated as A2.Clearance rate calculation formula: clearance rate=1-(A1-A2)/A0.
Result shows, as shown in Figure 10, four kinds of blue flavones of tiger fur all have scavenging(action) to DPPH.The half elimination ratio of Vc is 1.69mg/mL, and the half elimination ratio of TFS-b is 1.85mg/mL, and both are very close.Among four kinds of flavonoid components, the elimination effect of TFS-b to DPPH is best, and clearance rate is 78.6%, TFS-a be 59.2%, TFS-c be 48.7%, TFS-d is 31.2%.
(3) the superoxide anion scavenging capacity of the blue flavones of tiger fur
Get the blue flavones sterling TFS-a of Vc and 4 kind of tiger fur respectively, TFS-b, TFS-c, TFS-d be dissolved in 10mL distilled water, configuration strength of solution gradient is 2,4,6,8mg/mL.
Get 4.0mLTris-HCl damping fluid (0.5mol/L, pH8.2) in colorimetric cylinder, 25 DEG C of water-bath heating 20min, after adding the product to be tested 0.4mL of different concns respectively, add pyrogallol (being prepared by the 10mmol/LHCl) 0.6mL of 4.5mmol/L again, mix in rear 25 DEG C of water-baths and react, after 4min, add 10mmol/LHCl2 ~ 3 termination reaction immediately, with distilled water zeroing, measure absorbancy angle value A at 326nm place.Clearance rate calculation formula: clearance rate (%)=[1-(A
1-A
2)/A
0] × 100%
Wherein A
0: do not add absorbance when sample only adds pyrogallol
A
1: the absorbance of sample and pyrogallol full added-time
A
2: do not add absorbance when pyrogallol only adds sample
Result shows, as shown in figure 11, four kinds of blue flavonoid components of tiger fur all have scavenging(action) to ultra-oxygen anion free radical.The half elimination ratio of Vc is 1.64mg/mL, TFS-b is 1.85mg/mL, close to Vc level.When sterling concentration is 8.0mg/mL, TFS-b is 84.2% to the clearance rate of ultra-oxygen anion free radical, almost identical with Vc, and TFS-a, TFS-c, TFS-d clearance rate to ultra-oxygen anion free radical is respectively 72.4%, 50.3%, 41.5%.
the anti-tumor activity of the blue flavones raw product of embodiment 6 tiger fur
(1) cultivation of tumour cell
Human cervical cancer cell lines HeLa, Human Laryngeal Cancer Cell Hep-2, mouse myeloma cell line SP2/0, lung cancer cell line A549 are test cell, and Madin-Darby canine kidney(cell line) (MDCK) system DK is normal cell.What culturing cell was selected is RPMI-1640 substratum, adds 10mL cell culture fluid in 100mL culturing bottle, and often cross 24h and just change nutrient solution, 72h goes down to posterity once.When going down to posterity, old nutrient solution is discarded, and get 3mL trysinization liquid digestion attached cell 5min, jolting culturing bottle, makes attached cell come off completely, adds appropriate nutrient solution and goes down to posterity.At 37 DEG C, 5%CO
2cultivate under condition.After 3mL trypsin solution peptic cell 5min, basis of microscopic observation cell shape, outwell trypsinase, add 3 ~ 5mL substratum, 2000 leave heart 5min, abandon culture supernatants, new nutrient solution is added to bottom cell, piping and druming is dispersed into single cell suspension, joins in 96 porocyte culture plates, put into CO according to the amount of 5000 cells in every hole
2concentration is 5%, and temperature controls 2h in the incubator of 37 DEG C and makes it adherent.
(2) cell-specific toxicity test
The blue flavones raw product of tiger fur described in Example 2 and TFS-a, TFS-b, TFS-c, TFS-d tetra-components are the solution of 6mg/mL with aseptic PBS configuration concentration respectively, after above-mentioned cell cultures 24h, add flavones raw product and TFS-a, TFS-b, TFS-c, TFS-d tetra-components wherein respectively, arranging volume is 10 μ L and 20 μ L, two test group, often group arrange three parallel.37 DEG C, 5%CO
2cultivate 48h under condition, take out and be put in observations under inverted microscope.
Result shows, the growth of the blue flavones raw product of 10 μ L and 20 μ L tiger furs to Human cervical cancer cell lines Hela, mouse myeloma cell line SP2/0 all has significant restraining effect, and to the growth of human lung cancer cell line A549, Human Laryngeal Cancer Cell Hep-2 and normal cell system DK without significant difference.The growth of the blue flavones TFS-a high concentration components of tiger fur to Human cervical cancer cell lines Hela has restraining effect, and to the growth of human lung cancer cell line A549, Human Laryngeal Cancer Cell Hep-2, mouse myeloma cell line SP2/0 and normal cell system DK without significant difference.The growth of the blue flavones TFS-b high concentration components of tiger fur to Human cervical cancer cell lines Hela and mouse myeloma cell line SP2/0 has restraining effect, and to the growth of human lung cancer cell line A549, Human Laryngeal Cancer Cell Hep-2 and normal cell system DK without significant difference.The blue flavones TFS-c of tiger fur and the growth of TFS-d component to Human cervical cancer cell lines Hela, mouse myeloma cell line SP2/0 all have significant restraining effect, and to the growth of human lung cancer cell line A549, Human Laryngeal Cancer Cell Hep-2 and normal cell system DK without significant difference.As shown in table 3 and Figure 12 ~ 28.
the blue flavones raw product of table 3. tiger fur and each component are on the impact of tumour cell and normal cell growth
Claims (7)
1. the blue flavones of tiger fur, it is prepared by following method: by blue for tiger fur drying and crushing, extraction using alcohol; Petroleum ether extraction, abandons petroleum ether layer, extraction into ethyl acetate, volatilizes and obtains the blue flavones of tiger fur.
2. the blue flavones of tiger fur according to claim 1, is characterized in that: described extraction using alcohol be with volume fraction be the ethanol of 60% as extraction agent, preparation liquid ratio is 25:1, soaked overnight, at 80 DEG C, ultrasonic frequency 20-35Hz, power 90-100W, supersound extraction 34min, suction filtration.
3. the blue flavones of tiger fur according to claim 1 and 2, is characterized in that: the blue flavones of described tiger fur is dissolved in methanol solution, and add 15g silica gel mixed sample, after fully stirring, 48 DEG C-52 DEG C volatilize methyl alcohol in an oven; Silica gel containing sample being added to silicagel column capital, treating that silica-gel powder precipitation surface is smooth, take chloroform-methanol as elutriant, and flow velocity is that 1mL/min carries out gradient elution, obtains elution peak and is labeled as elution peak I, elution peak II, elution peak III, elution peak IV;
Merge and collect elution peak II elutriant, through concentrated, lyophilize, obtain the blue flavones TFS-b of tiger fur;
Merge and collect elution peak III elutriant, through concentrated, lyophilize, obtain the blue flavones TFS-c of tiger fur;
Merge and collect elution peak IV elutriant, through concentrated, lyophilize, obtain the blue flavones TFS-d of tiger fur.
4. the blue flavones of tiger fur according to claim 1 prepare anti-salmonella and or intestinal bacteria medicine in application.
5. the blue flavones TFS-b of tiger fur according to claim 3 applies the removing of hydroxy radical qiao, DPPH free radical, superoxide anion.
6. the application of the blue flavones of tiger fur according to claim 1 in the medicine of preparation treatment Human cervical cancer cell lines Hela or mouse myeloma cell line SP2/0.
7. the blue flavones TFS-c of tiger fur according to claim 3 or the application of the blue flavones TFS-d of tiger fur in preparation treatment Human cervical cancer cell lines Hela or mouse myeloma cell line SP2/0 medicine.
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| CN114208857A (en) * | 2021-12-31 | 2022-03-22 | 青岛农业大学 | Application of sansevieria trifasciata crude extract in preparing bactericide for preventing and treating plant diseases caused by downy mildew |
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| CN114208857A (en) * | 2021-12-31 | 2022-03-22 | 青岛农业大学 | Application of sansevieria trifasciata crude extract in preparing bactericide for preventing and treating plant diseases caused by downy mildew |
| CN114208857B (en) * | 2021-12-31 | 2022-08-30 | 青岛农业大学 | Application of sansevieria trifasciata crude extract in preparing bactericide for preventing and treating plant diseases caused by downy mildew |
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