CN107412300A - A kind of anti-inflammatory activity extract, anti-inflammatory composition and preparation method thereof - Google Patents
A kind of anti-inflammatory activity extract, anti-inflammatory composition and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of anti-inflammatory activity extract, anti-inflammatory composition and preparation method thereof.The extract that the active ingredient of the anti-inflammatory activity extract obtains after being extracted for the alcohol extract of white flower beggar-ticks Bidens alba (Linn.) DC herbs further across petroleum ether or ethyl acetate or chloroform.Its antiphlogistic effects is better than white flower beggar-ticks Bidens alba (Linn.) DC water extract or alcohol extract.After anti-inflammatory drug preparation is further made in the petroleum ether extract of white flower beggar-ticks, when drug concentration is respectively 2.4,12.1,60.7mg/kg, Dichlorodiphenyl Acetate is suitable when causing mouse peritoneal capillary permeability to increase inhibition with aspirin 20mg/kg；So the anti-inflammatory products of the present invention have wide market prospects.
Description
Technical field
The invention belongs to feverfew extracts active ingredients and isolate and purify field, and in particular to a kind of white flower beggar-ticks
Bidens alba (Linn.) DC anti-inflammatory activities extract, anti-inflammatory composition and preparation method thereof.
Background technology
The Chinese of white flower beggar-ticks is more chaotic.Version in 2007《Guangzhou wild plant》With version in 2005
《Guangdong Plants register》It is that Bidens alba (Linn.) DC. is referred to as white flower beggar-ticks by Latin literary fame, by Bidens pilosa
Linn.var.radiata Sch.-Bip. are referred to as bidens pilosa；And version in 1979《Chinese Plants will》With 1999
Version《Chinese book on Chinese herbal medicine》Bidens pilosa Linn.var.radiata Sch.-Bip. are referred to as white flower beggar-ticks, it is right
Bidens alba (Linn.) DC. is not recorded.The reason for possible is that Bidens alba (Linn.) DC. in China are invaders
Kind, and be distributed only over the tropical and subtropical region such as the Guangdong, Guangxi and Hainan in China, in addition, only in recent years due to
Bidens alba (Linn.) DC. cause serious influence to the ecology of locality, just draw attention.Although Chinese is chaotic,
But most scholars point out the specific classification of the species when making a search, and mark Latin literary fame.Feelings unless otherwise specified
Under condition, the involved all white flower beggar-ticks of this patent research refer both to Bidens alba (Linn.) DC.
White flower beggar-ticks (Bidens alba (Linn.) DC.) belongs to Dicotyledoneae (Dicotyledoneae) chrysanthemum mesh
(Asterales) one kind of composite family (Compositae) Bidens (Biens Linn.), 1 year or herbaceos perennial,
Originate in tropical America.In China, white flower Bidens is the pernicious evil that a kind of propagation is fast, prevention and control are difficult, harm is big in invasive species
Grass, by road, large area simple community is formed in mountain forest side, crops rand and black fallow, has a strong impact on other biological thing
The existence of kind, the anti-governor pressure of ecology are huge.
At present, white flower beggar-ticks Bidens alba (Linn.) DC is mainly distributed on the Lingnan area in China, including Guangdong,
The provinces such as Guangxi, Hainan, it is especially widely distributed (Tian Xingshan etc., 2010) in Guangdong Province.The pyrophilous high humidity weather of white flower beggar-ticks,
South of the Five Ridges northern area does not have white flower beggar-ticks Bidens alba (Linn.) DC growth substantially；And in contrast, Bidens bipinnata
Other species of grass category are then relatively fewer in Lingnan area.The significant differences such as its region grown determine white flower beggar-ticks
Bidens alba (Linn.) DC may with other species of Bidens in physiological property it is different.
Research about white flower beggar-ticks Bidens alba (Linn.) DC is less, and the research especially on biological utilisation is more
It is few.White flower beggar-ticks Bidens alba (Linn.) DC, if can be by its thoroughly research, and be provided as a kind of invasive species
Sourceization utilizes, and will be significant.
The content of the invention
It is an object of the invention to provide a kind of white flower beggar-ticks Bidens alba (Linn.) DC anti-inflammatory activities extract,
Anti-inflammatory composition and preparation method thereof.
The technical solution used in the present invention is：
A kind of anti-inflammatory activity extract, its active ingredient are the alcohol of white flower beggar-ticks Bidens alba (Linn.) DC herbs
The extract that extract obtains after being extracted further across organic solvent；Wherein, organic solvent includes petroleum ether, ethyl acetate, chlorine
Imitative, n-hexane, normal heptane, ether, benzene,toluene,xylene, pentane, hexane, octane, hexamethylene, cyclohexanone, chlorobenzene, dichloro
Benzene, dichloromethane, methanol, isopropanol, oxirane, methyl acetate, propyl acetate, acetone, glycol monoethyl ether, ethylene glycol list
Ether, acetonitrile, or its isomer.
Preferably, organic solvent includes petroleum ether, ethyl acetate, chloroform.
Applicant passes through experimental verification, if obtained with water extraction white flower beggar-ticks Bidens alba (Linn.) DC herbs
Extract, be substantially not detectable activity.If only extract white flower beggar-ticks Bidens alba (Linn.) DC with ethanol solution
Herb obtains extract, can detect weaker activity.And by alcohol extract further across petroleum ether or ethyl acetate or chlorine
The extract obtained after imitative extraction, its anti-inflammatory activity are stronger.
A kind of preparation method of anti-inflammatory activity extract, comprises the following steps：
White flower beggar-ticks Bidens alba (Linn.) DC herb is shone into dry grinding, adds ethanol solution, ultrasonic extraction,
Extract solution is obtained, centrifugation removes chlorophyll, is then concentrated under reduced pressure and removes ethanol；Suitable quantity of water is added, is extracted, obtained with organic solvent
Anti-inflammatory activity extract；
Wherein, organic solvent includes petroleum ether, ethyl acetate, chloroform, n-hexane, normal heptane, ether, benzene, toluene, diformazan
Benzene, pentane, hexane, octane, hexamethylene, cyclohexanone, chlorobenzene, dichloro-benzenes, dichloromethane, methanol, isopropanol, oxirane, vinegar
Sour methyl esters, propyl acetate, acetone, glycol monoethyl ether, ethylene glycol monoethyl ether, acetonitrile, or its isomer.
Preferably, the concentration of volume percent of ethanol solution is 50%-99%.
Preferably, the concentration of volume percent of ethanol solution is 75%-95%.
Preferably, white flower beggar-ticks Bidens alba (Linn.) DC herb is shone into dry grinding, is 1 according to solid-liquid ratio:
4-1:25 add ethanol solution.
Preferably, white flower beggar-ticks Bidens alba (Linn.) DC herb is shone into dry grinding, is 1 according to solid-liquid ratio:
5-1:20 add ethanol solution.
Preferably, the condition of ultrasonic extraction includes：Frequency is 20-1100KHz, and temperature is 20-50 DEG C, time 30-
120min, ultrasonic number are 1-5 times.
Preferably, organic solvent includes petroleum ether, ethyl acetate, chloroform.
Preferably, during extraction, it is the 1-5 times of petroleum ether measured or ethyl acetate or chloroform to add volume, is extracted 1-5 times, is closed
And extract, it is concentrated under reduced pressure, is dried in vacuo, obtains anti-inflammatory activity extract.
Preferably, during extraction, it is the 1-3 times of petroleum ether measured or ethyl acetate or chloroform to add volume, is extracted 1-3 times, is closed
And extract, it is concentrated under reduced pressure, is dried in vacuo, obtains anti-inflammatory activity extract.
A kind of anti-inflammatory composition, it is characterised in that：Its active ingredient is the anti-inflammatory activity extract described in any of the above-described.
Preferably, the anti-inflammatory composition also includes excipient substance.
Preferably, described auxiliary material includes at least one of following material：Solvent, propellant, solubilizer, stabilizer,
Glidant, flavouring, preservative, suspending agent, coating material, aromatic, anti-binder, integrated agent, penetration enhancer, pH value are adjusted
Section agent, buffer, plasticizer, cosolvent, emulsifying agent, colouring agent, binder, disintegrant, filler, lubricant, wetting agent, ooze
Pressure conditioning agent, surfactant, foaming agent, defoamer, thickener, inclusion agents, NMF, absorbent, diluent, flocculant thoroughly
With deflocculant, filter aid, release retarding agent.
Preferably, anti-inflammatory composition includes various formulations：
Classified according to the decentralized system of formulation, specifically, following formulation can be made：Solution-type, colloidal solution
Type, emulsion-type, suspension type, gas decentralized, microdispersed form, solid dispersing；
According to typoiogical classification, specifically, following formulation can be made：Liquid dosage form (such as aromatic waters, solution, note
Penetrate agent, mixture, lotion, liniment etc.), gas formulation (such as aerosol, spray), solid dosage forms (such as powder, pill, tablet,
Film etc.), semisolid dosage form (such as ointment, suppository, paste)；
Classify according to method of administration, specifically, following formulation can be made：Formulation through gastrointestinal administration, without stomach
The formulation of intestinal canal administration.
A kind of preparation method of anti-inflammatory composition, the anti-inflammatory activity extract hypotoxicity that any of the above-described is prepared
Solvent dissolves, and adds hypotoxicity auxiliary agent, anti-inflammatory composition is prepared.
Preferably, low-toxic solvent is dimethyl sulfoxide.
Preferably, hypotoxicity auxiliary agent is Tween-80.
Anti-inflammatory activity extract or anti-inflammatory composition described in any of the above-described is in treatment active chronic inflammation medicine is prepared
Application.
The active chronic inflammation is including involved by pneumonia, hepatitis, ephritis, gastroenteritis, appendicitis, upper respiratory tract infection
And inflammation.
The beneficial effects of the invention are as follows：
The invention discloses a kind of anti-inflammatory activity extract and anti-inflammatory composition, its active ingredient is white flower beggar-ticks
What the alcohol extract of Bidens alba (Linn.) DC herbs obtained after being extracted further across petroleum ether or ethyl acetate or chloroform
Extract, its effect are better than white flower beggar-ticks Bidens alba (Linn.) DC water extract or alcohol extract.
Present invention research finds that obtaining white flower beggar-ticks anti-inflammatory activity extract and its pharmaceutical preparation has preferable anti-inflammatory
Effect, when petroleum ether extract concentration is 12.6 μ g/ml, generation is discharged to the RAW264.7 macrophages NO of LPS inductions
96.9% suppression；After anti-inflammatory drug preparation is further made in the petroleum ether extract of white flower beggar-ticks, drug concentration difference
For 2.4,12.1,60.7mg/kg when, Dichlorodiphenyl Acetate cause mouse peritoneal capillary permeability increase inhibition and aspirin
It is suitable during 20mg/kg.It is huge to the RAW264.7 of LPS inductions when the chloroform extract concentration of white flower beggar-ticks is 16.7 μ g/mL
Phagocyte NO releases produce 85.8% suppression；Anti-inflammatory drug preparation is made in the chloroform extract of white flower beggar-ticks, in dosage
During 0.8mg/kg, it is suitable with 20mg/kg aspirin that Dichlorodiphenyl Acetate causes mouse peritoneal capillary permeability to increase inhibition.When
When the acetic acid ethyl ester extract concentration of white flower beggar-ticks is more than 0.8 μ g/mL, the RAW264.7 macrophages NO of LPS inductions is released
Put the suppression of generation more than 90%.Aspirin is a kind of traditional antipyretic, analgesia, anti-inflammatory drug, for curing cold, generating heat,
The diseases such as headache, toothache and rheumatoid arthritis.So the anti-inflammatory drug preparation of the present invention, its drug concentration is in 0.8-
The effect of 20mg/kg aspirin is obtained with during 12.1mg/kg, there are wide market prospects.
White flower beggar-ticks (Bidens alba (Linn.) DC.) is used as a kind of invasive species, have propagate fast, prevention and control are difficult,
Endanger the features such as big, the utilization of resources of research of the invention to white flower beggar-ticks (Bidens alba (Linn.) DC.), which melts, wards off
Road, is of great immediate significance.
Brief description of the drawings
Fig. 1 is cell of the white flower beggar-ticks anti-inflammatory activity extract (petroleum ether extract) to RAW264.7 of embodiment 1
Toxicity test result；
Fig. 2 is that the white flower beggar-ticks anti-inflammatory activity extract (petroleum ether extract) of embodiment 1 is induced lipopolysaccharides LPS
RAW264.7 NO release inhibiting rate experimental results；
Fig. 3 is that the white flower beggar-ticks anti-inflammatory drug preparation (petroleum ether extract) of embodiment 1 causes mouse peritoneal for acetic acid
Capillary permeability increases inhibition assay result；
Fig. 4 is cell of the white flower beggar-ticks anti-inflammatory activity extract (chloroform extract) for RAW264.7 of embodiment 2
Toxicity test result；
Fig. 5 is that the white flower beggar-ticks anti-inflammatory activity extract (chloroform extract) of embodiment 2 induces for lipopolysaccharides LPS
RAW264.7 NO release inhibiting rate experimental results；
Fig. 6 is that the white flower beggar-ticks anti-inflammatory drug preparation (chloroform extract) of embodiment 2 causes mouse peritoneal hair for acetic acid
Thin vasopermeability increases inhibition assay result；
Fig. 7 is the white flower beggar-ticks anti-inflammatory activity extract (acetic acid ethyl ester extract) of embodiment 3 for RAW264.7's
Cytotoxicity experiment result；
Fig. 8 is that the white flower beggar-ticks anti-inflammatory activity extract (acetic acid ethyl ester extract) of embodiment 3 lures for lipopolysaccharides LPS
Lead RAW264.7 NO release inhibiting rate experimental results；
Fig. 9 is cytotoxicity experiment result of the white flower beggar-ticks water extract to RAW264.7 of comparative example 1；
Figure 10 is cytotoxicity experiment result of the white flower beggar-ticks alcohol extract to RAW264.7 of comparative example 2.
Embodiment
In the case of unless otherwise specified, all white flower beggar-ticks involved by this patent refer both to Bidens alba
(Linn.)DC.The white flower beggar-ticks kind of this patent has already passed through South China Botanical Garden Chinese Academy of Sciences Yi Qifei assistant researcher mirror
It is fixed.
A kind of anti-inflammatory activity extract, its active ingredient are the alcohol of white flower beggar-ticks Bidens alba (Linn.) DC herbs
The extract that extract obtains after being extracted further across organic solvent；Wherein, organic solvent includes petroleum ether, ethyl acetate, chlorine
Imitative, n-hexane, normal heptane, ether, benzene,toluene,xylene, pentane, hexane, octane, hexamethylene, cyclohexanone, chlorobenzene, dichloro
Benzene, dichloromethane, methanol, isopropanol, oxirane, methyl acetate, propyl acetate, acetone, glycol monoethyl ether, ethylene glycol list
Ether, acetonitrile, or its isomer.
Preferably, organic solvent includes petroleum ether, ethyl acetate, chloroform.
A kind of preparation method of anti-inflammatory activity extract, is comprised the following steps that：
White flower beggar-ticks Bidens alba (Linn.) DC herb is shone into dry grinding, granularity is 10-40 mesh, according to feed liquid
Than 1:4-1:25 add the ethanol solution that concentration of volume percent is 50%-99%, and ultrasonic extraction is 20- including frequency
1100KHz, temperature are 20-50 DEG C, and time 30-120min, ultrasonic number is 1-5 times, obtains extract solution.Extract solution decompression is dense
Certain volume is reduced to, centrifugation removes chlorophyll, then proceedes to be concentrated into no alcohol taste, appropriate distilled water dissolving is added, with 1-5 times
Measure petroleum ether or ethyl acetate or chloroform equal solvent extracts 1-5 times, combining extraction liquid, be concentrated under reduced pressure, be dried in vacuo, obtain white flower
Beggar-ticks Bidens alba (Linn.) DC anti-inflammatory activity extracts.
A kind of anti-inflammatory composition, its active ingredient are above-mentioned anti-inflammatory activity extract.
Preferably, the anti-inflammatory composition also includes excipient substance；Described auxiliary material includes at least one of following material：
Solvent, propellant, solubilizer, stabilizer, glidant, flavouring, preservative, suspending agent, coating material, aromatic, anti-bonding
Agent, integrated agent, penetration enhancer, pH value regulator, buffer, plasticizer, cosolvent, emulsifying agent, colouring agent, binder, collapse
Solve agent, filler, lubricant, wetting agent, osmotic pressure regulator, surfactant, foaming agent, defoamer, thickener, inclusion
Agent, NMF, absorbent, diluent, flocculant and deflocculant, filter aid, release retarding agent.
The anti-inflammatory composition includes various formulations：
Classified according to the decentralized system of formulation, specifically, following formulation can be made：Solution-type, colloidal solution
Type, emulsion-type, suspension type, gas decentralized, microdispersed form, solid dispersing；
According to typoiogical classification, specifically, following formulation can be made：Liquid dosage form (such as aromatic waters, solution, note
Penetrate agent, mixture, lotion, liniment etc.), gas formulation (such as aerosol, spray), solid dosage forms (such as powder, pill, tablet,
Film etc.), semisolid dosage form (such as ointment, suppository, paste)；
Classify according to method of administration, specifically, following formulation can be made：Formulation through gastrointestinal administration, without stomach
The formulation of intestinal canal administration.
With reference to embodiment, the invention will be further described, but is not limited thereto.
Embodiment 1
White flower beggar-ticks Bidens alba (Linn.) DC herb is shone into dry grinding, granularity is 25 mesh, according to solid-liquid ratio
1:8 add the ethanol solution that concentration of volume percent is 95%, and ultrasonic extraction includes：Frequency is 800KHz, 30 DEG C of temperature, the time
60min, ultrasonic number 3 times, obtain extract solution.Extract solution is concentrated under reduced pressure into certain volume, and centrifugation removes chlorophyll, then proceeded to
No alcohol taste is concentrated into, adds appropriate distilled water dissolving, petroleum ether extraction is measured 3 times with 2 times, combining extraction liquid, is concentrated under reduced pressure, in
800Pa, 150min is dried in vacuo under the conditions of 45 DEG C, obtains white flower beggar-ticks anti-inflammatory activity extract.
White flower beggar-ticks anti-inflammatory activity extract powder is dissolved with dimethyl sulfoxide, recycles Tween-80 to be helped as medicine
Agent, it is prepared into the white flower beggar-ticks anti-inflammatory drug preparation of each concentration.
White flower beggar-ticks anti-inflammatory activity extract (i.e. the petroleum ether extract of alcohol extract) is obtained to embodiment 1 and carries out work(
The experimental study of energy aspect.
First, white flower beggar-ticks anti-inflammatory activity extract is tested to RAW264.7 Cytotoxic evaluations
Medicine dissolves and dilution：White flower beggar-ticks anti-inflammatory activity extract powder is dissolved in analytically pure DMSO, so
1/8 times is diluted to basal medium DMEM afterwards, DMSO concentration is reduced to 12.5%；Subsequent each secondary dilution, which uses, to be contained
12.5%DMSO basal medium two-fold dilution's medicines.
By RAW264.7 cells, (density is 2 × 105Individual/mL, 100 μ L) be inoculated in during 96 holes pull, after the completion of trained in constant temperature
37 DEG C of cultivation 24h in case are supported, 70-80% of bottom area or so is reached to attached cell, adds 3.3 μ L gradient concentration medicines,
It is 0.4% to make every hole DMSO contents.24h is cultivated, adds MTT (0.5mg/ml) 20 μ L, continues to cultivate 4h.Careful supernatant discarding
Afterwards and then 150 μ L DMSO dissolvings are added, shake 10min, after the first a ceremonial jade-ladle, used in libation of generation is crystallized fully dissolving, with ELIASA in 490nm
Place determines each hole absorbance, calculates cell inhibitory rate.Blank group (being not added with medicine, add equal cell nutrient solution) is set per plate, is adjusted
Zero group (be not added with cell, be not added with medicine, only adding 100 μ L basal mediums) and administration group, wherein each sample sets 6 again
Hole, test in triplicate altogether.As a result Fig. 1 is seen.
As can be known from Fig. 1, when extract concentrations are 25.5 μ g/ml, the inhibiting rate to RAW264.7 is 17.1%, is more than
During 25.5 μ g/ml, there is obvious cytotoxicity, during less than 25.5 μ g/ml, cytotoxicity is smaller.
2nd, white flower beggar-ticks anti-inflammatory activity extract discharges Inhibition test to lipopolysaccharide-induced RAW264.7 NO
By RAW264.7 cells, (density is 2 × 105Individual/mL, 100 μ L) be inoculated in during 96 holes pull, after the completion of in incubator
In 37 DEG C of cultivation 24h, reach 70-80% of bottom area or so to attached cell, add 3.3 μ L gradient concentration medicines, make every
Hole DMSO contents are 0.4%, and medicine final concentration is respectively 1.6,3.2,6.4,12.7 μ g/ml, continue after cultivating 6h, add eventually
The μ g/mL of concentration 2 LPS solution, continue to cultivate 24h.Then the μ L of cell supernatant 80 are transferred in 96 new orifice plates, add 20
μ LGriess reagents I and II.After vibrating 10min on constant-temperature mixer, NO absorbances are detected at 550 nm with ELIASA.
Blank group (being not added with LPS and medicine, add equal cell nutrient solution), model group (being not added with medicine, add equivalent LPS) and administration are set per plate
Group, each sample set 6 multiple holes, altogether 3 repeated experiments.As a result Fig. 2 is seen.
As can be known from Fig. 2, when the concentration of extract is 3.2-12.7 μ g/mL, LPS inductions can be significantly inhibited
RAW264.7 macrophages NO discharges, when reaching 12.7 μ g/mL, inhibiting rate 96.9%.
3rd, white flower beggar-ticks anti-inflammatory activity pharmaceutical preparation Dichlorodiphenyl Acetate causes mouse peritoneal capillary permeability to increase suppression real
Test
Kunming mice 60, male and female half and half, 20 ± 2g of body weight.It is randomly divided into normal group, negative control group (physiological saline
Group, this group are caused scorching but are not administered), positive drug control group (aspirin, 20mg/kg), the white flower beggar-ticks anti-inflammatory of embodiment 1
The high, medium and low dosage group of pharmaceutical preparation, every group 10.Pharmaceutical diluents, the administration of high, medium and low dosage group are used as by the use of Tween-80
Concentration is below Chinese medicine Conventional nontoxic Dose standard (5000mg/kg), and respectively 60.7,12.1,2.4mg/kg.
Each group mouse successive administration 3 days, the 0.5ml medicines of perfusion administration once a day.After last time administration 30min, press
The azovan blue of 10ml/kg dosage tail vein injection 0.5%, by the intraperitoneal injection of 10mg/kg dosage containing 0.6% vinegar after 60min
Sour physiological saline.After 50min, all mouse cervical dislocations are put to death, while to intraperitoneal injection 4ml physiological saline, gently rub it is several it is lower after,
Peritoneal exudate is collected, centrifuges (4 DEG C, 5min, 3000rpm/min), takes each group supernatant respectively, is detected in 590nm.To ooze out
The concentration that part is colored in liquid represents vasopermeability power.As a result Fig. 3 is seen.
As can be known from Fig. 3：When the concentration of anti-inflammatory component is respectively 2.4,12.1,60.7mg/kg, Dichlorodiphenyl Acetate causes mouse peritoneal
Capillary permeability is suitable when increasing inhibition with aspirin 20mg/kg.And the concentration of anti-inflammatory component is only
The effect suitable with aspirin 20mg/kg is obtained with during 2.4mg/kg, illustrates that the white flower beggar-ticks anti-inflammatory of the present invention is lived
Property pharmaceutical preparation has good market prospects.
Embodiment 2
White flower beggar-ticks Bidens alba (Linn.) DC herb is shone into dry grinding, granularity is 25 mesh, according to solid-liquid ratio
1:8 add the ethanol solution that concentration of volume percent is 95%, and ultrasonic extraction includes：Frequency is 800KHz, 30 DEG C of temperature, the time
60min, ultrasonic number 3 times, obtain extract solution.Extract solution is concentrated under reduced pressure into certain volume, and centrifugation removes chlorophyll, then proceeded to
No alcohol taste is concentrated into, adds appropriate distilled water dissolving, is extracted 3 times, combining extraction liquid, is concentrated under reduced pressure with 2 times of amount chloroforms, in
800Pa, 150min is dried in vacuo under the conditions of 45 DEG C, obtains white flower beggar-ticks anti-inflammatory activity extract.
White flower beggar-ticks anti-inflammatory extract powder is dissolved with dimethyl sulfoxide, recycles Tween-80 as pharmaceutical auxiliary agent, system
The standby white flower beggar-ticks anti-inflammatory drug preparation into each concentration.
White flower beggar-ticks anti-inflammatory activity extract (i.e. the chloroform extract of alcohol extract) is obtained to embodiment 2 and carries out function
The experimental study of aspect.
First, white flower beggar-ticks anti-inflammatory activity extract is tested to RAW264.7 Cytotoxic evaluations
Medicine dissolves and dilution：White flower beggar-ticks anti-inflammatory activity extract powder is dissolved in analytically pure DMSO, so
1/8 times is diluted to basal medium DMEM afterwards, DMSO concentration is reduced to 12.5%；Subsequent each secondary dilution, which uses, to be contained
12.5%DMSO basal medium two-fold dilution's medicines.
By RAW264.7 cells, (density is 2 × 105Individual/mL, 100 μ L) be inoculated in during 96 holes pull, after the completion of trained in constant temperature
37 DEG C of cultivation 24h in case are supported, 70-80% of bottom area or so is reached to attached cell, adds 3.3 μ L gradient concentration medicines,
It is 0.4% to make every hole DMSO contents.24h is cultivated, adds MTT (0.5mg/ml) 20 μ L, continues to cultivate 4h.Careful supernatant discarding
Afterwards, 150 μ L DMSO dissolvings are then added, shake 10min, after the first a ceremonial jade-ladle, used in libation of generation is crystallized fully dissolving, are existed with ELIASA
Each hole absorbance is determined at 490nm, calculates cell inhibitory rate.Blank group is set per plate (to be not added with medicine, add equal cell culture
Liquid), zeroing group (be not added with cell, be not added with medicine, only adding 100 μ L basal mediums) and administration group, wherein each sample sets 6
Individual multiple holes, test in triplicate altogether.As a result Fig. 4 is seen.
As can be known from Fig. 4, as 33.4 μ g/ml of extract concentrations, the inhibiting rate to RAW264.7 is 20.4%, is more than
During 33.4 μ g/ml, there is obvious cytotoxicity, during less than 33.4 μ g/ml, cytotoxicity is smaller.
2nd, white flower beggar-ticks anti-inflammatory activity extract discharges Inhibition test to lipopolysaccharide-induced RAW264.7 NO
By RAW264.7 cells, (density is 2 × 105Individual/mL, 100 μ L) be inoculated in during 96 holes pull, after the completion of in incubator
In 37 DEG C of cultivation 24h, reach 70-80% of bottom area or so to attached cell, add 3.3 μ L gradient concentration medicines, make every
Hole DMSO contents are 0.4%, and medicine final concentration is respectively 2.1,4.2,8.4,16.7 μ g/ml, continue after cultivating 6h, add eventually
The μ g/mL of concentration 2 LPS solution, continue to cultivate 24h.Then the μ L of cell supernatant 80 are transferred in 96 new orifice plates, add 20
μ LGriess reagents I and II.After vibrating 10min on constant-temperature mixer, NO absorbances are detected at 550 nm with ELIASA.
Blank group (being not added with LPS and medicine, add equal cell nutrient solution), model group (being not added with medicine, add equivalent LPS) and administration are set per plate
Group, each sample set 6 multiple holes, altogether 3 repeated experiments.As a result Fig. 5 is seen.
As can be known from Fig. 5, when the concentration of extract is in 2.1-16.7 μ g/mL, LPS inductions can be significantly inhibited
RAW264.7 macrophages NO discharges, when reaching 16.7 μ g/mL, inhibiting rate 85.8%.
3rd, white flower beggar-ticks anti-inflammatory activity pharmaceutical preparation Dichlorodiphenyl Acetate causes mouse peritoneal capillary permeability to increase suppression real
Test
Kunming mice 40, male and female half and half, 20 ± 2g of body weight.It is randomly divided into normal group, negative control group (physiological saline
Group, this group are caused scorching but are not administered), positive drug control group (aspirin, 20mg/kg), the white flower beggar-ticks anti-inflammatory of embodiment 2
Pharmaceutical preparation group, every group 10.Pharmaceutical diluents, administration concentration 0.8mg/kg, less than Chinese medicine usual non-toxic are used as by the use of Tween-80
Property Dose standard (5000mg/kg).
Each group mouse successive administration 3 days, the 0.5ml medicines of perfusion administration once a day.After last time administration 30min, press
The azovan blue of 10ml/kg dosage tail vein injection 0.5%, by the intraperitoneal injection of 10mg/kg dosage containing 0.6% vinegar after 60min
Sour physiological saline.After 50min, all mouse cervical dislocations are put to death, while to intraperitoneal injection 4ml physiological saline, gently rub it is several it is lower after,
Peritoneal exudate is collected, centrifuges (4 DEG C, 5min, 3000rpm/min), takes each group supernatant respectively, is detected in 590nm.To ooze out
The concentration that part is colored in liquid represents vasopermeability power.As a result Fig. 6 is seen.
As can be known from Fig. 6, when the concentration of anti-inflammatory component is in 0.8mg/kg, Dichlorodiphenyl Acetate causes mouse peritoneal capillary penetrating
Property is suitable when increasing inhibition with aspirin 20mg/kg.
Embodiment 3
White flower beggar-ticks Bidens alba (Linn.) DC herb is shone into dry grinding, granularity is 25 mesh, according to solid-liquid ratio
1:8 add the ethanol solution that concentration of volume percent is 95%, and ultrasonic extraction includes：Frequency is 800KHz, 30 DEG C of temperature, the time
60min, ultrasonic number 3 times, obtain extract solution.Extract solution is concentrated under reduced pressure into certain volume, and centrifugation removes chlorophyll, then proceeded to
No alcohol taste is concentrated into, adds appropriate distilled water dissolving, is extracted 3 times, combining extraction liquid, is concentrated under reduced pressure with 2 times of amount ethyl acetate,
150min is dried in vacuo under the conditions of 800Pa, 45 DEG C, obtains white flower beggar-ticks anti-inflammatory activity extract.
White flower beggar-ticks anti-inflammatory activity extract (i.e. the acetic acid ethyl ester extract of alcohol extract) progress is obtained to embodiment 3
Experimental study in terms of function.
First, white flower beggar-ticks anti-inflammatory activity extract is tested to RAW264.7 Cytotoxic evaluations
Medicine dissolves and dilution：White flower beggar-ticks anti-inflammatory activity extract powder is dissolved in analytically pure DMSO, so
1/8 times is diluted to basal medium DMEM afterwards, DMSO concentration is reduced to 12.5%；Subsequent each secondary dilution, which uses, to be contained
12.5%DMSO basal medium two-fold dilution's medicines.
By RAW264.7 cells, (density is 2 × 105Individual/mL, 100 μ L) be inoculated in during 96 holes pull, after the completion of trained in constant temperature
37 DEG C of cultivation 24h in case are supported, 70-80% of bottom area or so is reached to attached cell, adds 3.3 μ L gradient concentration medicines,
It is 0.4% to make every hole DMSO contents.24h is cultivated, adds MTT (0.5mg/ml) 20 μ L, continues to cultivate 4h.Careful supernatant discarding
Afterwards and then 150 μ L DMSO dissolvings are added, shake 10min, after the first a ceremonial jade-ladle, used in libation of generation is crystallized fully dissolving, with ELIASA in 490nm
Place determines each hole absorbance, calculates cell inhibitory rate.Blank group (being not added with medicine, add equal cell nutrient solution) is set per plate, is adjusted
Zero group (be not added with cell, be not added with medicine, only adding 100 μ L basal mediums) and administration group, wherein each sample sets 6 again
Hole, test in triplicate altogether.As a result Fig. 7 is seen.
As can be known from Fig. 7, when extract concentrations are 51.3 μ g/ml, the inhibiting rate to RAW264.7 is 18.0%, is more than
During 51.3 μ g/ml, there is obvious cytotoxicity；When extract concentrations are less than 51.3 μ g/ml, cytotoxicity is not shown.
2nd, white flower beggar-ticks anti-inflammatory activity extract discharges Inhibition test to lipopolysaccharide-induced RAW264.7 NO
By RAW264.7 cells, (density is 2 × 105Individual/mL, 100 μ L) be inoculated in during 96 holes pull, after the completion of in incubator
In 37 DEG C of cultivation 24h, reach 70-80% of bottom area or so to attached cell, add 3.3 μ L gradient concentration medicines, make every
Hole DMSO contents are 0.4%, and medicine final concentration is respectively 0.2,0.4,0.8,1.2 μ g/ml, continue after cultivating 6h, add eventually
The μ g/mL of concentration 2 LPS solution, continue to cultivate 24h.Then the μ L of cell supernatant 80 are transferred in 96 new orifice plates, add 20
μ LGriess reagents I and II.After vibrating 10min on constant-temperature mixer, NO absorbances are detected at 550 nm with ELIASA.
Blank group (being not added with LPS and medicine, add equal cell nutrient solution), model group (being not added with medicine, add equivalent LPS) and administration are set per plate
Group, each sample set 6 multiple holes, altogether 3 repeated experiments.As a result Fig. 8 is seen.
As can be known from Fig. 8, when the concentration of extract is 0.2-1.6 μ g/mL, LPS inductions can be significantly inhibited
RAW264.7 macrophages NO discharges, and when concentration is more than 0.8 μ g/mL, inhibiting rate is more than 90%.
Embodiment 4
White flower beggar-ticks Bidens alba (Linn.) DC herb is shone into dry grinding, granularity is 10 mesh, according to solid-liquid ratio
1:5 add the ethanol solution that concentration of volume percent is 65%, and ultrasonic extraction conditions include：Frequency is 100KHz, 25 DEG C of temperature,
Time 30min, ultrasonic number 5 times, obtain extract solution.Extract solution is concentrated under reduced pressure into certain volume, and centrifugation removes chlorophyll, then
Continue to be concentrated into no alcohol taste, add the dissolving of appropriate distilled water, with 3 times of amount petroleum ether extractions 2 times, be concentrated under reduced pressure, in 800Pa,
150min is dried in vacuo under the conditions of 45 DEG C, obtains white flower beggar-ticks anti-inflammatory activity extract.
Embodiment 5
White flower beggar-ticks Bidens alba (Linn.) DC herb is shone into dry grinding, granularity is 40 mesh, according to solid-liquid ratio
1:15 add the ethanol solution that concentration of volume percent is 85%, and ultrasonic extraction conditions include：Frequency is 1100KHz, temperature 50
DEG C, time 120min, ultrasonic number 1 time, obtain extract solution.Extract solution is concentrated under reduced pressure into certain volume, and centrifugation removes chlorophyll,
Then proceed to be concentrated into no alcohol taste, add appropriate distilled water dissolving, measure petroleum ether extraction 3 times with 1 times, combining extraction liquid, subtract
Pressure concentration, vacuum drying 150min obtains white flower beggar-ticks anti-inflammatory activity extract under the conditions of 800Pa, 45 DEG C.
Comparative example 1
White flower beggar-ticks Bidens alba (Linn.) DC herb is shone into dry grinding, granularity is 25 mesh, according to solid-liquid ratio
1:8 add ultra-pure waters, and then 110-120 DEG C is flowed back 2 hours, is repeated 2 times, and is merged Aqueous extracts, is concentrated under reduced pressure, in 800Pa, 45 DEG C
Under the conditions of be dried in vacuo 150min, obtain white flower beggar-ticks water extract.
Then as the experimental method of embodiment 1 carries out cytotoxicity experiment.As a result Fig. 9 is seen.
As shown in Figure 9, when the concentration of water extract is from 13 μ g/ml to 1669 μ g/ml, RAW264.7 inhibiting rate is occupied all the time
Between 9.6%-22.1%, and the increase with drug concentration is not occurred, the dose-dependant phenomenon of inhibiting rate increase.
It is generally acknowledged that cell inhibitory rate be less than 15% when, medicine be do not have it is cytotoxic.The possible cause for this phenomenon occur is water
Extract has weaker cytotoxicity to RAW264.7；Simultaneously because extract and separate is not carried out, carbohydrate, amino acid in water extract
Promote RAW264.7 growth Deng material, concrete reason needs further research.
Comparative example 2
White flower beggar-ticks Bidens alba (Linn.) DC herb is shone into dry grinding, granularity is 25 mesh, according to solid-liquid ratio
1:8 add the ethanol solution that concentration of volume percent is 95%, and ultrasonic extraction conditions are 800KHz, 30 DEG C of temperature, the time
60min, ultrasonic number 3 times, obtain extract solution.Extract solution is concentrated under reduced pressure into certain volume, and centrifugation removes chlorophyll, then proceeded to
800Pa is concentrated in, 150min is dried in vacuo under the conditions of 45 DEG C, obtains white flower beggar-ticks alcohol extract (the only first half of embodiment 1
Point, obtain alcohol extract part).
Experimental method with embodiment 1 carries out cytotoxicity experiment.As a result Figure 10 is seen.
From in Figure 10, the alcohol extract of white flower beggar-ticks is smaller to RAW264.7 cytotoxicity, when concentration is 305.5
There was only 12.9% to RAW264.7 inhibiting rate during μ g/ml, when concentration is 611.0 μ g/ml to RAW264.7 inhibiting rate
Have 17.4%.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
- A kind of 1. anti-inflammatory activity extract, it is characterised in that：Its active ingredient is white flower beggar-ticks Bidens alba (Linn.) The extract that the alcohol extract of DC herbs obtains after being extracted further across organic solvent；Wherein, organic solvent includes petroleum ether, second Acetoacetic ester, chloroform, n-hexane, normal heptane, ether, benzene,toluene,xylene, pentane, hexane, octane, hexamethylene, cyclohexanone, chlorine Benzene, dichloro-benzenes, dichloromethane, methanol, isopropanol, oxirane, methyl acetate, propyl acetate, acetone, glycol monoethyl ether, Ethylene glycol monoethyl ether, acetonitrile, or its isomer.
- 2. anti-inflammatory activity extract according to claim 1, it is characterised in that：Organic solvent includes petroleum ether, acetic acid second Ester, chloroform.
- 3. a kind of preparation method of anti-inflammatory activity extract, it is characterised in that comprise the following steps：White flower beggar-ticks Bidens alba (Linn.) DC herb is shone into dry grinding, ethanol solution is added, ultrasonic extraction, obtains Extract solution, centrifugation remove chlorophyll, are then concentrated under reduced pressure and remove ethanol；Suitable quantity of water is added, is extracted with organic solvent, obtains anti-inflammatory Activity extract；Wherein, organic solvent include petroleum ether, ethyl acetate, chloroform, n-hexane, normal heptane, ether, benzene,toluene,xylene, Pentane, hexane, octane, hexamethylene, cyclohexanone, chlorobenzene, dichloro-benzenes, dichloromethane, methanol, isopropanol, oxirane, tumer Ester, propyl acetate, acetone, glycol monoethyl ether, ethylene glycol monoethyl ether, acetonitrile, or its isomer.
- 4. preparation method according to claim 3, it is characterised in that：The concentration of volume percent of ethanol solution is 50%- 99%.
- 5. according to the preparation method described in any one of claim 3~4, it is characterised in that：By white flower beggar-ticks Bidens alba (Linn.) DC herb shines dry grinding, is 1 according to solid-liquid ratio:4~1:25 add ethanol solution.
- 6. preparation method according to claim 3, it is characterised in that the condition of ultrasonic extraction includes：Frequency is 20- 1100KHz, temperature are 20-50 DEG C, and time 30-120min, ultrasonic number is 1-5 times.
- 7. preparation method according to claim 3, it is characterised in that：Organic solvent includes petroleum ether, ethyl acetate, chlorine It is imitative.
- 8. preparation method according to claim 3, it is characterised in that：During extraction, addition volume is the 1-5 times of oil measured Ether, ethyl acetate or chloroform, extract 1-5 times, combining extraction liquid, be concentrated under reduced pressure, be dried in vacuo, obtain anti-inflammatory activity extract.
- A kind of 9. anti-inflammatory composition, it is characterised in that：Its active ingredient is that the anti-inflammatory activity described in claim any one of 1-2 carries Take thing.
- 10. prepared by the anti-inflammatory composition described in anti-inflammatory activity extract or claim 9 described in claim any one of 1-2 Treat the application in active chronic inflammation medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108079037A (en) * | 2017-12-11 | 2018-05-29 | 广东省农业科学院动物卫生研究所 | A kind of pharmaceutical composition with analgesic activity |
| CN108244345A (en) * | 2018-01-23 | 2018-07-06 | 广东省农业科学院动物卫生研究所 | Feed addictive of the beggar-ticks containing white flower and its preparation method and application |
-
2017
- 2017-07-31 CN CN201710635989.7A patent/CN107412300A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108079037A (en) * | 2017-12-11 | 2018-05-29 | 广东省农业科学院动物卫生研究所 | A kind of pharmaceutical composition with analgesic activity |
| CN108244345A (en) * | 2018-01-23 | 2018-07-06 | 广东省农业科学院动物卫生研究所 | Feed addictive of the beggar-ticks containing white flower and its preparation method and application |
| CN108244345B (en) * | 2018-01-23 | 2021-12-14 | 广东省农业科学院动物卫生研究所 | Feed additive containing bidens alba and preparation method and application thereof |
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