CN108619179A - Geranium extract and its medical usage - Google Patents
Geranium extract and its medical usage Download PDFInfo
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Abstract
The present invention provides Geranium extract and its medical usage, specifically, the present invention provides the four classification components and two activated monomers that extraction purification obtains from geranium wilfordii, shows preferable anti-TNF alpha activity and lower toxicity, can be used for the especially arthritic treatment of inflammation.
Description
Technical field
The present invention relates to the field of Chinese medicines, in particular it relates to Geranium extract and its inhibit TNF α preparing
Purposes in drug and anti-inflammatory drug.
Background technology
Geranium wilfordii (Geranium wilfordii) is MangNiu Er seedlings section Geranium plant, alias：Geranium wilfordii《Detailed outline picks up
It loses》, five leaves grass《The southern regions of the Yunnan Province book on Chinese herbal medicine》, old official grass《The southern regions of the Yunnan Province book on Chinese herbal medicine》, five-petaled flowers《The southern regions of the Yunnan Province book on Chinese herbal medicine collection of illustrative plates》, pass through grass always《This sketch of the southern regions of the Yunnan Province
Spectrum》, the Big Dipper grass《Classify herbal》.The main chemical compositions of geranium wilfordii include tannin, flavonoids, organic acid and volatile oil etc..
So far, it is known that the pharmacological action of geranium wilfordii includes antibacterial, antiviral, anti-oxidant, anti-hepatopathy, hemostasis and antitumor and other effects.
Tumor necrosis factor (Tumor necrosis factor alpha, TNF α) is a kind of multifunctional single nucleus element,
It is disengaged by macrophage, monocyte and natural killer cell and in acute and chronic inflammatory disease, antitumor reaction
And play pivotal player in infection.TNF α promotes inflammatory reaction, with many and relevant clinical problem of autoimmune disease
Development it is related, such as rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, psoriasis, psoriatic arthritis suppurates
Property mycoplasma and refractory asthma etc..And clinical research is it has been proven that TNF α is played the part of in the pathogenesis of rheumatoid arthritis
Drill key player.
These have all prompted an anti-inflammatory approach：Inhibit development of the activity of TNF α to control inflammation, the approach is to suffering from
The quality of life of person has good improvement result.TNF α antagonist treatment and inflammation related disease have been used at present.However,
TNF α antagonist Antiinflammatory Regimen haves the shortcomings that big toxicity, drug resistance or effect are relatively low.
Under the influence of today " back to nature " ideological trend, natural drug is developed into one powerful trend, and Chinese medicine
Cause the concern in the world with its abundant resource, unique curative effect, smaller toxic side effect again.Therefore, this field still needs to develop
With smaller toxic side effect, drug resistance and/or the efficient Chinese medicine with anti-inflammatory effect.
Invention content
In one aspect, the application provides Geranium extract A, is prepared by the following method：
(1) geranium wilfordii is extracted 1-5 times (such as 1,2,3,4 or 5 time) in ethyl alcohol or ethanol water, collects extracting solution
And concentrate, obtain heroubill total extract；
(2) the heroubill total extract is extracted with ethyl acetate 1-5 times (such as 1,2,3,4 or 5 time), collects acetic acid
Ethyl ester phase simultaneously concentrates, and gained concentrate is carried out gradient elution with water and methanol mixed solution on absorption resin, collects VWater：
VMethanol=(50:50)-(30:70) eluent when and concentration obtain the heroubill extract A.
Term " gradient elution " use herein refers to a kind of by constantly changing mobile phase concentration proportioning, so that
The component that property differs greatly in sample realizes the elution process of good separation.In elution process, can by the means such as TLC into
Line trace, with distinguish different component form and/or proportion of mobile phase under eluent.
In certain preferred aspects, the method has at least one of following features：
1) it in step (1), will be extracted in 5-12L ethyl alcohol or ethanol water per Kg geranium wilfordiis；
2) in step (1), geranium wilfordii is extracted in 30-85wt% ethanol waters；
3) in step (1), geranium wilfordii is extracted in room temperature；
4) in step (2), by the concentrate on macroporous absorbent resin gradient elution；
5) in step (2), by gained concentrate VWater：VMethanol=(95:5)-(30:70) mixed solution carries out gradient
Elution.
In certain preferred aspects, in step (1), geranium wilfordii is carried in 30-70wt% ethanol waters
It takes；Such as it is extracted in the ethanol water of 30wt%, 40wt%, 50wt%, 60wt% or 70wt%.
In certain preferred aspects, in step (2), by the concentrate on D101 type macroporous absorbent resins
Carry out gradient elution.
In certain preferred aspects, in step (2), V is collectedWater：VMethanol=40:Eluent when 60.
In certain preferred aspects, in step (2), according to concentrate described in following gradient elution：
VWater：VMethanol=95：5；
VWater：VMethanol=80：20；
VWater：VMethanol=70：30；
VWater：VMethanol=60：40；
VWater：VMethanol=50：50；
VWater：VMethanol=40：60；
VWater：VMethanol=30：70.
In certain preferred aspects, in step (2), according to concentrate described in following gradient elution：
VWater：VMethanol=95：5；
VWater：VMethanol=80：20；
VWater：VMethanol=60：40；
VWater：VMethanol=40：60.
On the other hand, the application provides Geranium extract A ', is prepared by the following method：
The Geranium extract A is purified using silica gel column chromatography method, is washed with chloroform and methanol system gradient
It is de-, collect VChloroform：VMethanol=(12:1)-(8:1) eluent when and concentration obtain the heroubill extract A '.
In certain preferred aspects, V is collectedChloroform：VMethanol=10:Eluent when 1.
In certain preferred aspects, according to Geranium extract A described in following gradient elution：
VChloroform：VMethanol=20：1；
VChloroform：VMethanol=15：1；
VChloroform：VMethanol=12：1；
VChloroform：VMethanol=10：1；
VChloroform：VMethanol=8：1.
In certain preferred aspects, according to Geranium extract A described in following gradient elution：
VChloroform：VMethanol=15：1；
VChloroform：VMethanol=10：1.
On the other hand, the application provides Geranium extract B, is prepared by the following method：
(1) geranium wilfordii is extracted 1-5 times (such as 1,2,3,4 or 5 time) in ethyl alcohol or ethanol water, collects extracting solution
And concentrate, obtain heroubill total extract；
(2) the heroubill total extract is extracted with ethyl acetate 1-5 times (such as 1,2,3,4 or 5 time), collects acetic acid
Ethyl ester phase simultaneously concentrates, and gained concentrate is carried out gradient elution with water and methanol mixed solution on absorption resin, collects VWater：
VMethanol=(70:30)-(50:50) eluent when and concentration obtain the heroubill extract B.
In certain preferred aspects, the method has at least one of following features：
1) it in step (1), will be extracted in 5-12L ethyl alcohol or ethanol water per Kg geranium wilfordiis；
2) in step (1), geranium wilfordii is extracted in 30-85wt% ethanol waters；
3) in step (1), geranium wilfordii is extracted in room temperature；
4) in step (2), by the concentrate on macroporous absorbent resin gradient elution；
5) in step (2), by gained concentrate VWater：VMethanol=(95:5)-(50:50) mixed solution carries out gradient
Elution.
In certain preferred aspects, in step (1), geranium wilfordii is carried in 30-70wt% ethanol waters
It takes.Such as it is extracted in 30wt%, 40wt%, 50wt%, 60wt% or 70wt% ethanol waters.
In certain preferred aspects, in step (2), V is collectedWater：VMethanol=60:Eluent when 40.
In certain preferred aspects, in step (2), by the concentrate on D101 type macroporous absorbent resins
Carry out gradient elution.
In certain preferred aspects, in step (2), according to concentrate described in following gradient elution：
VWater：VMethanol=95：5；
VWater：VMethanol=80：20；
VWater：VMethanol=70：30；
VWater：VMethanol=60：40；
VWater：VMethanol=50：50.
In certain preferred aspects, in step (2), according to concentrate described in following gradient elution：
VWater：VMethanol=95：5；
VWater：VMethanol=80：20；
VWater：VMethanol=60：40.
On the other hand, the application provides Geranium extract B ', is prepared by the following method：
The Geranium extract B is purified using silica gel column chromatography method, is washed with chloroform and methanol system gradient
It is de-, collect VChloroform：VMethanol=(50:1)-(20:1) eluent when and concentration obtain the heroubill extract B '.
In certain preferred aspects, V is collectedChloroform：VMethanol=30:Eluent when 1.
In certain preferred aspects, according to Geranium extract B described in following gradient elution：
VChloroform：VMethanol=100：1；
VChloroform：VMethanol=50：1；
VChloroform：VMethanol=30：1；
VChloroform：VMethanol=20：1；
VChloroform：VMethanol=15：1.
In certain preferred aspects, according to Geranium extract B described in following gradient elution：
VChloroform：VMethanol=30：1.
On the other hand, the application provides a kind of pharmaceutical composition, contains as above any geranium wilfordii extraction
Object optionally also contains one or more pharmaceutically acceptable carriers or excipient.
In certain preferred aspects, the carrier includes but not limited to：Aluminium oxide, aluminum stearate, lecithin,
Haemocyanin such as human serum albumin, buffer substance such as phosphate, glycerine, sorbic acid, potassium sorbate, the portion of saturated vegetable fatty acid
Point glyceride mixture, water, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, cabosil, three
Magnesium silicate, polyvinylpyrrolidone, cellulose, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, beeswax and lanolin.
The excipient refers to the additives in addition to main ingredient in pharmaceutical preparation.Its property is stablized, with main ingredient without compatibility
Taboo, does not generate side effect, does not affect the treatment, be unlikely to deform at normal temperatures, be dry and cracked, going mouldy, damaging by worms, is harmless, without life
Reason acts on, and is not acted on chemically or physically with main ingredient generation, does not influence the assay etc. of main ingredient.As in tablet binder, fill out
Fill agent, disintegrant, lubricant；Wine, vinegar, concoction in medicine pill etc.；Matrix portion in semisolid preparation ointment, creme
Point；Preservative, antioxidant, corrigent, aromatic, cosolvent, emulsifier, solubilizer, osmotic pressure regulator in liquid preparation,
Colorant etc. can be described as excipient.
Described pharmaceutical composition can be administered by following approach：Parenteral, local, intravenous, oral, subcutaneous, artery
In interior, intradermal, percutaneous, rectum, encephalic, peritonaeum, intranasal, intramuscular route or as inhalant.Described pharmaceutical composition can be with
Optionally at least other reagent administering drug combinations of certain effect in treating various diseases.
Various suitable dosage forms can be made in described pharmaceutical composition according to administration route.Such as tablet, capsule, particle
Agent, oral solution, oral suspensions, Orally taken emulsion, powder, tincture, syrup, injection, suppository, ointment, cream,
Paste, eye-drops preparations, pill, implant, aerosol, powder spray, spray etc..Wherein, the pharmaceutical composition or suitable
Dosage form can contain 0.01mg to 1000mg described in Geranium extract be preferably comprised suitable for containing 0.1mg to 800mg
0.5-500mg, further preferably 0.5 to 350mg, particularly preferred 1-250mg.
When oral medication, described pharmaceutical composition, which can be made into, arbitrarily takes orally acceptable dosage form, including but unlimited
In tablet, capsule, aqueous solution or water slurry.Wherein, the carrier that tablet uses generally comprises lactose and cornstarch, in addition
Lubricant such as magnesium stearate can be added.The diluent that capsule preparations use generally comprises lactose and dried corn starch.Aqueous suspension
Liquid formulation is typically then to be used in mixed way active constituent and suitable emulsifier and suspending agent.Optionally, the above oral preparation shape
Some sweeteners, aromatic or colorant can also be added in formula.
When topical application, described pharmaceutical composition can be made into ointment, lotion or cream formulation form appropriate,
It is middle that active constituent is suspended or dissolved in one or more carriers.Carrier workable for ointment formulation includes but not limited to：Mine
Object oil, Albolene, albolene, propylene glycol, polyethylene glycol oxide, polypropylene oxide, emulsifying wax and water；Lotion or creme can
The carrier used includes but not limited to：Mineral oil, sorbitan monostearate, polysorbate60, cetyl ester wax, 16 carbon
Alkene is fragrant and mellow, 2- octyldodecanols, benzyl alcohol and water.
Described pharmaceutical composition can the medication in the form of aseptic injection preparation, including aseptic injection water or oil suspension or nothing
Bacterium injects solution.Wherein, workable carrier and solvent include water, Ringer's solution and isotonic sodium chlorrde solution.In addition, sterilizing
Fixed oil also be used as solvent or suspension media, such as monoglyceride or two glyceride.
On the other hand, the application also provides a kind of pharmaceutical composition, contains progallin A and/or kaempferia galamga
Phenol -7-O- alpha-L-rhamnosides and one or more TNF α antagonists.
In certain preferred aspects, the TNF α antagonist is selected from Etanercept, the Xidans Ying Li resist, A Damu is mono-
Anti-, D2E7, CDP571 and CDP870.
On the other hand, the application provides as above any Geranium extract, progallin A or the kaempferia galamga
Purposes of the phenol -7-O- alpha-L-rhamnosides in preparing the drug for inhibiting TNF α.
On the other hand, the application provides as above any Geranium extract, progallin A or the kaempferia galamga
Purposes of the phenol -7-O- alpha-L-rhamnosides in preparing the drug for inhibiting inflammation.
In certain preferred aspects, the inflammation is selected from arthritis (such as rheumatoid arthritis), tatanic
Rachitis, inflammatory bowel disease (such as Crohn diseases), psoriasis, suppurative mycoplasma and refractory asthma.
Advantageous effect of the invention
The present invention provides four classification components isolated from Geranium extract and two activated monomers (not to eat
Sub- acetoacetic ester and Kaempferol -7-O- alpha-L-rhamnosides), with higher anti-TNF alpha activity and anti-inflammatory activity and/or relatively low
Toxic side effect, can be used for the treatment of the inflammation diseases such as arthritis.
Description of the drawings
Fig. 1 shows Geranium extract preparation flow figure.
Fig. 2 shows progallin A purified pool HPLC spectrograms.
Fig. 3 shows progallin A structure.
Fig. 4 shows progallin A Structural Identification spectrogram；Wherein figure (A) is progallin A mass spectrogram (EI+)；
Scheme (B) be progallin A nuclear magnetic resonance spectroscopy (1HNMR)；It is progallin A carbon-13 nmr spectra to scheme (C)
(13CNMR)。
Fig. 5 shows Kaempferol -7-O- alpha-L-rhamnoside structures.
Fig. 6 shows Kaempferol -7-O- alpha-L-rhamnoside Structural Identification spectrograms；Wherein figure (A) is Kaempferol -7-O- α -
L- rhamnosides mass spectrogram (EIC-N)；It is Kaempferol -7-O- alpha-L-rhamnosides mass spectrogram (EIC-P) to scheme (B)；Scheme (C) kaempferia galamga
Phenol -7-O- alpha-L-rhamnosides mass spectrogram (EIC-P ').
Fig. 7 shows Kaempferol -7-O- alpha-L-rhamnoside Structural Identification spectrograms；Wherein, figure (A) is Kaempferol -7-O-
Alpha-L-rhamnoside carbon-13 nmr spectra (13CNMR)；It is Kaempferol -7-O- alpha-L-rhamnoside nuclear magnetic resonance spectroscopies to scheme (B)
(1HNMR)。
Fig. 8 shows inhibiting effect of the Geranium extract to TNF α.
Fig. 9 shows toxic effect of the Geranium extract to L929 cells.
Figure 10 shows EA-3 and EA-4 antagonistic activity and cytotoxicity to TNF α；Wherein figure (A) and (C) is shown respectively
Toxicity of the EA-3 and EA-4 to L929 cells；Figure (B) and (D) respectively illustrates the antagonistic activity of EA-3 and EA-4 to TNF α.
Figure 11 shows antagonistic activity and cytotoxicity of the EA-3-1 to TNF α；Wherein figure (A) shows EA-3-1 pairs
The toxicity of L929 cells；Figure (B) shows antagonistic activities of the EA-3-1 to TNF α.
Figure 12 shows antagonistic activity and cytotoxicity of the progallin A to TNF α；Wherein figure (A), which is shown, does not eat
Toxicity of the sub- acetoacetic ester to L929 cells；Figure (B) shows antagonistic activity of the progallin A to TNF α.
Figure 13 shows antagonistic activity and cytotoxicity of the EA-4-2 to TNF α；Wherein figure (A) shows EA-4-2 pairs
The toxicity of L929 cells；Figure (B) shows antagonistic activities of the EA-4-2 to TNF α.
Figure 14 shows antagonistic activity and cytotoxicity of the Kaempferol -7-O- alpha-L-rhamnosides to TNF α；Wherein scheme (A)
Show toxicity of the Kaempferol -7-O- alpha-L-rhamnosides to L929 cells；Figure (B) shows Kaempferol -7-O- α-L- sandlwoods
Antagonistic activity of the glucosides to TNF α.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
Embodiment 1.The preparation method of Geranium extract
At room temperature by the herb 10kg of MangNiu Er seedlings section Geranium plant geranium wilfordii (Geranium wilfordii)
It is added in 50% ethanol-water solutions of 100kg, stirring is impregnated 8 hours, filtrate is collected by filtration, 80kg is added in filter residue at room temperature
In 50% ethanol-water solution, stirring is impregnated 8 hours, filtrate is collected by filtration, 50% alcohol-waters of 60kg are added in filter residue at room temperature
In solution, stirring is impregnated 8 hours, and filtrate is collected by filtration, and filtrate merging will be concentrated under reduced pressure into 1/30 volume, concentrate decompression three times
Dry or vacuum freeze-drying is up to Geranium extract (GWE).
Embodiment 2.Geranium extract is classified component
The Geranium extract that Example 1 obtains is appropriate, is extracted with ethyl acetate 4 times, and obtained extract liquor is concentrated,
Freeze-drying, obtains Geranium extract ethyl acetate extraction phase EA.By macroreticular resin on Geranium extract ethyl acetate extraction phase
Column (D101), successively with 95:5,80:20,60:40,40:60,20:80,0:100 water and methanol elution, TLC are monitored and are distinguished
The eluent under above-mentioned each ratio is collected, is concentrated, freeze-drying obtains EA-1, EA-2, EA-3, EA-4, EA-5, and EA-6 amounts to six
It is classified component.
Embodiment 3.EA-3 is classified component
Silicagel column on the EA-3 that embodiment 2 is obtained, successively with 30:1,9:1,0:100 chloroform and methanol elution, TLC
It monitors and collects the eluent under above-mentioned each ratio respectively, concentrate, freeze-drying obtains EA-3-1, and EA-3-2, EA-3-3 are three total
It is classified component.
Embodiment 4.The monomer progallin A obtained from EA-3-1
The EA-3-1 freeze-dried powders obtained in Example 3, which are dissolved in right amount in 50% ethanol water, to be obtained
The solution of 24mg/ml.The solution liquid is filtered with 0.45 μm of syringe filter disk.The filtrate is classified (spectrogram with HPLC
It is shown in Fig. 2), method is as follows：
High performance liquid chromatography：Agilent 1100；
Chromatographic column：Hypersil ODS(250x 4.6mm,5-Micron)；
Column temperature：30℃；
Detection wavelength：254nm；
Flow velocity：0.75ml/min；
Elution time：40min；
Maximum pressure：400bar；
Sample size：20μl；
Mobile phase：Water-ethanol；
Gradient program:
| Time (min) | Watr-proportion % | Ethyl alcohol Phase Proportion % |
| 0 | 100 | 0 |
| 40 | 0 | 100 |
The component that retention time is 16.32min is collected, the monomer that purity is 96% is obtained, is identified as gallic acid second
Ester (Fig. 3,4).
Embodiment 5.EA-4 is classified component
By silicagel column on the EA-4 of 2 gained of embodiment, successively with 15:1,10:1,7:1,5:1,0:100 chloroform and methanol
Elution, TLC are monitored and are collected the eluent under above-mentioned each ratio respectively, are concentrated, and freeze-drying obtains EA-4-1, EA-4-2, EA-4-
The total five classifications component of 3, EA-4-4, EA-4-5.
Embodiment 6.Monomer Kaempferol -7-O- the alpha-L-rhamnosides obtained from EA-4-2
Gel column (LH-20) on the EA-4-2 obtained in embodiment 5 is chromatographed, is eluted with methanol, obtaining purity is
97.33% monomer, the identified monomer are Kaempferol-7-O- alpha-L-rhamnosides (Fig. 5-7).
Embodiment 7.The antagonistic activity and cytotoxicity of Geranium extract and each classification component and monomer to TNF α
External L929 hyperplasias/cytotoxicity analysis carries out on droplet amount disk.In containing 10% cow's serum, 1%PBS
And 1% nonessential Amino acid the minimum minimal mediums of EagleShi (EMEM) in cultivate L929 cells.Use 2ml PBS solutions
It rinses the L929 cells converged and through trypsin treatment, is then resuspended in complete medium.Draw the suspension of 200 μ l cells
Liquid centrifuges remaining suspension up to 5 minutes to carry out cell density counting under 1500rpm.Removing supernatant is simultaneously complete by this
Whole culture medium adds in the diluting cells to form ultimate density as 1.5 × 105Cell/ml.100 μ l cell suspending liquids are added
Into each hole slot of the flat droplet amount disk of 96 hole slots, and in 37 DEG C and 5%CO2It is cultivated 24 hours under air.
(1) to the evaluation of TNF α antagonistic activity
The Geranium extract obtained in embodiment 1-6 and each classification component and monomer are dissolved in 1x PBS solutions respectively
In to obtain the solution for being equivalent to 1g crude drugs/ml solution.The solution dilutes to obtain 6.25,12.5,25 again through 1x PBS solutions,
50,100,200 μ g/ml solution.It is mixed respectively with isometric TNF α solution by the dilution of gained and through cultivating 1 hour.
It cultivates in advance within this 1 hour before terminating, training is removed from the flat droplet amount disk of 96 hole slots of the above-mentioned L929 cells cultivated through 24 hours
Base is supported, the fresh culture that 50 μ l contain 4 μ g/ml actinomycin Ds (Act D) is then added.Take each 50 μ of 6 kinds of mixed liquors
L, and added in the flat droplet amount disk of 96 hole slots of the L929 cells through culture 24 hours.Adjust actinomyces in culture solution
The ultimate density of plain D (Act D) is 2 μ g/ml, and the ultimate density of TNF α is 0.1ng/ml.Only be added 2 μ g/ml Act D or
The hole slot of 0.1ng/ml TNF αs is respectively as positive controls and blank control group.The flat droplet amount disk is through mildly rocking
After being mixed, in 37 DEG C and 5%CO2Continue to cultivate 24 hours under air.Using ELISA readers, in 490/630nm
Under, it carries out OD values reading and measures.The calculation formula of TNF α inhibiting effect is as follows：
(2) cytotoxicity of Geranium extract
The Geranium extract obtained in embodiment 1-6 and each classification component and monomer are dissolved in 1x PBS solutions respectively
In to obtain being equivalent to 1g crude drugs/ml solution.The solution dilutes to obtain 6.25,12.5,25,50,100 again through 1x PBS solutions,
200 μ g/ml solution.6 kinds of dilutions (50 μ l) of gained are added separately to the 96 of the above-mentioned L929 cells cultivated through 24 hours
In the flat droplet amount of hole slot, the fresh culture that 50 μ l contain 4 μ g/ml actinomycin Ds (Act D) is then added to adjust culture
The ultimate density of actinomycin D (Act D) is 2 μ g/ml in liquid.In 37 DEG C and 5%CO2It is flat micro- that 96 hole slot is cultivated under air
Drop amount 24,48,72 hours.Then by 50 μ l XTT (i.e. 2,3- bis- (2- methoxyl group -4- nitro -5- sulfophenyls) -5- [(phenyl amines
Base) carbonyl] -2H- tetrazolium hydroxides) it is added into each hole slot and cultivates again 4 hours.Using ELISA readers, in
Under 490/630nm, reading measurement is carried out.The calculating formula of cytotoxicity is as follows：
As a result as shown in 1,2 and Fig. 8-14 of table, wherein Geranium extract shows under the concentration of 200 μ g/ml solution
89.08% inhibitory activity.Also, detailed cytotoxicity is not shown under the concentration range.
Result above confirms that Geranium extract and each classification component and two monomers all have the activity of inhibition TNF α simultaneously
The antagonist of TNF α is can be used as, and does not show apparent cytotoxicity.
1 Geranium extract of table inhibits the activity of TNF α
2 Geranium extract cytotoxicity of table
Embodiment 8.The influence of Geranium extract and each classification component and monomer to inflammation swelling
(1) influence of rat paw edema caused by Geranium extract Carrageenan
SD rats 50,160~210g of weight is randomly divided into 3 groups, i.e. Geranium extract administration group by weight
(6.75gkg-1d-1), aspirin control group (0.1gkg-1d-1) and blank control group (give medicine group same volume
Product drinking water).Daily ig is administered once, continuous five days, and 1 hour in 1% jiao of Rat Right metapedes plantar subcutaneous injection after the last administration
Pitch dish glue 0.1ml, with volumetric method respectively measures injection carrageenan before and injection after 1,2,4,6 hour right metapedes volume, inject
The difference of forward and backward volume is swelling, carries out comparison among groups.The results are shown in Table 3, and 1 hour right foot starts to swell after injecting proinflammatory agent
It is swollen, peak is reached within 2~4 hours, Geranium extract group 2,4,6 hours right paw swellings after causing inflammation are substantially less than blank control group
(P<0.01)。
The influence of rat paw edema caused by 3 Geranium extract Carrageenan of table
N=10, x ± s, and same time point blank control group ratio,aP<0.01,bP<0.05
(2) influence of Geranium extract paraxylene induced mice auricle edema
ICR mouse 50 are selected,18~20g of weight is randomly divided into 3 groups by weight, and grouping and administration are same as above, last
It is two-sided 100% dimethylbenzene, 50 μ l to be uniformly applied to mouse right ear in 1 hour after administration, are put to death mouse cervical dislocation after 1 hour, immediately
Ears are cut from the ear portion, round auricle, electronics Libra difference are laid respectively at the same position of ears with a diameter of 8mm card punch
It weighs, every mouse auris dextra piece subtracts left auricle and represents auricle swelling degree again again.The results are shown in Table 4, shows Geranium extract
There is significant inhibition dimethylbenzene induced mice auricle edema effect (P<0.01).
The influence of 4 Geranium extract paraxylene induced mice auricle edema of table
N=10, x ± s, and blank control group ratio,bP<0.05
Although the specific implementation mode of the present invention has obtained detailed description, according to all introductions having disclosed, originally
Field technology personnel can carry out various modifications and replace to the details of technical solution of the present invention, these change the present invention's
Within protection domain.The full scope of the present invention is given by the appended claims and any equivalents thereof.
Claims (10)
1. Geranium extract A, is prepared by the following method：
(1) geranium wilfordii is extracted 1-5 times in ethyl alcohol or ethanol water, collect extracting solution and concentrated, obtained heroubill and always extract
Object；
(2) the heroubill total extract is extracted with ethyl acetate 1-5 times, collects ethyl acetate phase and concentrated, gained is dense
Contracting object is used on absorption resin and methanol mixed solution carries out gradient elution, collection VWater：VMethanol=(50:50)-(30:70) when
Eluent and concentration, obtain the heroubill extract A；Preferably, V is collectedWater：VMethanol=40:Eluent when 60.
2. Geranium extract A described in claim 1, wherein the method have at least one of following features：
1) it in step (1), will be extracted in 5-12L ethyl alcohol or ethanol water per Kg geranium wilfordiis；
2) in step (1), geranium wilfordii is extracted in 30-85wt% ethanol waters, preferably 30-70wt% ethyl alcohol is water-soluble
Liquid；Such as 30wt%, 40wt%, 50wt%, 60wt% or 70wt%；
3) in step (1), geranium wilfordii is extracted in room temperature；
4) in step (2), by the concentrate on macroporous absorbent resin gradient elution；Preferably, the concentrate is existed
Gradient elution is carried out on D101 type macroporous absorbent resins；
5) in step (2), by gained concentrate VWater：VMethanol=(95:5)-(30:70) mixed solution carries out gradient elution；
Preferably, according to concentrate described in following gradient elution：
VWater：VMethanol=95：5；
VWater：VMethanol=80：20；
VWater：VMethanol=70：30；
VWater：VMethanol=60：40；
VWater：VMethanol=50：50；
VWater：VMethanol=40：60；
VWater：VMethanol=30：70；
Preferably, according to concentrate described in following gradient elution：
VWater：VMethanol=95：5；
VWater：VMethanol=80：20；
VWater：VMethanol=60：40；
VWater：VMethanol=40：60.
3. Geranium extract A ', is prepared by the following method：
Geranium extract A described in claims 1 or 2 is purified using silica gel column chromatography method, with chloroform and methanol body
It is gradient elution, collects VChloroform：VMethanol=(12:1)-(8:1) eluent when and concentration obtain the heroubill extract A '；It is excellent
Selection of land collects VChloroform：VMethanol=10:Eluent when 1；
Preferably, according to Geranium extract A described in following gradient elution：
VChloroform：VMethanol=20：1；
VChloroform：VMethanol=15：1；
VChloroform：VMethanol=12：1；
VChloroform：VMethanol=10：1；
VChloroform：VMethanol=8：1；
Preferably, according to Geranium extract A described in following gradient elution：
VChloroform：VMethanol=15：1；
VChloroform：VMethanol=10：1.
4. Geranium extract B, is prepared by the following method：
(1) geranium wilfordii is extracted 1-5 times in ethyl alcohol or ethanol water, collect extracting solution and concentrated, obtained heroubill and always extract
Object；
(2) the heroubill total extract is extracted with ethyl acetate 1-5 times, collects ethyl acetate phase and concentrated, gained is dense
Contracting object is used on absorption resin and methanol mixed solution carries out gradient elution, collection VWater：VMethanol=(70:30)-(50:50) when
Eluent and concentration, obtain the heroubill extract B；Preferably, V is collectedWater：VMethanol=60:Eluent when 40.
5. Geranium extract B described in claim 1, wherein the method have at least one of following features：
1) it in step (1), will be extracted in 5-12L ethyl alcohol or ethanol water per Kg geranium wilfordiis；
2) in step (1), geranium wilfordii is extracted in 30-85wt% ethanol waters, preferably 30-70wt% ethyl alcohol is water-soluble
Liquid；Such as 30wt%, 40wt%, 50wt%, 60wt% or 70wt%；
3) in step (1), geranium wilfordii is extracted in room temperature；
4) in step (2), by the concentrate on macroporous absorbent resin gradient elution；Preferably, the concentrate is existed
Gradient elution is carried out on D101 type macroporous absorbent resins；
5) in step (2), by gained concentrate VWater：VMethanol=(95:5)-(50:50) mixed solution carries out gradient elution；
Preferably, according to concentrate described in following gradient elution：
VWater：VMethanol=95：5；
VWater：VMethanol=80：20；
VWater：VMethanol=70：30；
VWater：VMethanol=60：40；
VWater：VMethanol=50：50；
Preferably, according to concentrate described in following gradient elution：
VWater：VMethanol=95：5；
VWater：VMethanol=80：20；
VWater：VMethanol=60：40.
6. Geranium extract B ', is prepared by the following method：
The Geranium extract B of claim 4 or 5 is purified using silica gel column chromatography method, with chloroform and methanol body
It is gradient elution, collects VChloroform：VMethanol=(50:1)-(20:1) eluent when and concentration obtain the heroubill extract B '；It is excellent
Selection of land collects VChloroform：VMethanol=30:Eluent when 1；
Preferably, according to Geranium extract B described in following gradient elution：
VChloroform：VMethanol=100：1；
VChloroform：VMethanol=50：1；
VChloroform：VMethanol=30：1；
VChloroform：VMethanol=20：1；
VChloroform：VMethanol=15：1；
Preferably, according to Geranium extract B described in following gradient elution：
VChloroform：VMethanol=30：1.
7. pharmaceutical composition optionally also contains one containing claim 1-6 any one of them Geranium extracts
Kind or a variety of pharmaceutically acceptable carriers or excipient.
8. pharmaceutical composition contains progallin A and/or Kaempferol -7-O- alpha-L-rhamnosides, and one kind or more
Kind TNF α antagonist；Preferably, the TNF α antagonist be selected from Etanercept, the Xidans Ying Li anti-, adalimumab, D2E7,
CDP571 and CDP870.
9. the Geranium extract of any one of claim 1-6, progallin A or Kaempferol -7-O- alpha-L-rhamnosides exist
Prepare the purposes in the drug for inhibiting TNF α.
10. the Geranium extract of any one of claim 1-6, progallin A or Kaempferol -7-O- alpha-L-rhamnosides
Purposes in preparing the drug for inhibiting inflammation；Preferably, the inflammation is selected from arthritis (such as rheumatoid arthritis), strong
Straightforward rachitis, inflammatory bowel disease (such as Crohn diseases), psoriasis, suppurative mycoplasma and refractory asthma.
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Application publication date: 20181009 |