CN105833288B - A kind of gallic acid phosphatide complexes, preparation method and application - Google Patents
A kind of gallic acid phosphatide complexes, preparation method and application Download PDFInfo
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Abstract
The present invention relates to pharmaceutical technology fields, more particularly to a kind of phosphatide complexes, more particularly to a kind of gallic acid phosphatide complexes, preparation method and application, the gallic acid phosphatide complexes are made of gallic acid and phosphatide, and the molar ratio of the gallic acid and phosphatide is 1:(0.1-8).Gallic acid and phosphatide complexes of the invention, it is safe, simple using phosphatide small toxicity, synthetic method, in conjunction with female medicine nutgall acid, improve the bioactivity, bioavilability and pharmacological activity of gallic acid；And the gallic acid that is prepared of the present invention and phosphatide complexes have anti-oxidant and/or liver-protective effect.
Description
Technical field
The present invention relates to pharmaceutical technology fields, and in particular to a kind of phosphatide complexes more particularly to a kind of gallic acid phosphorus
Fat complexes, preparation method and application.
Background technique
Gallic acid also known as gallic acid, 3,4,5-trihydroxybenzoic acid of chemical name are a kind of Polyphenols chemical combination
Object.Gallic acid (C7H6O5) it is acicular crystal (by being crystallized in anhydrous methanol or chloroform), Duo Yiqi sulfuric monohydrate C7H6O5·
H2O form exists；235-240 degrees Celsius of its fusing point (decomposition), specific gravity 1.694 can lose when being heated to 100-120 degrees Celsius
The crystallization water.1 gram of gallic acid dissolve in 87 milliliters of water, 3 milliliters of boiling water, 6 milliliters of ethyl alcohol, 100 milliliters of ether, 10 milliliters of glycerol and
5 milliliters of acetone, are practically insoluble in benzene, chloroform and petroleum ether.Gallic acid is extremely unstable under alkalinity and neutrallty condition, in meta-acid
Then relatively stable under property, Qiang Guang, hot conditions, different antioxidants has certain protective effect to it, can enhance its stabilization
Property.
Gallic acid is widely present in Chinese gall, tower drawing, Lythrum salicaria, Fructus Corni, Coriaria sinica, pomegranate, penthorum chinense pursh, Arabic phase
Think in the plants such as tree, there is very high medical value.Research data confirms that gallic acid pharmacological activity is extensive, as effective
Active skull cap components have extremely strong antioxidation, can resist damage of the free radical to human body, can be with anti-inflammatory antibacterial, anti-swollen
Tumor, anti-radiation, protection angiocarpy and anti arteriosclerosis and thrombosis effect, can also prevent such as arthritis, cancer, sugar
A variety of diseases such as urine disease and heart disease.
But gallic acid as natural polyphenol monomer since the chemical changes such as oxidation, isomerization and degradation easily occur,
Degradation reaction especially easily occurs in alkaline environment, and loses its distinctive oxidation resistance, thus in intestinal environment its
Stability is lower.In addition, due to the polyphenol structure and fat-soluble difference of gallic acid, be very easy in vivo with protein,
Polymerization reaction occurs for the macromolecular substances such as polysaccharide, makes it be difficult to belong to the poor compound of absorption across cell membrane, thus significantly
Reduce its peculiar bioactivity and bioavilability.So selection small toxicity, synthetic method safety, simple pharmaceutical carrier
In conjunction with female medicine nutgall acid, with improve gallic acid bioavilability and pharmacological activity for improve gallic acid reality
Application value is particularly important.
Phosphatide, also referred to as phospholipid, Phospholipids are a kind of phosphorous lipid materials, are divided into glycerophosphatide and non-glycerol phosphatide
Two major classes.So far, it has been found that phosphatide is almost present in the cell of all animals and plants bodies, and source is very extensive.
Animal phospholipids are organized mainly from milk, yolk, animal brain, the dirty, muscle of liver etc., and plant phosphatide is then stored in oil plant more
In seed, nut and cereal, mainly exist with gel phase.From structure, in phosphatide on phosphorus atoms the oxygen atom of hydroxyl have compared with
Strong electronics tendency, and nitrogen-atoms has stronger betatopic to be inclined to, therefore in non-proton transport system solvent, phosphatide can be with
It is combined with compound with certain proportion relation, forms stable compound by charge transfer interaction, referred to as phosphatide is compound.
Phosphatide complexes are Italian scholar's novel drug-loading systems serendipitous when studying liposome in 1987.It grinds
The chromocor compound studied carefully combines with phosphatide under conditions of relatively simple and forms compound, and compound features go out and parent drug
Completely different biological characteristics and pharmacological activity, fat-soluble, saturating cell film property and bioavilability greatly improve.With
Deeply, scientific research personnel has found that many active skull cap components and plant extracts have compared with strong affinity phosphatide for research, can be with shape
At stable compound.Compared with single natural component, phosphatide complexes show different physicochemical properties, such as object phase
Change, fat-soluble or water-soluble enhancing, the change of fusing point, spectroscopic properties；And pharmacological action also greatly enhances, such as drug half
Decline the extension of phase, and adverse reaction reduces, and absorption in the gastrointestinal tract increases, and bioavilability improves etc..
In recent years, gradually increase about the research report of phosphatide complexes, be directed to active skull cap components both at home and abroad at present, such as
Silymarin, radix scutellariae, Puerarin, tea polyphenols phosphatide complexes oneself expanded extensively and furtherd investigate, which also starts
Applied to field of pharmaceutical preparations, such as traditional Chinese medicine extraction component, green-tea extract, grape seed extract, gadol extract, ginseng are mentioned
Take the phosphatide etc. of object.By literature search, there is not yet the report of gallic acid and phosphatide composition compound, there are no gallic acids
The report for generating compound is reacted with phosphatide.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of gallic acid phosphatide complexes, its preparation
Method and application, the compound can be improved the solvability of gallic acid, promote its bioavilability by the package of phosphatide,
The preparation method and application of gallic acid phosphatide complexes with good oxidation resistance and protection liver function.
In order to achieve that object of the invention, the invention adopts the following technical scheme:
In a first aspect, the present invention provides a kind of gallic acid phosphatide complexes, the gallic acid phosphatide complexes by
The molar ratio of gallic acid and phosphatide composition, the gallic acid and phosphatide is 1:(0.1-8)).
The molar ratio of the gallic acid and phosphatide such as can be 1:0.1,1:0.2,1:0.3,1:0.4,1:0.5,1:
0.6,1:0.8,1:1,1:1.5,1:2,1:3,1:4,1:5,1:6,1:7 or 1:8.
In the present invention, gallic acid is chemical since oxidation, isomerization and degradation etc. easily occurs as natural polyphenol monomer
Variation, especially easily occurs degradation reaction in alkaline environment, and loses its distinctive oxidation resistance, so in intestinal environment
In its stability it is lower.Due to the polyphenol structure and fat-soluble difference of gallic acid, it is very easy to and protein, more in vivo
Polymerization reaction occurs for the macromolecular substances such as sugar, makes it be difficult to belong to the poor compound of absorption across cell membrane, to drop significantly
Low its peculiar bioactivity and bioavilability.So selection phosphatide, small toxicity, synthetic method are safe, simple, with mother
Medicine nutgall acid combines, and improves the bioavilability and pharmacological activity of gallic acid.
In the present invention, gallic acid and the not simple physical mixed of phosphatide are interacted, and are formd multiple
Close object.
As optimal technical scheme, the molar ratio of the gallic acid and phosphatide is 1:(0.5-4), such as can be 1:
0.5、1:0.6、1:0.7、1:0.8、1:0.8、1:1、1:1.2、1:1.5、1:1.8、1:2、1:2.2、1:2.5、1:2.8、1:3、
1:3.2,1:3.5,1:3.8 or 1:4, preferably 1:1.
Preferably, the phosphatide is natural phospholipid and/or synthetic phospholipid.
Preferably, the natural phospholipid is soybean lecithin and/or egg yolk lecithin.
Preferably, the synthetic phospholipid puts phthalein phosphorus for two hard cruel phthalein Phosphatidylcholins, two hard extremely phthalein phosphatide phthalein glycerol, two palm fibres
Rouge phthalein choline, two oily phthalein Phosphatidylcholins or two palm fibres put the combination of any one or at least two in phthalein phosphatide phthalein ethanol amine,
Such as it can be two hard cruel phthalein Phosphatidylcholins and two hard cruel phthalein phosphatide phthalein glycerol, two hard cruel phthalein Phosphatidylcholins and two palm fibres put phthalein
Phosphatidylcholin, two hard cruel phthalein Phosphatidylcholins and two oily phthalein Phosphatidylcholins, two hard cruel phthalein Phosphatidylcholins and two palm fibres put phthalein
Phosphatide phthalein ethanol amine, two hard cruel phthalein phosphatide phthalein glycerol and two palm fibres put phthalein Phosphatidylcholin, two hard cruel phthalein phosphatide phthalein glycerol and two oil
Phthalein Phosphatidylcholin, two hard cruel phthalein phosphatide phthalein glycerol and two palm fibres put phthalein phosphatide phthalein ethanol amine, two palm fibres put phthalein Phosphatidylcholin and two
Oily phthalein Phosphatidylcholin, two palm fibres put phthalein Phosphatidylcholin and two palm fibres put phthalein phosphatide phthalein ethanol amine, two oily phthalein Phosphatidylcholins and two
Palm fibre puts phthalein phosphatide phthalein ethanol amine.
Preferably, the phosphatide is soybean lecithin.
Second aspect, the present invention provide a kind of preparation method of gallic acid phosphatide complexes as described in relation to the first aspect,
The following steps are included:
(1) gallic acid is dissolved in fat-soluble solvent, obtains clear liquid；
(2) phosphatide reaction, stirring is added by formula in step (1)；
(3) reaction dissolvent is removed, atent solvent is added and removes the drug not reacted with phosphatide, is done after removing atent solvent
It is dry to get gallic acid phosphatide complexes.
Preferably, fat-soluble solvent described in step (1) is tetrahydrofuran, methylene chloride, chloroform, n-hexane, second
In alcohol, ethyl acetate, hexamethylene, methanol, isopropanol, n-butanol or acetone any one or at least two mixture, it is excellent
It is selected as dehydrated alcohol.
Preferably, the temperature of step (2) described reaction be 20-80 DEG C, such as can be 20 DEG C, 21 DEG C, 22 DEG C, 23 DEG C,
24 DEG C, 25 DEG C, 26 DEG C, 28 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C or 70 DEG C, preferably 30-70
DEG C, further preferably 50 DEG C.
Preferably, step (2) reaction is connection reflux unit, and the stirring is magnetic agitation.
Preferably, the time of step (2) described stirring be 1-12h, such as can be 1h, 2h, 3h, 4h, 5h, 6h, 7h,
8h, 9h, 10h, 11h or 12h, preferably 2-8h, further preferably 3h.
Preferably, step (3) the removing reaction dissolvent is by being evaporated under reduced pressure and/or being dried in vacuo.
Preferably, step (3) temperature for removing reaction dissolvent is 30-70 DEG C, such as can be 30 DEG C, 31 DEG C, 32
DEG C, 33 DEG C, 34 DEG C, 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C or 70 DEG C, it is excellent
It is selected as 35-60 DEG C.
Preferably, atent solvent described in step (3) be methylene chloride, chloroform or acetic acid second it is cruel in any one
Or at least two mixture, preferably chloroform.
The third aspect, the present invention provide a kind of pharmaceutical composition, multiple comprising gallic acid phosphatide as described in relation to the first aspect
Close object.
Preferably, described pharmaceutical composition also includes the auxiliary material pharmaceutically received.
Preferably, the auxiliary material is any one in excipient, diluent, carrier, flavoring agent, adhesive and filler
Or at least two combination.
Preferably, described pharmaceutical composition is tablet, sustained-release tablet, capsule, suppository, freeze-dried powder, injection, compound
In tablet, gelling agent or emulsion any one or at least two combination.
Fourth aspect, the present invention provide a kind of pharmaceutical composition as described in the third aspect in anti-oxidant and/or protection liver
Application in terms of dirty function.
Compared with prior art, the invention has the following advantages:
(1) gallic acid and phosphatide complexes of the invention, it is safe, simple using phosphatide small toxicity, synthetic method, with mother
Medicine nutgall acid combines, and improves the bioactivity, bioavilability and pharmacological activity of gallic acid；
(2) gallic acid of the invention and the not simple physical mixed of phosphatide complexes, are interacted,
The fat-soluble of gallic acid is also improved in the case where not influencing the pharmacological activity of gallic acid, avoids it with protein, is more
Polymerization reaction occurs for the macromolecular substances such as sugar；
(3) gallic acid of the invention and phosphatide complexes have anti-oxidant and liver-protective effect.
Detailed description of the invention
The ultraviolet scanning spectrum result of Fig. 1 gallic acid compound of the present invention, gallic acid and phosphatide compares；
Fig. 2 infrared spectrogram compares, wherein Fig. 2 (a) gallic acid, Fig. 2 (b) lecithin, Fig. 2 (c) compound, Fig. 2
(d) physical mixture of gallic acid and lecithin；
Fig. 3 scanning electron microscope (SEM) photograph compares, wherein Fig. 3 (a) gallic acid, Fig. 3 (b) lecithin, Fig. 3 (c) compound, Fig. 3
(d) physical mixture of gallic acid and lecithin；
Fig. 4 gallic acid compound, gallic acid and vitamin C of the present invention to 1,1- diphenyl -2- trinitrophenyl-hydrazine from
Compared by the Scavenging activity of base；
Fig. 5 gallic acid compound, gallic acid and vitamin C of the present invention join (the 3- ethyl-benzo thiophene of nitrogen-two to 2,2-
Azoles -6- sulfonic acid) Scavenging activity of di-ammonium salts free radical compares；
The rejection ability of Fig. 6 gallic acid compound, gallic acid and vitamin C of the present invention to linoleic acid autoxidation system
Compare；
Fig. 7 gallic acid compound urgency toxicity test changes of weight statistics of the present invention.
Specific embodiment
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should be bright
, the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation of 1 gallic acid phosphatide complexes of embodiment
Gallic acid 188mg and soybean lecithin 750mg are accurately weighed, soybean lecithin is set in a round bottom flask, is added
After 25 DEG C of stirring and dissolvings of 15mL dehydrated alcohol, it is gradually added into gallic acid when whisking, is to slowly warm up to 50 DEG C.It persistently stirs cold
Solidifying back flow reaction 3h after 45 DEG C of dry solvents of rotary evaporation, adds chloroform and removes the drug not react with phosphatide, vacuum is done
It is dry to take out for 24 hours and cross 200 meshes to get gallic acid phosphatide complexes.
The gallic acid phosphatide complexes being prepared, gallic acid and lecithin are subjected to ultraviolet spectra, infrared spectroscopy
And electron-microscope scanning, as a result as shown in Figure 1, Figure 2 and Fig. 3.Wherein, ultra-violet absorption spectrum can reflect the variation of the unsaturated bond of compound
Situation；Infrared spectroscopy is the absorption spectrum for studying molecular motion, can be pushed away according to the location and shape of absorption peak in infrared spectroscopy
Functional group's information of seco compound.
(1) uv scan result:
Gallic acid, compound are dissolved in ethanol solution respectively, the ethanol solution of equal gallic acids concentration is made.
By the concentration of phosphatide in complex solution as reference, the phosphatide ethanol solution of isoconcentration is prepared.Three kinds of solution carry out ultraviolet
The analysis of 400-200nm length scanning.The uv-spectrogram of gallic acid, lecithin and compound is as shown in Figure 1.
From Fig. 1 result it is found that phosphatide in surface sweeping wave-length coverage there is no characteristic absorption peak, gallic acid is then in 272nm
There is maximum absorption band at place, and compound equally has maximum absorption band in the wavelength, but peak value does not have the gallic acid of isoconcentration
Height it is possible thereby to prove change of the compound there is no chemical structure, and mainly shows the chemical characteristic of gallic acid,
But because feature structure may participate in gallic acid with the combination of phosphatide, therefore the peak value of characteristic peak is declined.
(2) IR spectrum scanning result:
The physical mixture for taking suitable gallic acid, phosphatide, compound and gallic acid and phosphatide respectively, using bromine
Change potassium pressed disc method, using infrared spectrometer in 400-4000cm-1IR spectrum scanning is carried out in range.Results of IR
As shown in Figure 2.
By Fig. 2 result it is found that gallic acid has phenolic hydroxyl group (3283cm-1), phenyl ring (1469,1542,1613cm-1), carbonyl
Base (1702cm-1) characteristic absorption peak, lecithin have hydroxyl (3288cm-1), methylene (2924cm-1) and carbonyl
(1737cm-1) characteristic absorption peak.The infrared spectroscopy of gallic acid and phosphatide physical mixture substantially remains gallic acid
With the characteristic absorption peak of phosphatide, for the simple superposition of the two infrared spectroscopy, each spike number is held essentially constant, only relative intensity
Some variations have occurred, illustrate that there is no interact therebetween in mixture.Compared with physical mixture, the figure of compound
Apparent variation has occurred in spectrum, though remaining most of absorption peak of phosphatide, gallic acid is in 500-1750cm-1Between it is big
Partial Feature Absorption Characteristics peak disappears or occurs, and shows that gallic acid is interacted with lecithin, but compound exists
It is new absorption peak do not occur in scanning range, illustrates that the formation of compound does not generate new covalent bond, galla turcica
Acid remains respective chemical structure with lecithin.
(3) electron-microscope scanning result is as follows:
Respectively to metal spraying plated film under gallic acid, phosphatide, compound and gallic acid and phosphatide physical mixture vacuum after
It is scanned Electron microscopic study.As a result as shown in Figure 3.
By Fig. 3 result it is found that gallic acid has long variability crystal characteristic and particle is smaller, and phosphatide is then without fixation
Form, in the physical mixture of the two, the particle of two kinds of forms can be observed.And compound visible no food under Electronic Speculum
Sub- acid crystals volume morphing completely disappears particle and becomes larger, and completely in conjunction with phosphatide, significant change is had occurred in form.
The preparation of 2 gallic acid phosphatide complexes of experimental example
Gallic acid 600mg and soybean lecithin 400mg is weighed, is placed in conical flask, ethyl alcohol 60mL is added, in 30 DEG C of magnetic force
4h is stirred in blender, forms transparent deep yellow solution, decompressing and extracting organic solvent is added appropriate chloroform, clarifies solution,
Shake well, is filtered to remove precipitating, and filtrate is dry to get gallic acid phosphatide complexes.
The preparation of 3 gallic acid phosphatide complexes of experimental example
Gallic acid 200mg and soybean lecithin 800mg is weighed, is placed in conical flask, tetrahydrofuran 40mL is added, in 30 DEG C
3h is stirred in magnetic stirring apparatus, forms transparent deep yellow solution, and decompressing and extracting organic solvent is added appropriate chloroform, makes
Solution clarification, shake well are filtered to remove precipitating, and filtrate is dry to get gallic acid phosphatide complexes.
The preparation of 4 gallic acid phosphatide complexes of experimental example
Gallic acid 400mg and soybean lecithin 600mg is weighed, is placed in conical flask, methanol 40mL is added, in 50 DEG C of magnetic force
2h is stirred in blender, forms transparent brown yellow solution, and appropriate n-hexane is added in decompressing and extracting organic solvent, keeps solution clear
Clearly, shake well, is filtered to remove precipitating, and filtrate is dry to get gallic acid phosphatide complexes.
Scavenging activity of the 5 gallic acid phosphatide complexes of experimental example to 1,1- diphenyl -2- trinitrophenyl-hydrazine free radical
1,1- diphenyl -2- trinitrophenyl-hydrazine analytic approach is widely used in the antioxidant activity research of active constituent, and 1,1-
Diphenyl -2- trinitrophenyl-hydrazine is a kind of stable free radical in organic solvent, there is strong absorption near 517nm, in deep
Purple.In the presence of free radical scavenger, the lone pair electrons of 1,1- diphenyl -2- trinitrophenyl-hydrazine are paired, and 517nm absorbs
It disappears or weakens, the degree weakened is absorbed by measurement, the activity of free radical scavenger can be evaluated.
For the anti-oxidant test system of the present invention using ethyl alcohol as reaction system, all samples all have preferable dissolubility: using second
Alcohol dissolves 1,1- diphenyl -2- trinitrophenyl-hydrazine powder, is configured to 0.25mmol/L 1, the storage of 1- diphenyl -2- trinitrophenyl-hydrazine
Standby liquid.Gallic acid, gallic acid compound and vitamin C are matched into the stock solution for 0.16g/L, take 2,4,6,8mL difference
The gallic acid of volume and its stock solution of compound are diluted to 8.0mL with alcohol, and 2.0mL 0.25mmol/L 1,1- hexichol is added
The starting reaction of base -2- trinitrophenyl-hydrazine stock solution, makes the final concentration of of 1,1- diphenyl -2- trinitrophenyl-hydrazine in reaction system
0.05mmol/L, avoid light place 30min at room temperature after shaking up measure suction of the reaction solution at 517nm then using alcohol as reference
Luminosity, various concentration gallic acid and its compound can calculate the inhibiting rate of 1,1- diphenyl -2- trinitrophenyl-hydrazine with following formula:
Inhibiting rate (%)=(A0-ASample)/A0× 100%.
Fig. 4 is that gallic acid and its compound and common food antioxidant vitamin C remove 1,1- diphenyl -2- three
The ability of nitrophenyl hydrazine free radical compares.
By Fig. 4 result as it can be seen that the Scavenging activity of 1,1- diphenyl -2- trinitrophenyl-hydrazine free radical is successively under same concentrations
Are as follows: gallic acid > compound > vitamin C.1,1- diphenyl -2- trinitrophenyl-hydrazine the free radical scavenging ability of gallic acid is aobvious
It writes and is higher than compound.
6 gallic acid phosphatide complexes of experimental example join nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts to 2,2-
The Scavenging activity of free radical
2,2- connection nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts generate metastable blue-green after aoxidizing
2,2- join nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts+water-soluble free radical, have at 734nm maximum
Absorption peak.Under antioxidant effect, reaction system is faded, and feature light absorption value reduces.Solution decolourization, which is more obvious, to be shown to be examined
The oxidation resistance of sample is stronger.
By 5.0mL 7mmolL-12,2- connection (3- ethyl-benzothiazole -6- sulfonic acid) the diammonium saline solution of nitrogen-two and
88μL 140mmol·L-1Potassium persulfate mixing, be protected from light at room temperature reduction 12-16h after, obtain 2,2- connection (the 3- second of nitrogen-two
Base-benzothiazole -6- sulfonic acid) di-ammonium salts free radical stock solution.The stock solution is diluted using preceding with dehydrated alcohol, until dividing
Being measured to absorbance on light photometer at 734nm wavelength is 0.51 ± 0.02.
Take 0.6mL various concentration gallic acid, gallic acid phosphatide complexes and vitamin C sample respectively with 2.4mL 2,
2- connection nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts stock solution shakes mixing 10s at room temperature, is placed in dark place
6min reads absorbance under 734nm wavelength.
2,2- joins nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts free radical scavenging activity (I%)=(1-AS/A0)
× 100%, wherein ASFor the light absorption value of sample cell；A0For the light absorption value of control tube.
The 2,2- of gallic acid phosphatide complexes of the present invention joins nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts
The result of+free radical scavenging activity is as shown in figure 5, the compound joins (the 3- ethyl-benzothiazole -6- sulphur of nitrogen-two to 2,2-
Acid) di-ammonium salts+scavenging capacity in concentration dependent variation.
It can be seen that 2, the 2- under same concentrations from the result of Fig. 5 and join nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) two
The Scavenging activity of ammonium salt free radical is successively are as follows: gallic acid > compound > vitamin C.The 2,2- of gallic acid joins (the 3- second of nitrogen-two
Base-benzothiazole -6- sulfonic acid) di-ammonium salts free radical scavenging ability is significantly higher than compound.
Rejection ability of the 7 gallic acid phosphatide complexes of experimental example to linoleic acid autoxidation system
Linoleic acid is a kind of precursor of other unsaturated fatty acids in unsaturated fatty acid and compound body, is people and dynamic
The intracorporal essential fatty acid of object, easily generation lipid peroxidation.Lipid peroxidation is one and generates free radicals and free radical participation
Chain reaction process.The a variety of free radicals generated in reaction process can oxidation of divalent iron ion at ferric ion, and
Ferric ion can generate coloured ferric rhodanate in conjunction with thiocyanate radical, can be detected under certain wavelength, indirectly
Reflect the degree of lipid peroxidation.But in the presence of having antioxidant, the habituation of free radical and unsaturated fatty acid, lipid
Extent of peroxidation reduces, and the production quantity of ferric rhodanate will be reduced, measured OD value, that is, peroxide value in experimentation
It will reduce, illustrate that antioxidant reduces the level of lipid peroxidation of the unsaturated fatty acids such as linoleic acid.
It takes a certain concentration gallic acid, gallic acid phosphatide complexes and vitamin c solution 0.5mL to have in 25mL respectively to fill in
In test tube, be diluted to 4mL with ethyl alcohol, be added 2.5% linoleic acid ethanol solution 4mL, phosphate buffer (0.05mol/L,
PH7.0) 8mL and distilled water 4mL for reaction solution and is put into 40 DEG C of constant incubators and is kept in dark place after mixing.It is every to extract reaction solution for 24 hours
0.1mL is separately added into the ammonium thiocyanate solution 0.1mL of 75% ethyl alcohol 9.7mL and 30%, adds the chlorination of 0.02mol/L
Ferrous iron solution (containing 3.5%HCl) 0.1mL, making gallic acid, gallic acid phosphatide complexes and vitamin C final concentration is 200
μ g/mL, starting reaction accurate response 3min, measurement forms the absorbance of red compound until absorbance reaches at 500nm
Maximum value.
Gallic acid phosphatide complexes of the present invention are as shown in Figure 6 to linoleic rejection ability testing result.
It can be seen that linoleic induction period from Fig. 6 result to extend because antioxidant is added, the oxidation of blank group lures
Leading the phase is 11 days, and the oxidation induction period being added after extracting solution has been above 11 days.Although gallic acid is to sub- oil as the result is shown
The rejection ability of acid oxidase system also shows that certain oxidation resistance not such as the vitamin C under concentration, and compound
Rejection ability then significantly improve, be better than gallic acid.
Scavenging activity of the 8 gallic acid phosphatide complexes of experimental example to Fenton hydroxyl radical free radical
Successively precision weighs Phen solution 1.5mL, 0.01molL-1Phosphate buffer solution 4mL, ferrous sulfate are molten
Liquid 1mL is mixed spare as stock solution immediately.The gallic acid solution of different volumes is weighed in 10mL volumetric flask, and with storage
Standby liquid dilutes, and making its final concentration is respectively 0.04,0.08,0.12 and 0.16gL-1；Gallic acid phosphatide complexes and vitamin
The operation of C is same as above.Then to addition each 1mL of hydrogenperoxide steam generator in configured solution.All volumetric flasks are fixed with deionized water
Hold to scale, mixes, 37 DEG C of water-bath 1h.At ultraviolet specrophotometer, wavelength 536nm, with 1cm cuvette, with reagent blank
For control, its A value is surveyed.As a result it is repeated 3 times and is averaged.
As shown in Table 1, with reagent under concentration to the Scavenging activity of hydroxy radical successively are as follows: gallic acid > compound > dimension life
Plain C, compound have good rejection ability to the hydroxy radical release that Fenton's reaction causes.
The absorbance of 1 gallic acid of table, compound and vitamin C in Fenton hydroxy radical system
Blank control absorbance value is 1.2784 ± 0.0378.
9 gallic acid phosphatide complexes of experimental example are to Fe3+Reducing power
It is successively accurate that ferric chloride solution 0.5mL, potassium ferricyanide solution 2mL, 0.01molL is added-1PBS solution 2mL,
1.2×10-3mol·L-1Hydrochloric acid solution 0.5mL is mixed spare as stock solution.Weigh the gallic acid solution of different volumes in
In 10mL volumetric flask, and stand-by storage liquid dilutes, and making its final concentration is respectively 0.04,0.08,0.12 and 0.16gL-1；Galla turcica
Sour phosphatide complexes and ascorbic operation are same as above.All volumetric flasks are settled to scale with deionized water, mix, room temperature reaction
30min.At ultraviolet specrophotometer, wavelength 700nm, with 1cm cuvette, it is control with reagent blank, surveys its A value.Repeat 3
It is secondary to be averaged.
It as shown in Table 2, is 0.04gL in concentration-1When, gallic acid and compound restore Fe3+Ability better than dimension life
Plain C；Reagent is to Fe under other concentration3+Reducing power successively are as follows: vitamin C > gallic acid > compound, compound have one
Surely Fe is restored3+Ability.
2 gallic acid of table, compound and vitamin C are in the potassium ferricyanide-Fe3+A value in system
Blank control absorbance value is 0.0711 ± 0.0000
The acute toxicity testing (experiment of nursing in seven days) of 10 gallic acid phosphatide complexes of experimental example
Experimental animal and reagent: ICR mouse, 8 week old or so, weight 18-22g, half male and half female.Adaptability is raised before testing
It is 3 days feeding, 23-25 DEG C of temperature, humidity 60-70%.Gallic acid phosphatide complexes: self-control.
Administration mode: it is administered once in daily noon, is deprived of food but not water before administration.Every time with maximum administration concentration 0.25g/
ML is simultaneously administered by the maximum dosage-feeding of 0.2mL/10g weight, is equivalent to 5g/kg weight, successive administration stops after seven days
Administration, then observes activity, poisoning and the death condition of mouse, and records the weight, changes in diet, observing time seven of mouse
It.
Observation index: in terms of nervous system: (1) behavior and reaction (including abnormal cry, agitation, uneasiness etc.)；(2) it transports
Dynamic (including jerk, stiff, forced movement etc.)；(3) pupil and secretion (whether there is or not zoom in or out, shed tears for pupil).
Breathing and angiocarpy: being short of breath or too slow, nose is secreted, touches pareordia heart rate speed etc..In terms of stomach and intestine: abdomen flatulence or receipts
Contract, defecate it is hard or wet etc..Urogenital system: labia, mammary gland swelling, perineum are dirty.Skin and hair: color, integrality,
Whether hyperemia, dermexanthesis etc..Eye: ptosis, is trembled at exophthalmos.It weighs and appetite daily.
Experimental result: after administration, being observed continuously 7 days, and death condition does not occur in mouse, also without there is apparent poisoning
Phenomenon.The heart, liver, spleen, lung, kidney are dissected and observed after execution without special change.Changes of weight is as shown in fig. 7, mouse as the result is shown
Weight rule increases, it is seen that compound has no obvious toxic-side effects to mouse.
11 gallic acid phosphatide complexes of experimental example are to murine chronic alcoholic liver injury protective effect
60 ICR mouse are randomly divided into 6 groups: blank group, model group, positive controls and compound administration group (it is low, in,
High three dosage groups).Chronic alcohol liver injury model is made with 30% ethyl alcohol, the intracorporal triglycerides of detection animal subject,
Superoxide dismutase in total cholesterol, serum aspartate transaminase, alanine aminotransferase and hepatic tissue and the third two
The content of aldehyde, defencive function of the observation gallic acid to murine chronic alcoholic liver injury.
1, administration and the preparation of model
(1) 60 mouse are grouped and are numbered at random by adaptable fed 1 week: low dose of blank group, model group, gallic acid
Amount group, middle dose group, high dose group and positive control drug bifendate group.
(2) daily morning 10-11 presses 0.01mL/g and carries out stomach-filling: blank group and model group fill distilled water, compound is low,
It is 0.01 that middle and high dosage group, which is filled respectively with concentration, the suspension of 0.025,0.05g/mL；Positive controls fill 0.15g/mL biphenyl
Dibasic acid esters suspension.After successive administration 4 weeks, start after each administration 4 hours, in addition to blank group is to distilled water, remaining each component
The ethyl alcohol for being not 30% with concentration after 0.012mL/g stomach-filling dilution.
After (3) 2 weeks, 2% lidocaine abdomen of whole mouse is anaesthetized, heart extracting blood is carried out, after being rapidly separated liver
Weighing.Whole blood is taken to separate serum, hepatic tissue is put into -80 DEG C of refrigerators and freezes, and detects indices.
2, liver index measures
Mouse weight and liver weight are weighed before putting to death, by formula: liver index (%)=liver weight (g)/weight (g)
× 100%, the liver grams of every gram of weight is calculated as liver index.
3, Biochemical Indexes
(1) it takes whole blood to separate serum, alanine aminotransferase and serum aspartate transaminase is detected using reitman-frankel method
Content；Serum is taken to measure total cholesterol, content of triglyceride
(2) it takes mouse to weigh with position liver, 10% liver homogenate is made simultaneously in physiological saline is added in tissue homogenizer
4000r/min is centrifuged 10min, takes supernatant, detects superoxide dismutase activity using xanthine oxidase, is compared with thio bar
Appropriate acid colorimetric method detects mda content.
4, it statisticallys analyze
Experimental data is indicated with x ± s, carries out one-way analysis of variance using 13.0 software package of SPSS, comparison among groups use
T is examined, and is statistically significant with P≤0.05.
5, result
(a) ordinary circumstance
At the end of experiment, 60 rats share 50 survivals.During experiment, the mouse of compound middle dose group is all deposited
It is living；Model group has 3 mouse because of alcohol intolerance death, remaining mouse weight loss compared to the blank group, hair luster degree compared with
Difference is slow in action and slow in reacting；Each dosage group relative model group mouse of compound, diet is more, more active, integrality
Preferably.Each group mouse weight, liver index the results are shown in Table 3.
3 each group mouse weight of table, liver index compare
* P < 0.05 compared to the blank group；Compared with model group, #P < 0.05
(b) compound turns mice serum triglycerides, total cholesterol, serum aspartate transaminase, alanine amino
Move the influence of enzyme
As shown in Table 4, compare with blank group result: model group triglycerides, total cholesterol, serum aspartat turn ammonia
Enzyme, alanine aminotransferase content obviously increase (P≤0.05), illustrate that the experimental mice liver cell has damage；Compound
Low dose group, middle dose group, high dose group serum aspartate transaminase, alanine aminotransferase content have different degrees of increasing
The variation of height, triglycerides, total cholesterol level is not obvious.It is compared with model group, it is compound high dose group triglycerides, total
Cholesterol, serum aspartate transaminase, the reduction of alanine aminotransferase content are most obvious, and middle dose group is secondly, low dosage
Group total cholesterol, content of triglyceride are without significant changes
4 each group mice serum triglycerides of table, total cholesterol, serum aspartate transaminase, alanine aminotransferase
Content
* P < 0.05 compared to the blank group；Compared with model group, #P < 0.05
(c) measurement of murine liver tissue superoxide dismutase, malonaldehyde
As shown in Table 5, compared to the blank group: model group mouse liver, superoxide dismutase content are decreased obviously, and third
Dialdehyde content obviously increases (P≤0.05), compared with model group: the superoxide dismutase content of each dosage group of compound has not
It is reduced with degree, mda content then rises, and wherein the result of gallic acid high dose group and bifendate positive controls are most
Be it is close, middle dose group secondly.
The content of 5 each group murine liver tissue superoxide dismutase of table, malonaldehyde
* P < 0.05 compared to the blank group；Compared with model group, #P < 0.05
The basic, normal, high amount group triglycerides of compound, total cholesterol, serum aspartate transaminase, phenylalanine ammonia
Based transferase and mda content and model group, which have been compared, reduces (P < 0.05) in various degree, but compares with positive controls without bright
Significant difference is different；Superoxide dismutase activity is increased (P < 0.05) compared with model group, but compared with the control group without significant difference
(P > 0.05).
As it can be seen that compound has protective effect to murine chronic alcoholic liver injury.
The Applicant declares that the present invention illustrates the process method of the present invention through the above embodiments, but the present invention not office
It is limited to above-mentioned processing step, that is, does not mean that the present invention must rely on the above process steps to be carried out.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to raw material selected by the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
Claims (27)
1. a kind of gallic acid phosphatide complexes, which is characterized in that the gallic acid phosphatide complexes are by gallic acid and phosphorus
The molar ratio of rouge composition, the gallic acid and phosphatide is 1:(0.1-8)；
The preparation methods of the gallic acid phosphatide complexes the following steps are included:
(1) gallic acid is dissolved in fat-soluble solvent, obtains clear liquid；
(2) phosphatide reaction, stirring is added by formula in step (1)；
(3) reaction dissolvent is removed, atent solvent is added and removes the drug not reacted with phosphatide, removes drying after atent solvent,
Up to gallic acid phosphatide complexes.
2. gallic acid phosphatide complexes according to claim 1, which is characterized in that the gallic acid and phosphatide rub
You are than being 1:(0.5-4).
3. gallic acid phosphatide complexes according to claim 2, which is characterized in that the gallic acid and phosphatide rub
You are than being 1:1.
4. gallic acid phosphatide complexes according to claim 1, which is characterized in that the phosphatide is natural phospholipid.
5. gallic acid phosphatide complexes according to claim 4, which is characterized in that the natural phospholipid is soybean lecithin
Rouge and/or egg yolk lecithin.
6. gallic acid phosphatide complexes according to claim 4, which is characterized in that the phosphatide is soybean lecithin.
7. a kind of preparation method of such as gallic acid phosphatide complexes of any of claims 1-6, which is characterized in that
The following steps are included:
(1) gallic acid is dissolved in fat-soluble solvent, obtains clear liquid；
(2) phosphatide reaction, stirring is added by formula in step (1)；
(3) reaction dissolvent is removed, atent solvent is added and removes the drug not reacted with phosphatide, removes drying after atent solvent,
Up to gallic acid phosphatide complexes.
8. preparation method according to claim 7, which is characterized in that fat-soluble solvent described in step (1) is tetrahydro furan
It mutters, in methylene chloride, chloroform, n-hexane, ethyl alcohol, ethyl acetate, hexamethylene, methanol, isopropanol, n-butanol or acetone
Any one or at least two mixture.
9. preparation method according to claim 8, which is characterized in that the ethyl alcohol is dehydrated alcohol.
10. preparation method according to claim 9, which is characterized in that fat-soluble solvent described in step (1) is anhydrous second
Alcohol.
11. preparation method according to claim 7, which is characterized in that the temperature of step (2) described reaction is 20-80 DEG C.
12. preparation method according to claim 11, which is characterized in that the temperature of step (2) described reaction is 30-70
℃。
13. preparation method according to claim 12, which is characterized in that the temperature of step (2) described reaction is 50 DEG C.
14. preparation method according to claim 7, which is characterized in that step (2) reaction is connection reflux unit,
The stirring is magnetic agitation.
15. preparation method according to claim 7, which is characterized in that the time of step (2) described stirring is 1-12h.
16. preparation method according to claim 15, which is characterized in that the time of step (2) described stirring is 2-8h.
17. preparation method according to claim 16, which is characterized in that the time of step (2) described stirring is 3h.
18. preparation method according to claim 7, which is characterized in that step (3) the removing reaction dissolvent is by subtracting
Pressure distillation and/or vacuum drying.
19. preparation method according to claim 7, which is characterized in that step (3) it is described remove reaction dissolvent temperature be
30-70℃。
20. preparation method according to claim 19, which is characterized in that step (3) temperature for removing reaction dissolvent
It is 35-60 DEG C.
21. preparation method according to claim 7, which is characterized in that atent solvent described in step (3) is methylene chloride
And/or chloroform.
22. preparation method according to claim 21, which is characterized in that atent solvent described in step (3) is three chloromethanes
Alkane.
23. a kind of pharmaceutical composition, which is characterized in that comprising such as gallic acid phosphatide of any of claims 1-6
Compound.
24. pharmaceutical composition according to claim 23, which is characterized in that described pharmaceutical composition also includes pharmaceutically to connect
The auxiliary material received.
25. pharmaceutical composition according to claim 23, which is characterized in that the auxiliary material is diluent, carrier, seasoning
In agent, adhesive and filler any one or at least two combination.
26. pharmaceutical composition according to claim 23, which is characterized in that described pharmaceutical composition be tablet, capsule,
In suppository, injection, gelling agent or emulsion any one or at least two combination.
27. a kind of pharmaceutical composition as described in any one of claim 23-26 is preparing anti-oxidant and/or protection liver function
Application in the drug of energy aspect；
Drug in terms of the protection liver function is the drug for protecting chronic alcohol liver injury.
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| CN101199508B (en) * | 2007-11-23 | 2010-11-10 | 复旦大学 | Salvianic phenol acid or salt phospholipid compound and preparing method thereof |
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