CN103222997B - Traditional Chinese medicine epimedium total flavonoid phosphorylation molecular modification method - Google Patents
Traditional Chinese medicine epimedium total flavonoid phosphorylation molecular modification method Download PDFInfo
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- CN103222997B CN103222997B CN201310147904.2A CN201310147904A CN103222997B CN 103222997 B CN103222997 B CN 103222997B CN 201310147904 A CN201310147904 A CN 201310147904A CN 103222997 B CN103222997 B CN 103222997B
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- herba epimedii
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Abstract
The invention relates to a traditional Chinese medicine epimedium total flavonoid phosphorylation molecular modification method. The invention belongs to the technical field of veterinary drug preparation. According to the invention, epimedium total flavonoid is subjected to phosphorylation molecular modification by using a sodium tripolyphosphate-sodium trimetaphosphate method. With total flavonoid content and phosphate content in the product as indexes, levels of three factors of pH, reaction temperature, and reaction time are researched with an orthogonal design method, and the factors are optimized. Phosphorylated epimedium total flavonoid is prepared under determined optimal modifications of: reaction temperature of 70 DEG C, reaction time of 6h, and reaction pH value of 9. Through infrared spectroscopy, the obtained epimedium total flavonoid phosphorylated compound is epimedium total flavonoid phosphate ester. The contents of total flavonoid and phosphate radical are high, a product yield is high, and the product can be instantly dissolved in water. Safe concentration of liver cell in-vitro culturing is substantially increased. The epimedium total flavonoid phosphate provided by the invention has good water solubility, high yield, and good safety.
Description
One, technical field
The present invention relates to a kind of Herba Epimedii total flavones phosphorylated molecules method of modifying, belong to herbal medicine preparing technical field.
Two, background technology
Herba Epimedii is the most frequently used simply medea, has kidney invigorating and YANG supporting, strengthening the tendons and bones, and the effects such as expelling wind and removing dampness play an important role in the control of renal function deficiency, rheumatic arthralgia, yang deficiency hypertension and osteoporosis etc. various diseases.Flavone is the topmost effective ingredient of Herba Epimedii, mainly from stem and leaf extract and obtain.Existing a large amount of modern studies show that epimedium flavone has the multiple pharmacological effect such as good enhancing immunity, antiviral, antiinflammatory, antioxidation, blood pressure lowering, antitussive, increase body's hypoxia tolerance, has certain preventive and therapeutic effect to many epidemic diseases.But because epimedium flavone is difficult to water-solublely, it is all very inconvenient that many preparation types use, and absorb hardly at gastric when oral, be also easy to be subject to the impact of gastric strong acid environment and destroyed, make its bioavailability obviously low.And due to the development of intensive culture, veterinary clinic medication is substantially all added to master with spice or drinking-water.Therefore, this has just greatly limited the exploitation of epimedium flavone on veterinary clinic.So research improves epimedium flavone water solublity method, improves its bioavailability very necessary.
Chinese medicinal compound structural modification is to improve one of its water solublity and bioactive Main Means.In recent years, many scholars find, after the composition sulphations such as herbal polysaccharide composition, saponin or phosphorylation modification, make it become carboxylate, and its water solublity generally increases, and ImmunopharmacologicaAction Action obviously strengthens; Soybean isoflavone is after sulphation modification, and its biological activity also increases.In epimedium flavone structure, contain phenolic hydroxyl group group, can be phosphorylated or sulphation, thereby increase its water solublity, and improve its biological activity.
The present invention modifies Herba Epimedii total flavones phosphorylated molecules first, set up Herba Epimedii total flavones phosphorylated molecules method of modifying, optimize modification condition, find that phosphorylated molecules modification can significantly increase the water solublity of Herba Epimedii total flavones, reduce its cytotoxic effect, improve its bioavailability, and obtain higher efficiency of pcr product.
Three, summary of the invention
Technical problem the present invention is directed to that Herba Epimedii total flavones water solublity is low, gastric absorbs hardly, easily by stomach acids destroy, reduce the problems such as its bioavailability, a kind of Herba Epimedii total flavones phosphorylated molecules method of modifying is provided, reach and significantly improve Herba Epimedii total flavones water solublity and safety, be convenient to the object of preparation and raising bioavailability, to be finally applied to livestock and poultry clinical prevention.
Technical scheme
With orthogonal experiment optimization Herba Epimedii total flavones phosphorylated molecules method of modifying.After optimizing, Herba Epimedii total flavones phosphorylated molecules method of modifying is: precision takes 2.5000g sodium tripolyphosphate and 1.0000g sodium trimetaphosphate (5: 2), is dissolved in 50ml distilled water; Precision takes 500mg Herba Epimedii total flavones (general flavone content 62.22%) and adds in 100ml distilled water, after fully mixing, add the sodium tripolyphosphate-sodium trimetaphosphate mixed liquor 50ml preparing, regulate pH=9, put in thermostat water bath, 70 DEG C of reaction temperatures, response time 6h, after reaction finishes, by reactant first with the tap water 48h that dialyses, use again distill water dialysis 24h, after lyophilization, obtaining phosphorylation Herba Epimedii total flavones, is Herba Epimedii total flavones phosphate ester through the analysis of Nicolet FT-IR200 type Fourier transformation infrared spectrometer.The according to said method Herba Epimedii total flavones phosphate ester of preparation, its general flavone content reaches 36.41%, and phosphate content can reach 34.85%, and efficiency of pcr product reaches 124.18%, and it is instant to enter water, and the hepatocellular toxicity of duck embryo of In vitro culture is significantly reduced.
Beneficial effect
Viral disease has become one of biggest threat of current human and animal's health, and has caused huge Socie-economic loss.Wreak havoc in recent years bird flu, swine flu, acquired immune deficiency syndrome (AIDS), SARS, influenza, enterovirus EV 71 type of the mankind etc., wreak havoc bird flu, newcastle, reproductive and respiratory syndrome, circovurus type 2, pseudorabies, parvovirus infection, duck viral hepatitis of animal etc., all make human and animal's life and health suffer serious threat.The major reason that viral disease is wreaked havoc people and animals is the mankind still do not have clinical specially good effect at present medicine for these potent virus.The resisiting influenza virus specific drug " oseltamivir phosphate capsule " of Zeng Jin is now but no longer so effective to H5N1, H7N9.Although the antiviral agents such as ribavirin, rimantadine hydrochloride, acycloguanosine also have certain clinical efficacy to virosis, what but it caused feel sick, abalienation, diarrhoea, hepatic and renal function damage, dizzy etc. untoward reaction and drug residue, these are iatrogenic, the continuous increase of drug-induced disease makes people start to refuse these antiviral Western medicine, then select the little Chinese herbal medicine of toxic and side effects, Chinese medicine or combination therapy of Chinese and western have been selected by existing more than 40% consumer at present.And in aquaculture, viral disease remains current aviculture and endangers one of maximum epidemic disease, comprehensively disabled along with the antiviral such as amantadine, ribavirin Western medicine, there is neutral gear in the Western medicine for the treatment of animal virosis on animal drug market.
Animal epidemic especially viral disease mainly relies on vaccine to prevent, but a lot of vaccine exists the shortcomings such as less immunogenic, needs immunostimulant to assist and could produce powerful immunne response.Now clinical conventional immunostimulant as oily adjuvant, aluminium glue adjuvant etc. often exist local irritation strong, have shortcomings such as residual.Chinese medicine ingredients due to safety, efficiently and gradually become the focus of immunostimulant research and development.
Herba Epimedii total flavones is the main pharmacodynamics composition of Herba Epimedii, has the effects such as good enhancing immunity and antiviral, antiinflammatory, antioxidation, and laboratory research finds that it all has good preventive and therapeutic effect to many animal epidemics.But because Herba Epimedii total flavones is insoluble in water, it is all very inconvenient that many preparation types use, and absorb hardly at gastric when oral, be also easy to be subject to the impact of gastric strong acid environment and destroyed, make its bioavailability obviously low.And due to the development of intensive culture, veterinary clinic medication is substantially all added to master with spice or drinking-water.Therefore, this has just greatly limited the exploitation of Herba Epimedii total flavones on veterinary clinic.So research improves the water miscible method of Herba Epimedii total flavones, improves its bioavailability very necessary.
The present invention adopts sodium tripolyphosphate-sodium trimetaphosphate method phosphorylated molecules to modify Herba Epimedii total flavones, the Herba Epimedii total flavones phosphate ester forming not only phosphate radical and general flavone content high, efficiency of pcr product is also very high, and water solublity is good, safety, preparation uses more convenient.
Compared with prior art, advantage of the present invention is as follows:
1. the method that Herba Epimedii total flavones phosphorylated molecules is modified has no report both at home and abroad, the invention provides a kind of preparation method of Herba Epimedii total flavones phosphate ester and the condition of optimization thereof, has filled up the blank of domestic and international research.
2. the Herba Epimedii total flavones phosphate ester obtaining by the invention process, not only total flavones and phosphate content are high, and efficiency of pcr product is high, and has good water solublity, enters water instant, and preparation is easy to use.
3. the Herba Epimedii total flavones phosphate ester obtaining by the invention process significantly reduces compared with Herba Epimedii total flavones the hepatocellular toxicity of duck embryo of In vitro culture.
Four, detailed description of the invention
1. the phosphorylated molecules of Herba Epimedii total flavones is modified and condition optimizing
Adopt sodium tripolyphosphate-sodium trimetaphosphate method phosphorylated molecules to modify Herba Epimedii total flavones.Precision takes 2.5000g sodium tripolyphosphate and 1.0000g sodium trimetaphosphate (5: 2), is dissolved in 50ml distilled water; Precision takes 500mg Herba Epimedii total flavones (general flavone content 62.22%) and adds in 100ml distilled water, adds the sodium tripolyphosphate-sodium trimetaphosphate mixed liquor 50ml preparing after fully mixing.According to single factor trial test result, adopt orthogonal experiment design method, select reaction system pH (A), reaction temperature (B), response time (C) three factors of the Index Influence maximums such as general flavone content, phosphate content, efficiency of pcr product in product to set respectively three levels, according to orthogonal table L
9(3
4) (table 1) design carries out orthogonal test.After reaction finishes, reactant, first with the tap water 48h that dialyses, then is used to distill water dialysis 24h, after lyophilization phosphorylation Herba Epimedii total flavones.Result of the test is in table 2.
Table 1 orthogonal test factor and level design
Table 2L
9(3
4) orthogonal experiments
From table 2, general flavone content from 21.35% to 36.41% in product, phosphate content is in 25.53%～34.85% scope, and efficiency of pcr product is between 69.64%～122.68%.According to the intuitive analysis method of orthogonal design, respectively taking general flavone content and phosphate content, efficiency of pcr product as index, first calculate the meansigma methods (K value) of each factor same level, calculate extreme difference R value according to K value again, finally judge the primary and secondary order of each factor impact according to the size of R value.R value is larger, and the impact of this factor is also larger; Otherwise impact is less.Taking general flavone content as index, best of breed is A
2b
1c
2, general flavone content is up to 36.41%, can find out from extreme difference R, and each factor is C > B > A, i.e. time having the greatest impact to general flavone content on the impact sequence of general flavone content.Taking phosphate content as index, best of breed is A
28
1c
2, the high-load of phosphate radical reaches 34.85%, can find out from extreme difference R, and each factor is B > C > A, i.e. temperature having the greatest impact to phosphate content on the impact sequence of phosphate content.Taking efficiency of pcr product as index, best of breed is A
28
2c
1, yield reaches 122.68%, can find out from R, and each factor is B > A > C, i.e. temperature having the greatest impact to efficiency of pcr product on the impact sequence of phosphate content.Comprehensive analysis, best of breed is A
2b
1c
2, regulate reaction system pH=9,70 DEG C of reaction temperatures, response time 6h.
2. Herba Epimedii total flavones phosphorylated molecules modified outcome infrared spectrum analysis
Adopt KBr pressed disc method to analyze with Nicolet FT-IR200 type Fourier transformation infrared spectrometer the Herba Epimedii total flavones phosphorylated molecules modified outcome (seeing Fig. 1) that above-mentioned optimum condition is modified, basically identical with the rear infrared absorption spectroscopy of modification before modifying, show that the two structure is substantially similar, essence does not occur and change; Both all show the characteristic absworption peak of some flavone compounds simultaneously: in flavone, carbonyl absorption peak is about 1650cm
-1; The absworption peak of phenolic hydroxyl group is about 3200-3650cm
-1; The skeleton of phenyl ring shakes at 1450cm
-1, 1500cm
-1, 1600cm
-1there is absworption peak etc. left and right.Spectrum after modification is except flavone parent characteristic peak obtains retaining, at 1160.61cm
-1there is the characteristic absorption peak of a phosphate ester, at 918.15cm
-1the phosphite ester characteristic absorption peak occurring obviously, than becoming large before modifying, proves that the product after modifying is Herba Epimedii total flavones phosphate ester.
Brief description of the drawings Fig. 1 Herba Epimedii total flavones phosphorylated molecules is modified front and back infrared spectrum
1. modifying front infrared spectrogram for Herba Epimedii total flavones phosphorylated molecules, is 2. infrared spectrogram after Herba Epimedii total flavones phosphorylated molecules is modified.1. and 2. infrared absorption spectroscopy is basically identical, shows that the two structure is substantially similar, essence does not occur and change.1. at 3396.29cm
-12. at 3384.52cm
-1the absworption peak occurring is phenolic hydroxyl group characteristic absorption peak; 1. at 1651.00cm
-12. at 1651.00cm
-1the absworption peak that left and right occurs is carbonyl characteristic absorption peak in flavone; 1. at 1453.81cm
-1, 1500.90cm
-1, 1600.97cm
-1the skeleton vibrations characteristic absorption peak that the absworption peak that left and right occurs is phenyl ring, these are some characteristic absworption peaks of flavone compound.2. except characteristic peak 1. obtains retaining, at 1160.61cm
-1there is the characteristic absorption peak of a phosphate ester, at 918.15cm
-1the phosphite ester characteristic absorption peak that place occurs, obviously than 1. becoming large, proves that the product after modifying is Herba Epimedii total flavones phosphate ester.
3. Herba Epimedii total flavones phosphate ester water solublity is analyzed
First by etc. the Herba Epimedii total flavones of quality and Herba Epimedii total flavones phosphate ester dissolve with distilled water, PBS and cell culture fluid respectively, paired observation modify before the dissolving situation of latter two medicine in these three kinds of solvents.Found that, Herba Epimedii total flavones all dissolves hardly in 3 kinds of solvents, when adding again therein the ethanol of 10% left and right, and repeatedly when supersound process 2h left and right, just substantially without observing visible precipitate thing; And Herba Epimedii total flavones phosphate ester is while adding this three kinds of solvents, slight jolting, all dissolves at once, and solution is homogenizing light brown, light is observed, without obvious visual precipitate.Show, Herba Epimedii total flavones is modified and is formed after Herba Epimedii total flavones phosphate ester through phosphorylated molecules, and water solublity significantly increases, and preparation uses more convenient.
4. Herba Epimedii total flavones phosphate ester is to the hepatocellular safety analysis of In vitro culture duck embryo
Get 14-16 age in days duck embryo, under aseptic condition, take out liver.The duck embryo liver of taking-up is removed to gallbladder, with D-Hank ' s cleaning 2 times, shred rear with D-Hank ' s cleaning 3 times.Add 0.25% trypsin, 37 DEG C of digestion 8～15min, sop up trypsin, with D-Hank ' s cleaning 3 times, add the DMEM culture medium containing 1% benzylpenicillin potassium, 1% streptomycin sulfate, 1%L-glutamine, 10% calf serum.Counting makes cell concentration 0.8 × 10
6-1.2 × 10
6within the scope of individual/mL, put 37 DEG C, 5%CO
2in incubator, cultivate, after cell forms complete monolayer, discard cell culture fluid, wash 2 times with D-Hank`s liquid, add respectively different dilution Herba Epimedii total flavones and Herba Epimedii total flavones phosphate ester, 100 μ L/ holes, the each concentration of every medicine repeats 4 holes, establishes cell control well simultaneously.Continue to cultivate 68h, add the blue 30 μ L of 2mg/mL tetramethyl azo azoles, then continue to cultivate after 4h, after the centrifugal 10min of 3500rpm, tipping supernatant, each hole adds dimethyl sulfoxine 100 μ L, vibration 5-10min, reads 570nm absorbance (OD) value with microplate reader.OD value is larger, represents that cell number is more, and cell viability is stronger; Otherwise, represent that cell number is few, a little less than cell viability.Significantly do not evaluate the safety of medicine to cultured cell in vitro lower than the highest drug level of cell control well OD value to add medicine hole OD value.The results are shown in Table 3.
Table 3 Herba Epimedii total flavones phosphorylated molecules is modified front and back cell safe concentration measurement result
As can be seen from Table 3, Herba Epimedii total flavones maximum cell safe concentration is 500 μ g/mL, demonstrates the effect of Promote cell's growth in the time that concentration is 500～125 μ g/mL; And the maximum cell safe concentration of Herba Epimedii total flavones phosphate ester is 2500 μ g/mL, in the time that being 1250～39.06 μ g/mL, concentration demonstrates the effect of Promote cell's growth.The maximum cell safe concentration of Herba Epimedii total flavones phosphate ester is 5 times of Herba Epimedii total flavones, and it is also significantly larger to show as the activity scope of Promote cell's growth.Show that Herba Epimedii total flavones phosphate ester significantly reduces compared with Herba Epimedii total flavones hepatocellular toxicity, phosphorylated molecules is modified the cytotoxicity that has reduced Herba Epimedii total flavones, has improved its safety.
Claims (1)
1. a Herba Epimedii total flavones phosphorylated molecules method of modifying, adopts sodium tripolyphosphate-sodium trimetaphosphate method phosphorylated molecules to modify Herba Epimedii total flavones:
(1) by sodium tripolyphosphate: sodium trimetaphosphate=5: 2 2.5000g sodium tripolyphosphate and the 1.0000g sodium trimetaphosphate of taking accurate in scale, is dissolved in 50ml distilled water; It is that 62.22% Herba Epimedii total flavones adds in 100ml distilled water that precision takes 500mg general flavone content, after fully mixing, add the sodium tripolyphosphate-sodium trimetaphosphate mixed liquor 50ml preparing, regulate pH=8～10, put in thermostat water bath, 70 DEG C～90 DEG C of reaction temperatures, response time 4～8h, after reaction finishes, by reactant first with the tap water 48h that dialyses, use again distill water dialysis 24h, after lyophilization, obtain phosphorylation Herba Epimedii total flavones;
(2) condition of said method phosphorylated molecules modification Herba Epimedii total flavones is: reaction pH=9,70 DEG C of reaction temperatures, response time 6h;
(3) phosphorylated molecules is modified Herba Epimedii total flavones under these conditions, and in its product, general flavone content reaches 36.41%, and phosphate content can reach 34.85%, and efficiency of pcr product reaches 124.18%;
(4) phosphorylated molecules is modified Herba Epimedii total flavones under these conditions, the infrared spectrogram basic configuration of its product is consistent with before modification, essence does not occur and change, and all has flavone characteristic absorption peak, except flavone parent characteristic peak obtains retaining, at 1160.61cm
-1there is the characteristic absorption peak of a phosphate ester, at 918.15cm
-1the phosphite ester characteristic absorption peak of locating to occur obviously becomes large than the peak before modifying, and shows that in this law, phosphorylated molecules modification Herba Epimedii total flavones product is Herba Epimedii total flavones phosphate ester;
(5) the Herba Epimedii total flavones phosphate ester of preparation has good water solublity under these conditions, enters water instant;
(6) the Herba Epimedii total flavones phosphate ester of preparation significantly reduces compared with Herba Epimedii total flavones the hepatocellular toxicity of duck embryo of In vitro culture under these conditions.
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