CN102145066B - Preparation method for extracting general flavone from Chinese medicinal herbs - Google Patents
Preparation method for extracting general flavone from Chinese medicinal herbs Download PDFInfo
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Abstract
The invention relates to a preparation method for extracting general flavone from Chinese medicinal herbs, belonging to the technical field of plants. The preparation process comprises the following steps of: percolating and extracting dense cornel as a raw material by using aqueous ethanol as a solvent; flashing the extract, concentrating and recovering the solvent; separating and enriching the concentrated solution through macroporous resin columns, carrying out gradient elution with an aqueous solvent, mixing a certain proportion of aqueous solvent eluate, flashing, concentrating and recovering the solvent; and continuously drying in vacuum to form dry powder. The invention has the advantages of simple process, superior extraction and concentration methods, convenience for operation, environmental protection and high efficiency; the extractive contains 6.12-15.58 percent of general flavone, the extraction rate of the general flavone is up to 5.25-10.43 percent, and the transfer rate of the general flavone is up to 45.63-62.88 percent. Proved by primary in-vitro bioassay tests, the dense cornel general flavone extractive has certain activities, such as oxidation resistance, bacteriostasis, and the like, can be used as a food additive, an antioxidant, a preservative, and the like and has certain development and application values.
Description
Technical field
The invention belongs to the plant technology field, be specially a kind of method for preparing of from Chinese herbal medicine, extracting total flavones.
Background technology
Flavone compound is one type of important substance that is present in botanic biologically active, extensively is present in the not equal platymiscium, mainly is present in Rutaceae, Labiatae, pulse family, Umbelliferae and the Compositae.Research report has approximately in 20% the Chinese herbal medicine and contains flavone compound, and the resource of visible flavone compound is quite abundant.Flavone compound can be taken the photograph from food; Also can from the plant of being rich in flavone compound, extract, it can not only and have tangible anti peroxidation of lipid, defying age, removing free radical as the medicine of control cardiovascular and cerebrovascular disease; Blood fat reducing, cholesterol reducing, blood sugar lowering; Physiologically active such as anti-cancer and cancer-preventing, immunomodulating is one type of natural component with broad prospect of application, also is application prospect natural inhibitor widely; Antiseptic can be used for developing plurality kinds of health care food, food additive and cosmetics.Therefore has good development and application values.
The method of from natural plants, extracting flavone compound in recent years adopts conventional method for distilling more, comprises cold-maceration, ultrasonic method, circumfluence method etc.These methods have that solvent load is big, extraction time length, troublesome poeration, efficient drawback such as lower.And the separation and concentration flavones ingredient adopts macroporous resin column chromatography more, the method for aqueous solvent gradient elution in varing proportions.Because a large amount of extracting solution and eluent need concentrate, the improper effective ingredient that often causes easily of method for concentration destroys, and reduces extraction ratio of effective constituents, often contains some beavy metal impurities in the flavone extract simultaneously, and often exceeds standard.
Melicope patulinervia (Merr.& Chun) Huang is the close Fructus Evodiae platymiscium of Rutaceae, is shrub or arbor, be distributed in the China's Mainland Hainan etc. ground, be grown in the hillside fields sparse woods of 900～1200 meters of height above sea level, be Hainan endemic plant.According to bibliographical information, contained chemical constituent is mainly flavonoid, alkaloids and terpenoid etc. in this section plant, has significant antioxidation, antibacterial parasite killing, heat-clearing and toxic substances removing, promoting the circulation of QI to relieve pain, effects such as dampness removing to stop itchin.Be Chinese herbal medicine commonly used among the people simply, the treatment laryngopharynx swelling and pain that is mainly used among the people, hot carbuncle toxin, rheumatic arthralgia, wet fiery osteodynia, diseases such as treating swelling and pain by traumatic injury, external curing skin herpes simplex rash, skin pruritus, hemorrhoid.At present its The Chemical Constituents and Application and Development thereof are not seen that any report is arranged.
Summary of the invention
To the problems referred to above that exist in the prior art; The objective of the invention is to design provides a kind of technical scheme of from Chinese herbal medicine, extracting the method for preparing of total flavones; Its technology is simple, superior, easy to operate, the green high-efficient of extraction method for concentration, and the yield of extractive of general flavone is higher.
Described a kind of method for preparing of from Chinese herbal medicine, extracting total flavones is characterized in that may further comprise the steps:
1) raw material is handled: close Fructus Evodiae medical material is selected the back break into coarse powder with pulverizer, cross 20～40 mesh sieves;
2) percolation extracts: close Fructus Evodiae coarse powder is packed in the percolator; After soaking 8～12 hours with 50%～80% aquiferous ethanol, carry out percolation with 50%～80% aquiferous ethanol and extract, as if raw material weight be 1kg then the consumption of aquiferous ethanol be 8～16 liters; Flow velocity is 5～8 mL/min, collects percolate;
3) flash concentration: 60 ℃～90 ℃ of bath temperatures, vacuum is 0.090 Mpa～0.095Mpa with percolate, and the feed liquor flow velocity is 200～350 mLmin
-1Condition under carry out vacuum film and concentrate, reclaim ethanol, obtain every mL medicinal liquid and amount to the concentrated solution that contains 1.0～2.0g raw material;
4) macroporous resin adsorption purification: through the macroporous adsorbent resin separation and concentration, water, 10% aquiferous ethanol, 20%～70% aquiferous ethanol carry out gradient elution successively, use 70% acetone eluting at last with the concentrated solution that obtains, and elution flow rate is 5～15 mL min
-1, collect each position eluents of 3 times of column volumes respectively, collect the eluent that merges 20%～60% each position of aquiferous ethanol, under 60 ℃～90 ℃, vacuum is 0.090 Mpa～0.095Mpa with the eluent that merges, the feed liquor flow velocity is 250～350 mL min
-1Condition under carry out the vacuum film concentrate drying, promptly get close Fructus Evodiae extractive of general flavone dry powder.
Described a kind of method for preparing of from Chinese herbal medicine, extracting total flavones is characterized in that step 2) in: the aquiferous ethanol concentration that is used to soak is 60%～70%, and soak time is 9-10 hour; The aquiferous ethanol concentration that is used for the percolation extraction is 60%～70%, and flow velocity is 6～7 mL/min.
Described a kind of method for preparing of from Chinese herbal medicine, extracting total flavones, it is characterized in that in the step 3): bath temperature is 70 ℃～80 ℃, and vacuum is 0.093Mpa, and the feed liquor flow velocity is 250～300mLmin
-1
Described a kind of method for preparing of from Chinese herbal medicine, extracting total flavones; It is characterized in that in the step 4): water, 10% aquiferous ethanol, 20% aquiferous ethanol, 40% aquiferous ethanol, 60% aquiferous ethanol, 70% aquiferous ethanol carry out gradient elution successively; Use 70% acetone eluting at last, elution flow rate is 8～12mLmin
-1, collect each position eluents of 3 times of column volumes respectively, collect the eluent that merges 20% aquiferous ethanol, 40% aquiferous ethanol and 60% each position of aquiferous ethanol.
Described a kind of method for preparing of from Chinese herbal medicine, extracting total flavones; It is characterized in that in the step 4): water, 10% aquiferous ethanol, 25% aquiferous ethanol, 35% aquiferous ethanol, 45% aquiferous ethanol, 55% aquiferous ethanol, 70% aquiferous ethanol carry out gradient elution successively; Use 70% acetone eluting at last; Collect each position eluent of 3 times of column volumes respectively, collect the eluent at merging, 25% aquiferous ethanol, 35% aquiferous ethanol, 45% aquiferous ethanol and 55% each position of aquiferous ethanol.
Described a kind of method for preparing of from Chinese herbal medicine, extracting total flavones, it is characterized in that in the step 4): the temperature of the eluent concentrate drying of merging is under 70 ℃～80 ℃, and vacuum is 0.093 Mpa, and the feed liquor flow velocity is 280～320 mLmin
-1
Described macroporous adsorbent resin is a Dianon HP-20 macroporous adsorbent resin.
Above-mentioned a kind of method for preparing of from Chinese herbal medicine, extracting total flavones has following beneficial effect:
1, the present invention adopts the aquiferous ethanol percolation to extract to the close Fructus Evodiae, and vacuum flashing concentrates, and has that the solvent load of extraction is few, extraction efficiency is higher than cold-maceration, the room temperature extraction does not destroy effective ingredient; During flash concentration low, time of concentrated solution heating temperature short, concentrate that speed is fast, efficient is high, advantages such as simple to operate, easy to use, energy-conserving and environment-protective.
2, the above-mentioned method for preparing total flavones of from the close Fructus Evodiae, extracting, technology is simple, the extraction method for concentration is advanced, easy to operate, cost is low, green high-efficient, the extractive of general flavone yield is high.Adopt the resulting extractive of general flavone of determined by ultraviolet spectrophotometry, content of total flavone is 6.12%～15.58% in the extract, and the extraction ratio of total flavones is 5.25%～10.43%, and the rate of transform of total flavones is up to 45.63%～62.88%.Show that through preliminary external biological activity test close Fructus Evodiae extractive of general flavone has certain antibiotic, antioxidant activity, can be used as food additive, antioxidant, antiseptic, medicine material etc.Therefore has good development and application values.
3, measure the content of heavy metal in the extractive of general flavone through the ICP-MS method; Content of beary metal is very low in the close Fructus Evodiae total flavones of this inventive method preparation of discovery employing, meets " the standard of Chinese pharmacopoeia and " medicinal plants and preparation thereof are imported and exported green industry standard ".
The percentage composition that relates in the present specification unless otherwise indicated, the percentage composition of liquid is a volume ratio, solid percentage composition is a weight ratio.
Description of drawings
Fig. 1 is a 5-hydroxyl-6,7.8-trimethoxy-3 ', 4 '-the hydrogen nuclear magnetic resonance spectrogram of methylene-dioxy flavone;
Fig. 2 is a 5-hydroxyl-6,7.8-trimethoxy-3 ', 4 '-the carbon-13 nmr spectra figure of methylene-dioxy flavone;
Fig. 3 is 5,6,7.8-tetramethoxy-3 ', 4 '-the hydrogen nuclear magnetic resonance spectrogram of methylene-dioxy flavone;
Fig. 4 is 5,6,7.8-tetramethoxy-3 ', 4 '-the carbon-13 nmr spectra figure of methylene-dioxy flavone.
The specific embodiment
Below in conjunction with specific embodiment the present invention is described further.
Embodiment 1:
1) raw material is handled: close Fructus Evodiae medical material is selected the back break into coarse powder with pulverizer, cross 20 mesh sieves;
2) percolation extracts: close Fructus Evodiae coarse powder is packed in the percolator, soak 10 hours with 60% aquiferous ethanol after, carry out percolation with 70% aquiferous ethanol and extract, as if raw material weight be 1kg then the consumption of aquiferous ethanol be 12 liters, flow velocity is 6 mL/min, the collection percolate;
3) flash concentration: 70 ℃ of bath temperatures, vacuum is 0.095Mpa with percolate, and the feed liquor flow velocity is 250 mL min
-1Condition under carry out vacuum film and concentrate, reclaim ethanol, obtain every mL medicinal liquid and amount to the concentrated solution that contains the 2.0g raw material;
4) macroporous resin adsorption purification: the concentrated solution that obtains is passed through Diaion HP-20 macroporous adsorbent resin separation and concentration; Water, 10% aquiferous ethanol, 20% aquiferous ethanol, 40% aquiferous ethanol, 60% aquiferous ethanol, 70% aquiferous ethanol carry out gradient elution successively, and elution flow rate is 10 mLmin
-1, collect each position eluents of 3 times of column volumes respectively, collect the eluent that merges 20% aquiferous ethanol, 40% aquiferous ethanol and 60% each position of aquiferous ethanol, under 70 ℃, vacuum is 0.095Mpa with the eluent that merges, the feed liquor flow velocity is 300 mLmin
-1Condition under carry out vacuum film and concentrate vacuum concentration, drying, promptly get close Fructus Evodiae extractive of general flavone dry powder.
In method for preparing of the present invention:
In the step 1) close Fructus Evodiae medicinal material coarse powder is crossed 40 mesh sieves;
Step 2) the aquiferous ethanol concentration that is used to soak in is 50%, 70% or 80%, and soak time is 8 hours, 9 hours or 12 hours; The aquiferous ethanol concentration that is used for the percolation extraction is 50%, 60% or 80%, and flow velocity is that flow velocity is 5 mL/min, 7 mL/min or 8 mL/min;
Bath temperature is 60 ℃, 80 ℃ or 90 ℃ in the step 3), and vacuum is 0.093Mpa, and the feed liquor flow velocity is 200mLmin
-1, 300mLmin
-1Or 350mLmin
-1, obtain every mL medicinal liquid and amount to the concentrated solution that contains 1.0g, 1.3g, 1.5g, 1.8g raw material;
Water, 10% aquiferous ethanol, 25% aquiferous ethanol, 35% aquiferous ethanol, 45% aquiferous ethanol, 55% aquiferous ethanol, 70% aquiferous ethanol carry out gradient elution successively in the step 4); Use 70% acetone eluting at last; Collect each position eluent of 3 times of column volumes respectively, collect the eluent at merging, 25% aquiferous ethanol, 35% aquiferous ethanol, 45% aquiferous ethanol and 55% each position of aquiferous ethanol.
In the step 4): the temperature of the eluent concentrate drying of merging is 60 ℃, 70 ℃ or 80 ℃, and vacuum is 0.093 Mpa, and elution flow rate is 5mLmin
-1, 8mLmin
-1, 12mLmin
-1Or 15mLmin
-1, the feed liquor flow velocity is 250 mLmin
-1, 320 mLmin
-1Or 350 mLmin
-1Use H in the above-mentioned steps
2O and 10% aquiferous ethanol eluting all use 70% acetone eluting to remove all dirt for removing water-solubility impurity at last, the regeneration macroporous adsorbent resin;
Other can obtain beneficial effect of the present invention with embodiment 1.
Below through corresponding test the extractive of general flavone through the present invention preparation is carried out the assay of total flavones and heavy metal, carry out the test of in-vitro antibacterial and antioxidant activity simultaneously.
1, content of total flavone is measured in the close Fructus Evodiae extract
Condition determination: UV-2102PCS ultraviolet-uisible spectrophotometer (going up Solenognathus Ni Ke Instr Ltd.); Quercetin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute provides), the maximum absorption wavelength of Quercetin: 510 nm.
The drafting of standard curve: accurately take by weighing Quercetin reference substance 5.5 mg of dry constant weight, add 75% dissolve with ethanol and be settled in the measuring bottle of 50 mL, shake up to such an extent that concentration is 0.110 mg mL
-1Reference substance solution.Get above-mentioned Quercetin standard solution 0,0.5,1.0,1.5,2.0,2.5,3.0 respectively in 10 mL measuring bottles, be supplemented to 5 mL with 75% ethanol, each adds 0.5 mL, 5% sodium nitrite, shakes up, and respectively adds 10% aluminum nitrate, 0.5 mL again after placing 5 min, shakes up.Add 4% sodium hydroxide solution 4 mL behind 5 min again, mixing, with 75% ethanol dilution to scale.Survey absorbance behind 15 min in 510 nm places; Reagent is blank reference; With the Quercetin mass concentration is vertical coordinate, and absorbance is an abscissa drawing standard curve, carries out linear regression with method of least square; Get the regression equation between quercetin content and the absorbance: y=12.93x+0.44, γ=0.9980.
The assay of sample: accurate claim that fixed exsiccant close Fructus Evodiae extract dry powder (embodiment 1) is an amount of, with 75% dissolve with ethanol and be settled in the 25 mL measuring bottles.Draw a certain amount of above-mentioned solution in 10 mL measuring bottles; Add 75% alcoholic solution and make volume be about 5 mL, the method according to above-mentioned drafting Quercetin standard curve adds sodium nitrite, aluminum nitrate and sodium hydroxide solution successively; Mixing, with 75% ethanol dilution to scale.Measure absorbance after leaving standstill 20 min.Repeating 3 times measures; And converse content of total flavone in the sample according to standard curve; The average content that calculates total flavones in the close Fructus Evodiae extract is 16.12%～21.58%, and the extraction ratio of total flavones is 5.25%～10.43%, and the rate of transform of total flavones is up to 45.63%～62.88%.
Through system's extraction separation, obtained several flavone monomers from the difference of the close Fructus Evodiae 70% ethanol extraction extraction position, identified their structure through physicochemical identification and spectral technique.Wherein isolation identification goes out at the ethyl acetate extraction position 5,6,7.8-tetramethoxy-3 ', 4 '-the methylene-dioxy flavone, 5-hydroxyl-6,7.8-trimethoxy-3 ', 4 '-amount of methylene-dioxy flavone is very big.Through the HPLC assay, 5-hydroxyl-6 in the close Fructus Evodiae extractive of general flavone, 7.8-trimethoxy-3 ', 4 '-methylene-dioxy flavone and 5,6,7.8-tetramethoxy-3 ', 4 '-content of Ya dimethoxy flavone reaches 3.12% and 5.37% respectively.
2, the assay of heavy metal element in the close Fructus Evodiae extractive of general flavone
(1) instrument and running parameter: adopt micro-wave digestion-inductively coupled plasma mass spectrometry to measure the content of heavy metal element in the close Fructus Evodiae extractive of general flavone.X-Series type icp ms (U.S. power & light company); MARS-5 type microwave dissolver (U.S. CE M company) attaches RTP-300 Plus temperature control; Milli-Q Academic ultra-pure water processor (U.S. Millipore company).Nitric acid is that top grade is pure, and other reagent are analytical pure.Instrument working parameter such as following table 1.
Table 1 ICP-MS running parameter
| Running parameter | Setting value | Running parameter | Setting value |
| Forward power/(W) | 1200 | Sampling depth | 130 |
| Scan pattern | Jump the peak | Cooling gas flow/(L min -1) | 13.02 |
| |
100 | Secondary air amount/(L min -1) | 0.70 |
| Residence time/(μ s) | 10000 | Atomization gas flow/(L min -1) | 0.84 |
| The sampling time/(s) | 49 | Sample injection time/(s) | 45 |
| Sample lifting rate/(mLmin -1) | 1.0 | Scavenging period/(s) | 60 |
(2) The pretreatment and micro-wave digestion: precision takes by weighing close Fructus Evodiae extractive of general flavone powder (embodiment 1) 0.1 g (claiming accurate to 0.0001 g), places interior jar of politef micro-wave digestion jar, adds 5 mL concentrated nitric acids; 0.5 h is left standstill in slight vibration, puts into microwave dissolver, clears up by the micro-wave digestion program; Clear up the postcooling that finishes to room temperature; Digestion solution shifted in taking out jar and standardize solution in 50 mL volumetric flasks, be equipped with a blank solution simultaneously and do contrast, see table 2.
Table 2 micro-wave digestion program
| Clear up program | Power/W | Heating-up time/min | Controlled pressure/kPa | Temperature/℃ | Persistent period/min |
| 1 | 1200 | 5 | 340 | 120 | 2 |
| 2 | 1200 | 3 | 689 | 150 | 3 |
| 3 | 1200 | 3 | 1000 | 180 | 6 |
In clearing up margin of safety; Pressure in the counteracting tank, temperature, heating-up time are carried out preferably; Find to adopt intensification gradually, the boosting mode of three programs to carry out micro-wave digestion, obtained the Digestive system of no residue, clear, whole microwave treatment only needs 11 min.
For Volatile Elements such as arsenic and lead, in digestion process, need strict control temperature.Can keep digestion solution to be in approximate slight boiling condition during about 150 ℃ of experiment proof digestion condition, just reach HNO
3Boiling point.Simultaneously for preventing the sample carbonization, nitric acid is taked the form that drips, and notes temperature controlling after adding nitric acid, makes digestion solution keep slight boiling condition, to guarantee that element loss to be measured is minimum in the sample.
(3) standard solution and the range of linearity: chromium, arsenic, cadmium, lead, copper standard stock solution (U.S. SPEX CertiPrep Inc.), concentration is 10 mg L
-1, be stored in respectively in the vinyon bottle, be made into the mixed standard solution (containing 1% nitric acid) of debita spissitudo then according to the needs of measuring.
From standard reserving solution, shift out a certain amount of standard solution with dilution method progressively, be mixed with standard solution with the high purity water dilution, the standard series concentration of chromium, arsenic, cadmium, lead, copper is 2.0,5.0,10.0,20.0 μ g L
-1, contain 1%HNO in the standard solution
3Add inner mark solution respectively, under selected optimal conditions, adopt ICP-MS to measure, after standard solution got into ICP-MS, instrument had provided the working curve and the linear relationship of each element.The equation of linear regression of 5 kinds of elements, linearly dependent coefficient, the range of linearity and detection limit such as following table 3.
Table 3 linear equation and correlation coefficient
| Element | Equation of linear regression | Linearly dependent coefficient | The range of linearity (μ g L -1) | Detection limit (μ g L -1) |
| Cr | Y=3.53×10 2 X+1.21×10 3 | 0.999 8 | 0～1000 | 0.20 |
| As | Y=4.57×10 2 X+9.57×10 -1 | 0.999 7 | 0～1000 | 5.65×10-2 |
| Cd | Y=9.48×10 2 X+4.11 | 0.999 8 | 0～1000 | 5.13×10-3 |
| Pb | Y=1.02×10 4 X+9.80×10 2 | 0.999 8 | 0～1000 | 1.57×10-2 |
| Cu | Y=6.54×10 2 X+2.57×10 2 | 0.999 6 | 0～1000 | 7.53×10 -2 |
(3) accuracy of method experiment:, adopt standard addition method to measure the response rate of each element in order to estimate the accuracy of this method.Replication is 5 times under selected condition determination; The response rate and the relative standard deviation RSD of 5 kinds of elements have been measured respectively; 5 heavy metal element average recovery rates are between 99..2%～103.3% as a result; RSD shows the method accurately and reliably between 0.69%～2.04%, can satisfy the assay determination of each element in the sample.
(4) sample determination result: the instrument igniting, pump into high purity water, detect the blank counts of each element, pump into 2 μ g L then
-1Mixed standard solution, the sensitivity of detecting instrument.Behind the normal and instrument stabilizer of above-mentioned each step, can begin according to the running parameter of instrument to measure.To clear up good sample solution and suck the ICP-MS appearance successively, measure each constituent content in the solution, while working sample blank and standard solution, the content of Cu is 12.47 μ g g in the results sample
-1The content of Cr is 0.62 μ g g
-1The content of As is 0.30 μ g g
-1The content of Cd is 0.16 μ g g
-1The content of Pb is 4.21 μ g g
-1
Chromium, cadmium, lead, copper, arsenic belong to heavy metal and arsenic salt, are harmful element, can cause retention toxicosis after being absorbed by the body.Though it is copper is human essential elements, excessive also harmful.The limit index of these heavy metals and arsenic salt is main according to " Chinese pharmacopoeia is estimated with " medicinal plants and preparation thereof are imported and exported green industry standard ".With reference to this standard, i.e. Cu≤20.0 μ g g
-1, Cd≤0.3 μ g g
-1, Pb≤5.0 μ g g
-1, As≤2.0 μ g g
-1, do not have chromium in this standard, therefore with reference to green food standard (Cr≤1.0 μ g g
-1).Above-mentioned test determination result shows, toxic heavy metal element Cd in the close Fructus Evodiae extractive of general flavone, and Cr, Pb, the content of Cu, As all is lower than limit standard, meets medicinal plants and preparation thereof and imports and exports green industry standard and green food standard.
3, antioxidation result of the test
1, (1,1-Diphenyl-2-picryl-hydrazyl DPPH) is a kind of stable organic free radical to 1-diphenyl picryl phenylhydrazine, can represent the power of its non-oxidizability to the removing ability of DPPH free radical through detection of biological reagent.In recent years, Chinese scholars utilizes the DPPH solution absorbency to change as the spectrophotometry of removing the free radical ability, and is proved to be a kind of sensitivity, simple effective ways.This test adopts the DPPH spectrophotography in order to estimate the oxidation resistance of close Fructus Evodiae extractive of general flavone.
DPPH in organic solvent be a kind of stable be the center free radical with nitrogen, DPPH can generate the stabilizing solution with typical purple in solution, and at the 517nm place strong absorption is arranged.When it and free radical scavenger are done the time spent,, the typical purple of solution is shoaled owing to scavenger and the pairing of DPPH lone pair electrons reduce its absorbance at the 517nm place., the variation of its absorbance becomes quantitative relationship with its electronics of accepting.Be that absorbance is more little, the ability that free radical scavenger is removed free radical is strong more, thereby available spectrophotography carries out quantitative analysis.
(1) experiment condition: Tecan Infinite M200 ELIASA (Switzerland); UV-2102 PCS ultraviolet-uisible spectrophotometer (Shanghai You Nike Instr Ltd.); 101-3 electric heating air blast thermostatic drying chamber (blue sky, Hangzhou assay apparatus factory); KQ-250B type ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); R201B Rotary Evaporators (bio tech ltd is won in the Shen, Shanghai).1, and the bitter diazanyl free radical of 1-diphenyl-2-(1,1-Diphenyl-2-picryl-hydrazyl, DPPH) available from sigma company, ELISA Plate: 96 microwell plates, other reagent are analytical pure.
(2) preparation of DPPH solution and need testing solution: the preparation of DPPH solution: accurately take by weighing DPPH reagent 30.4 mg; Use 75% dissolve with ethanol, and quantitatively change in the 100 mL measuring bottles, therefrom precision is measured 4.5 mL; Be settled to 25 mL with 75 % ethanol, shake up to such an extent that mass concentration is 54 μ g mL
-1The DPPH stock solution, it is subsequent use to place refrigerator and cooled to hide.
The preparation of need testing solution: take by weighing accurately respectively that to extract the close Fructus Evodiae extractive of general flavone dry powder (embodiment 1) that purification obtains through the inventive method an amount of, with 75% dissolve with ethanol and be settled in the 100 mL measuring bottles.Get 50,100,200,400,600,800 μ L sample solutions then respectively, be settled in the 1 mL measuring bottle with 75% ethanol, obtain the need testing solution of variable concentrations, it is for use to place 4 ℃ of refrigerators to preserve.
(3) assay method and result: the need testing solution 20 μ L and the DPPH test solution 200 μ L that in 96 hole ELISA Plates, add variable concentrations with point sample with liquid-transfering gun respectively.Sample adds back concussion 30s; Behind 26 ℃ of insulation 30 min; Under 517 nm wavelength, measuring its light absorption value (Ap) with Infinite M 200 ELIASAs, measuring sample blank (the replacing DPPH) light absorption value (Ac) do not add DPPH simultaneously and add DPPH but do not add the light absorption value (Amax) of sample (with 20 μ L, 75% ethanol replacement sample) with 200 μ L75% ethanol.Make positive control with Trolox in the sample determination process, and converse the total oxidation resistance of sample with this, the mensuration result is expressed as and reaches the needed Trolox concentration of the suitable oxidation resistance of finite concentration test substances.Claim that this method is TEAC (Trolox equivalent antioxidant capacity) method.The TEAC value representation be the concentration of Trolox, Trolox concentration is big more, shows that to remove the free radical ability strong more.With Trolox (X) concentration is abscissa, is vertical coordinate drawing standard curve with the free radical scavenging activity (Y) that records, Y=7.261X+0.535, r=0.9970.
The sample oxidation resistance is with TEAC (trolox equivalent antioxidant capacity) expression, the clearance rate=1-of free radical (Ap-Ac)/Amax * 100%.
The sample of result of calculation embodiment 1 is 73.56 (mg GAE/g DW) to the TEAC value that DPPH removes ability, and the result shows that the confession test agent has certain clearance rate to the DPPH free radical, and clearance rate improves with the increase that supplies the test agent solution concentration.。Show that the extractive of general flavone that the present invention prepares has certain antioxidant activity.
Above result shows that close Fructus Evodiae total flavones is higher to the clearance rate of DPPH free radical, shows that the total flavones in the close Fructus Evodiae extract has certain antioxidant activity, and in the compound concentration scope, strengthens with concentration rising oxidation resistance.
4, bacteriostatic test result
The mensuration of bacteriostatic activity adopts agar diffusion filter paper method, judges bacteriostasis according to inhibition zone diameter.Filter paper diameter 6.0 mm, subsequent use behind the sterilizing-drying.Each sample is done repeated experiments 3 times, and The data SPSS1010 analyzes, and relatively adopts between group
tCheck.
(1) preparation of confession test agent solution: take by weighing a certain amount of close Fructus Evodiae extractive of general flavone (embodiment 1) respectively by the present invention's preparation; Parallelly take by weighing 3 parts; Adding distil water is settled in the volumetric flask, and dilution is solution in different concentration successively then, and does blank with sterilized distilled water.
(2) actication of culture and sterilization treatment: escherichia coli and staphylococcus aureus cold preservation strain are inserted 2 nutrient agar slant medium activation respectively, cultivate 24 h for 30 ℃.Culture medium, other test used equipment and reagent all at 120 ℃ of damp and hot autoclaving 2 h down.
(3) mensuration of bacteriostatic experiment and inhibition zone: the inhibition zone measurement is to utilize antibacterial substance globulate stereo structure diffusion in the agar culture medium of coating special test bacterium, the breeding of inhibition test bacterium, the transparent circle that around antibacterial substance, forms.With subsequent use behind the aseptic little filter paper high-temperature steam sterilizing-drying.Add in the sterile petri dish with the various bacteria suspensions of aseptic pipette, extract, soak into the filter paper of sample solution, place it in media surface with the tweezers gripping.The bacterium culture dish that contains of putting filter paper well is cultivated in calorstat respectively, then taken out, measure and write down the diameter of the antibacterial ring that forms.Test repetition 3 times for every group, experimental result is averaged.
(4) minimal inhibitory concentration (MIC) is measured the mensuration employing doubling dilution of result: MIC.The sample solution of variable concentrations is moved in each plate with pipet respectively, pour in the culture medium of high temperature sterilize fully mixing into, after the cooled and solidified, add bacteria suspension in every ware, be coated with aseptic spreader and evenly cultivate.Take out, observed result, with the solution concentration of long bacterium not as minimum inhibitory concentration MIC, the result supply test agent to the bacteriostatic test of staphylococcus aureus as a result MIC be 37.22 μ g mL
-1, inhibition zone diameter 18.47 mm; To colibacillary bacteriostatic test as a result MIC be 64.19 μ g mL
-1, inhibition zone diameter 11.55mm.
This result of the test shows, supplies the aqueous solution of test agent all to have stronger inhibitory action to supplying examination strain staphylococcus aureus, escherichia coli, and strengthens with the increase fungistatic effect of concentration.Obviously greater than escherichia coli, this result of the test shows that close Fructus Evodiae total flavones has certain bacteriostatic activity to the confession test agent to aureus with inhibition under the same concentrations condition.
Show that through preliminary activity test in vitro close Fructus Evodiae total flavones has certain antioxidation and antibacterial activity, therefore have good development and application values.
Claims (6)
1. method for preparing of from the close Fructus Evodiae, extracting total flavones is characterized in that may further comprise the steps:
1) raw material is handled: close Fructus Evodiae medical material is selected the back break into coarse powder with pulverizer, cross 20～40 mesh sieves;
2) percolation extracts: close Fructus Evodiae coarse powder is packed in the percolator; After soaking 8～12 hours with 50%～80% aquiferous ethanol, carry out percolation with 50%～80% aquiferous ethanol and extract, as if raw material weight be 1kg then the consumption of aquiferous ethanol be 8～16 liters; Flow velocity is 5～8 mL/min, collects percolate;
3) flash concentration: 60 ℃～90 ℃ of bath temperatures, vacuum is 0.090 Mpa～0.095Mpa with percolate, and the feed liquor flow velocity is 200～350 mLmin
-1Condition under carry out vacuum film and concentrate, reclaim ethanol, obtain every mL medicinal liquid and amount to the concentrated solution that contains 1.0～2.0g raw material;
4) macroporous resin adsorption purification: through the macroporous adsorbent resin separation and concentration, water, 10% aquiferous ethanol, 20%～70% aquiferous ethanol carry out gradient elution successively, use 70% acetone eluting at last with the concentrated solution that obtains, and elution flow rate is 5～15 mL min
-1, collect each position eluents of 3 times of column volumes respectively, collect the eluent that merges 20%～60% each position of aquiferous ethanol, under 60 ℃～90 ℃, vacuum is 0.090 Mpa～0.095Mpa with the eluent that merges, the feed liquor flow velocity is 250～350 mL min
-1Condition under carry out the vacuum film concentrate drying, promptly get close Fructus Evodiae extractive of general flavone dry powder.
2. a kind of method for preparing of from the close Fructus Evodiae, extracting total flavones as claimed in claim 1 is characterized in that step 2) in: the aquiferous ethanol concentration that is used to soak is 60%～70%, and soak time is 9-10 hour; The aquiferous ethanol concentration that is used for the percolation extraction is 60%～70%, and flow velocity is 6～7 mL/min.
3. a kind of method for preparing of from the close Fructus Evodiae, extracting total flavones as claimed in claim 1, it is characterized in that in the step 3): bath temperature is 70 ℃～80 ℃, and vacuum is 0.093Mpa, and the feed liquor flow velocity is 250～300mLmin
-1
4. a kind of method for preparing of from the close Fructus Evodiae, extracting total flavones as claimed in claim 1; It is characterized in that in the step 4): water, 10% aquiferous ethanol, 20% aquiferous ethanol, 40% aquiferous ethanol, 60% aquiferous ethanol, 70% aquiferous ethanol carry out gradient elution successively; Use 70% acetone eluting at last, elution flow rate is 8～12mLmin
-1, collect each position eluents of 3 times of column volumes respectively, collect the eluent that merges 20% aquiferous ethanol, 40% aquiferous ethanol and 60% each position of aquiferous ethanol.
5. a kind of method for preparing of from the close Fructus Evodiae, extracting total flavones as claimed in claim 1; It is characterized in that in the step 4): water, 10% aquiferous ethanol, 25% aquiferous ethanol, 35% aquiferous ethanol, 45% aquiferous ethanol, 55% aquiferous ethanol, 70% aquiferous ethanol carry out gradient elution successively; Use 70% acetone eluting at last; Collect each position eluent of 3 times of column volumes respectively, collect the eluent that merges 25% aquiferous ethanol, 35% aquiferous ethanol, 45% aquiferous ethanol and 55% each position of aquiferous ethanol.
6. a kind of method for preparing of from the close Fructus Evodiae, extracting total flavones as claimed in claim 1, it is characterized in that in the step 4): the temperature of the eluent concentrate drying of merging is under 70 ℃～80 ℃, and vacuum is 0.093 Mpa, and the feed liquor flow velocity is 280～320 mLmin
-1
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| CN103881577B (en) * | 2014-03-19 | 2016-03-16 | 湖州百胜涂料有限公司 | A kind of natural reflective coating |
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| CN107712551A (en) * | 2017-11-21 | 2018-02-23 | 成都新柯力化工科技有限公司 | A kind of method that general flavone food antioxidant is extracted from bark of dahurian larch |
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