CN102716275A - Detection method of anti-anxiety pharmaceutical composition - Google Patents
Detection method of anti-anxiety pharmaceutical composition Download PDFInfo
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Abstract
The invention discloses a detection method of an anti-anxiety pharmaceutical composition and a detection method of capsules of the anti-anxiety pharmaceutical composition. The method is characterized by using high performance liquid chromatography (HPLC) to detect and carrying out linear relation test, precision test, repeatability test, stability test and recovery test and a series of tests. The detection method of the invention is accurate and effective, and can well control the product quality.
Description
Technical field
The present invention relates to a kind of detection method of pharmaceutical composition, particularly a kind of detection method with pharmaceutical composition of angst resistance effect.
Background technology
Anxiety neurosis is that a kind of the outbreak repeatedly with acute anxiety is the neurosis of characteristic; Comprise generalized anxiety disorder and panic disorder; Outbreak often with dizzy, uncomfortable in chest, cardiopalmus, dyspnea, xerostomia, frequent micturition, urgent micturition, perspire, tremble and mobility uneasiness; And sleep disorder etc., and with the autonomic nervous dysfunction.Along with development of modern society, people's rhythm of life is generally accelerated, and the pressure that comes from aspects such as society, live and work is increasing, so anxiety patient day by day increases.
Clinical antianxiety drugs commonly used is divided into Benzodiazepines and non-Benzodiazepines at present.Benzodiazepines such as Decacil (chlordiazepoxide), diazepam and nitrazepam etc.; Non-benzene phenodiazine leather class is like buspirone, deanxit and antidepressants etc.Life-time service is prone to addiction, tolerance, has untoward reaction such as hypomnesis, gastrointestinal reaction, curative effect reduction.
The present invention screens, provides the Chinese medicine composition that a kind of effectiveness is high, safety is good to be used to treat anxiety neurosis on the basis of tcm clinical practice.
Summary of the invention
The object of the present invention is to provide a kind of detection method with pharmaceutical composition of angst resistance effect, another object of the present invention is to provide a kind of detection method of capsule preparations of the pharmaceutical composition with angst resistance effect.
The objective of the invention is to realize through following technical scheme:
A kind of detection method with pharmaceutical composition of angst resistance effect, this method adopt HPLC to detect:
Chromatograph and testing conditions: Agilent C18 chromatographic column; Acetonitrile-0.3% phosphoric acid solution with 10-30: 70-90 is a mobile phase; Detect wavelength 280nm;
Be dissolved in organic solvent with Hesperidin and prepare reference substance solution;
Organic solvent extraction said composition preparation, the preparation sample solution;
Draw reference substance solution and sample solution respectively and inject chromatograph of liquid, measure.
The detection method of pharmaceutical composition of the present invention is preferably:
HPLC chromatograph and testing conditions: Agilent C18 chromatographic column; Acetonitrile-0.3% phosphoric acid solution with 10-30: 70-90 is a mobile phase; 35 ℃ of column temperatures; Flow velocity 1mL/min detects wavelength 280nm;
The preparation of reference substance solution: get Hesperidin and use dissolve with methanol;
The preparation of sample solution: get this drug combination preparation, pulverize, make with the methanol eddy extraction;
Algoscopy: accurate respectively reference substance solution and the sample solution injection chromatograph of liquid drawn, measure, promptly get.
Content of hesperidin is 0.4-0.8mg/g in the pharmaceutical composition of the present invention.
The detection method of pharmaceutical composition of the present invention is preferably:
Chromatograph and testing conditions: Agilent C18 chromatographic column (4.6mm * 250mm, 5 μ m); With 20: 80 acetonitriles-0.3% phosphoric acid solution is mobile phase; 35 ℃ of column temperatures; Flow velocity 1mL/min detects wavelength 280nm.
The preparation of reference substance solution: precision takes by weighing Hesperidin 9.8mg, with dissolve with methanol and be settled to the 25mL volumetric flask, shakes up, and promptly gets.
The preparation of sample solution: get drug combination preparation of the present invention, pulverize, take by weighing 5g, put in the apparatus,Soxhlet's; Add an amount of methanol, putting in the water-bath refluxes is extracted into extracting liquid colourless, puts coldly, filters; Filtrating is concentrated near doing, and places 100mL volumetric flask bottle, and methanol constant volume is to 100mL.
Algoscopy: accurate respectively reference substance solution 5 μ l and the sample solution 15 μ l of drawing, inject chromatograph of liquid, measure, promptly get.
Content of hesperidin is 0.68mg/g in the pharmaceutical composition of the present invention.
The detection method of medicament composition capsule preparation of the present invention is:
Chromatograph and testing conditions: Agilent C18 chromatographic column (4.6mm * 250mm, 5 μ m); Acetonitrile-0.3% phosphoric acid solution with 10-30: 70-90 is a mobile phase; 35 ℃ of column temperatures; Flow velocity 1mL/min detects wavelength 280nm.
The preparation of reference substance solution: precision takes by weighing Hesperidin 9.8mg, with dissolve with methanol and be settled to the 25mL volumetric flask, shakes up, and promptly gets.
The preparation of sample solution: get medicament composition capsule preparation of the present invention and shell, take by weighing 2-8 weight portion granule, put in the apparatus,Soxhlet's; Add an amount of methanol, putting refluxes in the water-bath is extracted into extracting liquid colourless, puts cold; Filter; Filtrating is concentrated near doing, and places 100mL volumetric flask bottle, and methanol constant volume is to 100mL.
Algoscopy: accurate respectively reference substance solution 0.002-0.008 parts by volume and the sample solution 0.01-0.02 parts by volume drawn, inject chromatograph of liquid, measure, promptly get.
Content of hesperidin is 0.4-0.8mg/g in the medicament composition capsule preparation of the present invention.
The detection method of pharmaceutical composition of the present invention is preferably:
Chromatograph and testing conditions: Agilent C18 chromatographic column (4.6mm * 250mm, 5 μ m); With 20: 80 acetonitriles-0.3% phosphoric acid solution is mobile phase; 35 ℃ of column temperatures; Flow velocity 1mL/min detects wavelength 280nm.
The preparation of reference substance solution: precision takes by weighing Hesperidin 9.8mg, with dissolve with methanol and be settled to the 25mL volumetric flask, shakes up, and promptly gets.
The preparation of sample solution: get medicament composition capsule preparation of the present invention and shell, take by weighing 5.00 weight portion granules, put in the apparatus,Soxhlet's; Add an amount of methanol, putting refluxes in the water-bath is extracted into extracting liquid colourless, puts cold; Filter; Filtrating is concentrated near doing, and places 100mL volumetric flask bottle, and methanol constant volume is to 100mL.
Algoscopy: accurate respectively reference substance solution 0.005 parts by volume and sample solution 0.015 parts by volume drawn, inject chromatograph of liquid, measure, promptly get.
Content of hesperidin is 0.68mg/g in the medicament composition capsule preparation of the present invention.
Weight portion of the present invention and parts by volume are the relation of g/ml.
The crude drug of pharmaceutical composition of the present invention consists of:
Rhizoma valerianae latifoliae 5-20 weight portion Cortex Albiziae 5-15 weight portion
Semen Ziziphi Spinosae 5-15 weight portion Medulla Junci 0.5-4 weight portion.
The crude drug composition of pharmaceutical composition of the present invention is preferably:
Rhizoma valerianae latifoliae 8-16 weight portion Cortex Albiziae 6-12 weight portion
Semen Ziziphi Spinosae 6-14 weight portion Medulla Junci 0.6-2 weight portion.
The crude drug composition of pharmaceutical composition of the present invention is preferably:
Rhizoma valerianae latifoliae 12 weight portion Cortex Albiziaes 9 weight portions
Semen Ziziphi Spinosae (parched) 9 weight portion Medulla Juncies 1 weight portion.
The crude drug composition of pharmaceutical composition of the present invention is preferably:
Rhizoma valerianae latifoliae 8 weight portion Cortex Albiziaes 12 weight portions
Semen Ziziphi Spinosae (parched) 14 weight portion Medulla Juncies 0.8 weight portion.
The crude drug composition of pharmaceutical composition of the present invention is preferably:
Rhizoma valerianae latifoliae 18 weight portion Cortex Albiziaes 6 weight portions
Semen Ziziphi Spinosae (parched) 8 weight portion Medulla Juncies 1.2 weight portions.
Preparation of drug combination method of the present invention comprises the steps:
Step 1: choose the aforementioned proportion crude drug;
Step 2: get Rhizoma valerianae latifoliae and be ground into coarse powder, use ethanol extraction;
Step 3: Semen Ziziphi Spinosae and Cortex Albiziae are ground into coarse powder, water extraction;
Step 4: Medulla Junci is used ethanol extraction;
Above extracting solution is reclaimed solvent respectively, be condensed into dried cream, process the acceptable any conventional dosage form of pharmaceutics, comprise capsule, tablet, granule, gel, slow releasing agent, oral liquid by the preparation process of routine.
In the above-mentioned steps 2, the Rhizoma valerianae latifoliae medical material is used the 15-55% alcohol reflux; The preferred extraction 2-5 time, each 0.5-1.5 hour; Ethanol preferred 35%;
In the above-mentioned steps 3, Semen Ziziphi Spinosae and Cortex Albiziae add the water of 5-15 times of weight, reflux, extract, 2-5 time, each 1-3 hour;
In the above-mentioned steps 4, Medulla Junci was with the 60-99% alcohol reflux of 40-60 times of weight 2-5 time, each 0.5-1.5 hour; Ethanol preferred 95%.
For above-mentioned dosage form can be realized, need when these dosage forms of preparation, to add the pharmacy acceptable auxiliary, for example: filler, disintegrating agent, lubricant, suspending agent, binding agent, sweeting agent, correctives, antiseptic, substrate etc.Filler comprises: starch, pregelatinized Starch, lactose, mannitol, chitin, microcrystalline Cellulose, sucrose etc.; Disintegrating agent comprises: starch, pregelatinized Starch, microcrystalline Cellulose, carboxymethyl starch sodium, crospolyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, cross-linking sodium carboxymethyl cellulose etc.; Lubricant comprises: magnesium stearate, sodium lauryl sulphate, Pulvis Talci, silicon dioxide etc.; Suspending agent comprises: polyvinylpyrrolidone, microcrystalline Cellulose, sucrose, agar, hydroxypropyl emthylcellulose etc.; Binding agent comprises, starch slurry, polyvinylpyrrolidone, hydroxypropyl emthylcellulose etc.; Sweeting agent comprises: saccharin sodium, Aspartane, sucrose, cyclamate, enoxolone etc.; Correctives comprises: sweeting agent and various essence; Antiseptic comprises: parabens, benzoic acid, sodium benzoate, sorbic acid and its esters, benzalkonium bromide, fixed, the Folium eucalypti globueli (Eucalyptus globulus Labill.) wet goods of acetic acid chloroethene; Substrate comprises: PEG6000, PEG4000, insect wax etc.For making above-mentioned dosage form can realize pharmacy of Chinese materia medica, need when these dosage forms of preparation, to add acceptable other adjuvant of pharmacy (adjuvant of each dosage form record among the Fan Biting " pharmacy of Chinese materia medica ", Shanghai Science Press December in 1997 the 1st edition).
The method for preparing of medicament composition capsule agent of the present invention is:
Get Rhizoma valerianae latifoliae and be ground into coarse powder, with 35% ethanol that is equivalent to Rhizoma valerianae latifoliae medical material 5-15 times of weight, reflux, extract, 2-5 time, each 0.5-1.5 hour;
Semen Ziziphi Spinosae and Cortex Albiziae are ground into coarse powder, add the water of 5-15 times of weight, reflux, extract, 2-5 time, each 1-3 hour;
Medulla Junci is ground into coarse powder, with 95% ethanol of 40-60 times of weight, reflux, extract, 2-5 time, each 0.5-1.5 hour;
Above extracting solution is reclaimed solvent respectively, be condensed into dried cream, the dried cream of each composition adds the dextrin of the dried cream amount of 0.2-0.8 times of weight by the prescription mixed; Mixing, 80-95% ethanol is granulated as wetting agent; Cross 20 mesh sieves, granule should be kept at below the critical relative humidity 70-90%, promptly gets.
The method for preparing of medicament composition capsule agent of the present invention is preferably:
Get Rhizoma valerianae latifoliae and be ground into coarse powder, with 35% ethanol that is equivalent to Rhizoma valerianae latifoliae medical material 10 times of weight, reflux, extract, 3 times, each 1 hour; Semen Ziziphi Spinosae and Cortex Albiziae are ground into coarse powder, add the water of 10 times of weight, reflux, extract, 3 times, each 2 hours; Medulla Junci is ground into coarse powder, with 95% ethanol of 50 times of weight, reflux, extract, 3 times, each 1 hour; Above extracting solution is reclaimed solvent respectively, be condensed into dried cream, the dried cream of each composition adds the dextrin of the dried cream amount of 0.5 times of weight by the prescription mixed; Mixing, 90% ethanol is granulated as wetting agent; Cross 20 mesh sieves, granule should be kept at critical relative humidity below 80%, promptly gets.
Through a series of investigations such as linear relationship investigation, precision test, replica test, stability test and recovery tests, the detection method of pharmaceutical composition of the present invention is accurate and effective, can control the quality of product preferably.
Experimental example 1: medicament composition capsule agent pharmacodynamic experiment of the present invention
1 materials and methods
1.1 material
1.1.1 laboratory animal
The SD rat, male, initial body weight 140～180g is divided into 5 groups, 10 every group at random.
1.1.2 medicine and reagent
Diazepam, morphine: with the required concentration solution of normal saline furnishing.
Medicament composition capsule of the present invention: press the preparation of embodiment 1 method, with the required concentration solution of normal saline furnishing.
1.1.3 experimental apparatus
The pain instrument is brought out in the overhead cross of rat labyrinth, Vogel drinking-water conflict experimental provision and radiant heat stimulation.
1.2 experimental technique
1.2.1 rat elevated plus-maze test
The SD male rat is divided into totally 5 groups of the high, medium and low dose groups of medicament composition capsule of the present invention, diazepam group and blank control group at random; Pharmaceutical composition low dose group wherein of the present invention, middle dose groups, high dose group are irritated stomach by crude drug in whole 0.75g/kg/d, 1.5g/kg/d and 3g/kg/d respectively; Diazepam is pressed the 1mg/kg/d administration, and blank control group is irritated the isometric normal saline of clothes.Continuous 10d gastric infusion, the administration volume is 1ml/100g.Behind 10d Chinese drug-treated group and saline control group last administration 1h, behind the diazepam group administration 0.5h, do performance testing in 8:00～14:00Am.Overhead cross labyrinth comprises that two are opened arm (50.8cm * 10.2cm * 1.3cm), two close arm (50.8cm * 10.2cm * 40.6cm) and central area (10.2cm * 10.2cm), the 72.4cm apart from ground.Before the test of labyrinth every rat is put into 1 45cm * 30cm * 15cm plastic casing; Let alone freely to probe into the central platform place that places EPM behind the 5min rapidly; Make its head over against 1 open arms wherein; Adopt in the infrared technology record 5min animal to get into to open the arm number of times (open arm entry, OE), close the arm number of times (close arm entry, CE) and the number of times in the central area of labyrinth and opening arm and closing the movement time in the arm.Open arm number of times and percentage ratio (the percentage of entries to the open arms that always goes into the arm number of times with entering; OE%) (the percentage of time spent in the open arms OT%) represents the angst resistance effect index to movement time with opening the percentage ratio that arm closes the total time in the arm and in opening arm.With wet cloth wiping labyrinth, remove feces after each test is accomplished, after cleaning, carry out the test of next rat again with dried cloth.
1.2.2 rat Vogel drinking-water conflict experiment
Rat divides into groups and the same 1.2.1 of dosage, and rat Vogel drinking-water conflict experiment is carried out according to following minute two stages at daylight lamp.Phase I, the training of non-punishment drinking-water: rat is prohibited the single control box that places behind the water 24h, let it fully probe into, up to finding bottle neck and begin to lick water, record rat 3min licks the waterside number, eliminates and licks the rat that water is less than 300 times.Second stage, the punishment experiment: the above-mentioned rat that is not eliminated continues to prohibit water 24h (48h altogether) and is placed on control box.Rat can be found bottle neck very soon and begin to lick water, licks enough No. 20 instruments and picks up counting automatically and give once to shock by electricity (ratio of licking water and number of shocks is 20: 1), and electric shock intensity is generally 0.2-0.5mA (punishment phase), continues 2s.Record rat 3min licks the waterside number.
1.2.3 the spontaneous drinking-water experiment of rat
With method 1.2.2, just in the punishment phase, remove electric shock, the record rat licks the waterside number in the 3min when not having electric shock.
1.2.4 threshold of pain experiment
Rat divides into groups and the same 1.2.1 of dosage, and other adds 10 morphine groups, carries out the radiant heat test behind the lumbar injection 30min.
Bulb through one 100 watts provides; Aperture by diameter 4mm after the light line focus casts out; See through the thick poly (methyl methacrylate) plate vertical irradiation rat vola of 1mm, manual-lock radiant heat lamp after the elusive behavior appears in animal, in behavior pharmacological experiment process; Need the foot of lifting of normal rat is adjusted to about 8s incubation period, setting the maximum concluding time is 22s.Can stop tissue injury like this, wherein lift foot be meant incubation period begin at every turn to and the vola thermostimulation lift the interval between the foot up to rat.Every foot of every rat is given and is stimulated for four times, 5min at least at interval between twice heat radiation of same rat stimulates, and the result that getting last three heat radiations stimulates averages and is used for statistical test.
1.2.5 data
With SAS8.2 software data are added up; Relatively, the result representes with
between organizing with t check.* P＜0.05, * * P＜0.01, there is significant difference #P＜0.05.
2. experimental result
2.1 rat elevated plus-maze test
Table 1 medicament composition capsule of the present invention is to the influence (
n=9-10) of the overhead cross of rat labyrinth model
Annotate: compare * P＜0.05 with blank control group.
Can find out from table 1 result, compare that diazepam group and medicament composition capsule high dose group of the present invention can obviously increase rat OE% and OT% (P＜0.05) with the blank group; Compare with blank control group simultaneously, middle dose groups can increase rat OT% (P＜0.05), always goes into the arm number of times and does not then have significant change, does not have significant difference between other each group.Experimental result shows when medicament composition capsule dosage of the present invention is 3g/kg/d on this model, to have angst resistance effect.
2.2 rat Vogel drinking-water conflict experiment
Table 2 medicament composition capsule of the present invention is to the influence (
n=10) of rat Vogel drinking-water conflict model drinking times
Annotate: compare * P＜0.05 with blank control group.
Table 2 result shows that compare with matched group, dosage and high dose group can significantly increase the drinking times of rat under the electric shock situation in diazepam group, the medicament composition capsule of the present invention, and statistical significance (P＜0.05) is arranged, and does not have significant difference between other each group.Experimental result shows when medicament composition capsule dosage of the present invention is 1.5g/kg/d and 3g/kg/d, to have angst resistance effect at this model.
2.3 the spontaneous drinking-water experiment of rat
Table 3 medicament composition capsule of the present invention is to the influence (
n=10) of the spontaneous amount of drinking water of rat
Annotate: compare * P＜0.05 with blank control group.
Table 3 result shows that compare with matched group, each organizes equal not statistically significant (P＞0.05).
2.4 threshold of pain experiment
Table 4 medicament composition capsule of the present invention stimulates radiant heat and brings out the preclinical influence of pain (
n=10)
Annotate: compare * P＜0.05 with blank control group.
Table 4 result shows that compare with matched group, the morphine group has significant analgesia role (P＜0.05), and medicament composition capsule of the present invention is respectively organized equal not statistically significant (P＞0.05).Spontaneous drinking-water experiment and threshold of pain description of test: the spontaneous amount of drinking water of medicine and nonintervention and the threshold of pain, got rid of the interference of false positive factor.
3. conclusion
Draw through experiment: on the model of the overhead cross of rat labyrinth, medicament composition capsule of the present invention is being opened movement time and the percentage rate of number of times in the arm significantly increasing rat under the dosage of 3g/kg/d; In the Vogel drinking-water conflict experiment, same dosage can significantly increase rat and lick the waterside number, on these two models, all demonstrates clear and definite angst resistance effect, and effect is optimum, and the 1.5g/kg/d angst resistance effect takes second place.
Experimental example 2: medicament composition capsule agent Study on Preparation of the present invention
One, Rhizoma valerianae latifoliae extraction process orthogonal test
1 instrument and reagent
1.1 instrument
RE-52A type Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant)
SHZ-II water cycle vacuum pump (English valley, Gongyi City, Henan Province Electronic Instruments Plant)
KQ-500DE type numerical control supersonic cleaning apparatus (Kunshan Ultrasonic Instruments Co., Ltd.)
SARTORIUS-BS124S type electronic analytical balance (German Sartorius AG)
Agilent1100 type high performance liquid chromatograph (Agilent Technologies);
Agilent C18 chromatographic column (4.6mm * 250mm, 5 μ m)
FW-177 type pulverizer (Tianjin Tai Site Instr Ltd.)
HH-4 digital display thermostat water bath (Jie Ruier of Jintan City Electrical Appliances Co., Ltd)
The 98-1-B type electric jacket (Tianjin Tai Site Instr Ltd.) that adjusts the temperature electronically
98-1-C type numeral control-temperature electric heating cover (Tianjin Tai Site Instr Ltd.)
1.2 reagent
Hesperidin standard substance Nat'l Pharmaceutical & Biological Products Control Institute
Acetonitrile (chromatographically pure, U.S. Fisher company);
Methanol (chromatographically pure, U.S. Fisher company);
Watson distilled water (Beijing Watson's Distilled Water Co., Ltd.)
95% ethanol (Beijing Chemical Plant)
Rhizoma valerianae latifoliae: dry rhizome and the root of Valerianaceae plant Rhizoma valerianae latifoliae Valerian jatamansi Jones.(the place of production: Yunnan)
2 methods
2.1 orthogonal design
Utilize the optimization of orthogonal test technological parameter.Through the The selection result of list of references and preliminary experiment, finally selecting degree of grinding, extraction time, extraction time and solvent multiple is the investigation factor, and each adopts 3 levels, carries out L9 (34) orthogonal experiment, and the investigation index is a Determination of Hesperidin Content.
Table 5 Rhizoma valerianae latifoliae extracts orthogonal test factor level table
Coarse powder: refer to and to be no more than 40% powder through No. four sieves all through No. two sieves but be mixed with;
Fine powder: refer to and all to sieve, and contain and to be no less than 95% powder through No. six sieves through No. five;
No. six sieves of No. two sieve-24 orders No. four sieves-65 orders No. five sieves-80 orders-100 orders
2.2 Determination of Hesperidin Content is measured
Get every part of each 50.00g of Rhizoma valerianae latifoliae medical material, press L9 (34) orthogonal table and use 35% alcohol reflux.
2.2.1 chromatograph and testing conditions
Hesperidin HPLC measures chromatographic condition: Agilent C18 chromatographic column (4.6mm * 250mm, 5 μ m); Mobile phase: 20: 80 v/v of acetonitrile-0.3% phosphoric acid solution.35 ℃ of column temperatures, flow velocity 1mL/min detects wavelength 280nm.
2.2.2 the preparation of solution
The preparation of reference substance solution: precision takes by weighing Hesperidin 9.8mg, with dissolve with methanol and be settled to the 25mL volumetric flask, shakes up, and promptly gets.
The preparation of sample solution: take by weighing the dried cream that is equivalent to 2.5g Rhizoma valerianae latifoliae crude drug, place 25mL volumetric flask bottle, methanol constant volume is to 25mL, the ultrasonic 45min of hot bath, and it is subsequent use that supernatant is got in cooling.(the 0.1g crude drug/mL)
2.3.3 linear relationship is investigated
Reference substance solution under the accurate successively absorption 2.3.2 item; With 1 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L, 8 μ L volume sample introductions, measuring by above-mentioned chromatographic condition respectively, is abscissa (X) with the sampling volume; With the peak area is that vertical coordinate (Y) carries out linear regression; Getting regression equation is y=1625.5x+45.087, R=0.9999, and the range of linearity is 0.392 μ g～3.136 μ g.
Table 6 linear relationship is investigated the result
2.2.4 precision test
Get Hesperidin reference substance solution under the 2.3.2 item, continuous sample introduction 6, each 5 μ L, the record peak area calculates peak area RSD=0.34%, shows that this method precision is good.(n＝6)。
2.2.5 replica test
Press method under the 2.3.2 item, 6 parts of preparation sample solutions, the difference sample introduction, sample size 5 μ L record peak area, calculate RSD=1.27% (n=6), show that this method repeatability is better.
2.2.6 stability test
The accurate absorption with a collection of need testing solution 5 μ L, every interval certain hour sample introduction is measured 6 times, as a result Hesperidin RSD=0.72% altogether.Show that need testing solution is stable in 24h.
2.2.7 recovery test
6 parts of the sample storing solutions of accurate absorption known content, every part of about 3mL puts respectively in the 10mL volumetric flask, and contain the standard solution 1mL that Hesperidin is 0.392mg/mL accurate respectively again the adding, and behind the ultrasonic 45min of hot bath methanol, it is test liquid that dilution is settled to scale.Sample thief liquid 10 μ L inject high performance liquid chromatograph, measure by the assay method of sample.Average recovery rate is 99.94%, RSD=0.9% (n=6).
2.2.8 sample determination
Get sample solution sample introduction under above-mentioned chromatographic condition under the 2.2.2 item, each 10 μ L, the substitution Equation for Calculating gets content of hesperidin in the sample.Concrete outcome is seen table 7,8.
3 results
Table 7 orthogonal experiments
It is A that intuitive analysis gets optimum process
2B
3C
1D
3
Table 8 analysis of variance table
A, B, C, D have utmost point significant difference (P＜0.01).Influence degree A＞B＞D＞C.
4 conclusions
Final when confirming to be index with the Hesperidin, the optimised process of the extraction of Rhizoma valerianae latifoliae is A
2B
3C
1D
3, promptly Rhizoma valerianae latifoliae is ground into coarse powder, 10 times of amount 35% ethanol, reflux, extract, 3 times, each 1 hour.
Two, Semen Ziziphi Spinosae and Cortex Albiziae extraction process orthogonal test
1 instrument and reagent
1.1 instrument
RE-52A type Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant)
SHZ-II water cycle vacuum pump (English valley, Gongyi City, Henan Province Electronic Instruments Plant)
KQ-500DE type numerical control supersonic cleaning apparatus (Kunshan Ultrasonic Instruments Co., Ltd.)
SARTORIUS-BS124S type electronic analytical balance (German Sartorius AG)
Agilent1100 type high performance liquid chromatograph (Agilent Technologies);
FW-177 type pulverizer (Tianjin Tai Site Instr Ltd.)
HH-4 digital display thermostat water bath (Jie Ruier of Jintan City Electrical Appliances Co., Ltd)
The 98-1-B type electric jacket (Tianjin Tai Site Instr Ltd.) that adjusts the temperature electronically
98-1-C type numeral control-temperature electric heating cover (Tianjin Tai Site Instr Ltd.)
1.2 reagent
Jujuboside A standard substance Nat'l Pharmaceutical & Biological Products Control Institute
Acetonitrile (chromatographically pure, U.S. Fisher company);
Methanol (chromatographically pure, U.S. Fisher company);
Watson distilled water (Beijing Watson's Distilled Water Co., Ltd.)
95% ethanol (Beijing Chemical Plant)
Cortex Albiziae: the dry bark (place of production: Hebei) of leguminous plant Herba Albiziae Albizzia julibrissin Durazz.
Semen Ziziphi Spinosae: the dry mature seed of Rhamnaceae plant Ziziphi Spinosae (Ziziphus jujuba Mill.var.spinosa (Bunge) Hu ex H.F.Chou).(the place of production: Hebei)
2 methods
2.1 orthogonal design
Utilize the optimization of orthogonal test technological parameter.The selection result through list of references and preliminary experiment; Final selection degree of grinding, extraction time, extraction time and solvent multiple are the investigation factor; Each adopts 3 levels, carries out L9 (34) orthogonal experiment, and investigating index is the content of jujuboside A in Cortex Albiziae and the Semen Ziziphi Spinosae extract.
Table 9 Cortex Albiziae, Semen Ziziphi Spinosae extract orthogonal test factor level table
2.2 the assay of jujuboside A
Get Cortex Albiziae and each 50.00g of every part every kind medical material of Semen Ziziphi Spinosae medical material, press L9 (3
4) orthogonal table merges water and carry.
2.2.1 chromatograph and testing conditions
Chromatographic column: Agilent C
18Post (4.6mm * 250mm, 5 μ m); Mobile phase: second cyanogen-water (32: 68v/v); Flow velocity 1.0mL/min; Column temperature is 25 ℃; Detect wavelength: 204nm.
2.2.2 the preparation of solution
The preparation of reference substance solution: precision takes by weighing jujuboside A 8.2000mg, with dissolve with methanol and be settled to the 25mL volumetric flask, shakes up, and promptly gets.
The preparation of sample solution: take by weighing the dried cream that is equivalent to 10g Semen Ziziphi Spinosae crude drug, place the 50mL conical flask, pipette 25mL methanol, ultrasonic 45min, it is subsequent use to get supernatant.(the 0.4g crude drug/mL)
2.2.3 linear relationship is investigated
Reference substance solution under the accurate successively absorption 2.3.2 item; With 1 μ L, 2 μ L, 5 μ L, 8 μ L, 10 μ L, 15 μ L volume sample introductions, measuring by above-mentioned chromatographic condition respectively, is abscissa (X) with the sampling volume; With the peak area is that vertical coordinate (Y) carries out linear regression; Getting regression equation is y=259.35x+1.5521, R=0.9998, and the range of linearity is 0.328 μ g～4.9 μ g.
Table 10 linear relationship is investigated the result
2.2.4 precision test
Get jujuboside A reference substance solution under the 2.2.2 item, continuous sample introduction 6, each 10 μ L, the record peak area, calculating peak area RSD value is 0.50%, shows that this method precision is good.(n＝6)。
2.2.5 replica test
Press method under the 2.3.2 item, 6 parts of preparation sample solutions, the difference sample introduction, sample size 15 μ L record peak area, and calculating the RSD value is 0.54% (n=6), shows that this method repeatability is better.
2.2.6 stability test
The accurate absorption with a collection of need testing solution 10 μ L, every interval certain hour sample introduction is measured 6 times altogether, jujuboside A RSD=1.211%.Show that need testing solution is stable in 12h.
2.2.7 recovery test
Measure 6 parts of sample solutions under the 2.2.2 item, every part of 3mL, the accurate respectively jujuboside A reference substance solution 3mL that adds; Methanol constant volume is to 10mL; The HPLC method is measured the content of valepotriate, and calculate recovery rate, average recovery rate are 100.0209%; The RSD value is 0.4676% (n=6), shows that this method has the good response rate.
2.2.8 sample determination
Get sample solution sample introduction under above-mentioned chromatographic condition under the 2.2.2 item, each 10 μ L, the substitution Equation for Calculating gets jujuboside A content in the sample.
3 results
Table 11 sample size is measured the result
It is A that intuitive analysis gets optimum process
3B
3C
2D
3
Table 12 analysis of variance table
A, B, C, D have utmost point significant difference (P＜0.01).Influence degree B＞C＞D＞A.
4 conclusions
Because Semen Ziziphi Spinosae contains more oils and fats, be difficult to be ground into fine powder, consider to produce actual, final when confirming to be index with jujuboside A, the optimised process of Semen Ziziphi Spinosae and Cortex Albiziae co-extracted is A
2B
3C
2D
3, promptly Semen Ziziphi Spinosae and Cortex Albiziae are ground into coarse powder, and 10 times of water gagings extract each 2 hours 3 times.
Three, Medulla Junci extraction process orthogonal test
1 instrument and reagent
1.1 instrument
RE-52A type Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant)
SHZ-II water cycle vacuum pump (English valley, Gongyi City, Henan Province Electronic Instruments Plant)
KQ-500DE type numerical control supersonic cleaning apparatus (Kunshan Ultrasonic Instruments Co., Ltd.)
SARTORIUS-BS124S type electronic analytical balance (German Sartorius AG)
FW-177 type pulverizer (Tianjin Tai Site Instr Ltd.)
HH-4 digital display thermostat water bath (Jie Ruier of Jintan City Electrical Appliances Co., Ltd)
The 98-1-B type electric jacket (Tianjin Tai Site Instr Ltd.) that adjusts the temperature electronically
98-1-C type numeral control-temperature electric heating cover (Tianjin Tai Site Instr Ltd.)
1.2 reagent
Acetonitrile (chromatographically pure, U.S. Fisher company);
Methanol (chromatographically pure, U.S. Fisher company);
Watson distilled water (Beijing Watson's Distilled Water Co., Ltd.)
95% ethanol (Beijing Chemical Plant)
Medulla Junci: the dry stem pith of rush family plant Medulla Junci Juncus effusus L..
2 methods
2.1 orthogonal design
Consider the character of Medulla Junci and effective ingredient, in conjunction with the lot of documents report, according to the result of Preliminary screening, selecting degree of grinding, extraction time, extraction time and solvent multiple is the investigation factor, and each adopts 3 levels, carries out L
9(3
4) orthogonal experiment, the investigation index is a yield of extract.Investigate factor level and see table 13.
Table 13 Medulla Junci is extracted orthogonal test factor level table
2.2 yield of extract is measured
Get respectively 3 parts of Medulla Junci crude drug, coarse powder, fine powders, every part of 10.00g is with concentrating under reduced pressure behind 95% alcohol reflux; Concentrated solution places the evaporating dish that is dried to constant weight, and water-bath is concentrated into the bone dry constant weight, deducts the weight of evaporating dish; Get extractum weight, calculate yield of extract.
3 results
Table 14 Medulla Junci yield of extract result
Intuitive analysis learns that optimum process is A
3B
3C
3D
3
Table 15 analysis of variance table
The influence degree of four factors is B＞A＞D＞C; Extraction time (B) has utmost point significant difference (P＜0.01); Degree of grinding (A) has significant difference (P＜0.05), and solvent multiple (D) difference is remarkable (P＞0.05) not, extraction time (C) difference minimum (P＞0.2).
4 conclusions
The Medulla Junci quality is loose, during meticulous pulverizings of granularity loss many, so combine actual production, final definite when being index with the paste-forming rate, the optimised process of Medulla Junci is A
2B
3C
1D
1Be that Medulla Junci is ground into coarse powder, 50 times of amount 95% ethanol, reflux, extract, three times, each 1 hour.
Experimental example 3: Determination of Hesperidin Content assay method in the medicament composition capsule preparation of the present invention
1 instrument and reagent
1.1 instrument
RE-52A type Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant)
SHZ-II water cycle vacuum pump (English valley, Gongyi City, Henan Province Electronic Instruments Plant)
KQ-500DE type numerical control supersonic cleaning apparatus (Kunshan Ultrasonic Instruments Co., Ltd.)
SARTORIUS-BS124S type electronic analytical balance (German Sartorius AG)
Agilent1100 type high performance liquid chromatograph (Agilent Technologies);
Agilent C18 (Agilent C18 chromatographic column) chromatographic column (4.6mm * 250mm, 5 μ m)
HH-4 digital display thermostat water bath (Jie Ruier of Jintan City Electrical Appliances Co., Ltd)
1.2 reagent
Hesperidin standard substance Nat'l Pharmaceutical & Biological Products Control Institute
Acetonitrile (chromatographically pure, U.S. Fisher company);
Methanol (chromatographically pure, U.S. Fisher company);
Watson distilled water (Beijing Watson's Distilled Water Co., Ltd.)
95% ethanol (Beijing Chemical Plant)
Medicament composition capsule of the present invention (pressing the preparation of embodiment 1 method)
2 Determination of Hesperidin Content assay methods
2.1 chromatograph and testing conditions
Hesperidin HPLC measures chromatographic condition: Agilent C18 chromatographic column (4.6mm * 250mm, 5 μ m); Mobile phase: acetonitrile-0.3% phosphoric acid solution 20: 80v/v.35 ℃ of column temperatures, flow velocity 1mL/min detects wavelength 280nm.
2.2 the preparation of reference substance solution solution
Precision takes by weighing Hesperidin 9.8mg, with dissolve with methanol and be settled to the 25mL volumetric flask, shakes up, and promptly gets.
2.3 the preparation of sample solution
Capsule shells, and takes by weighing the 5.00g granule, puts in the apparatus,Soxhlet's, adds an amount of methanol, and putting in the water-bath refluxes is extracted into extracting liquid colourless, puts coldly, filters, and filtrating is concentrated near doing, and places 100mL volumetric flask bottle, and methanol constant volume is to 100mL.
2.4 linear relationship is investigated
2.2 following reference substance solution of accurate successively absorption; With 1 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L, 8 μ L volume sample introductions, measuring by above-mentioned chromatographic condition respectively, is abscissa (X) with the sampling volume; With the peak area is that vertical coordinate (Y) carries out linear regression; Getting regression equation is y=1625.5x+45.087, R=0.9999, and the range of linearity is 0.392 μ g～3.136 μ g.
Table 16 linear relationship is investigated the result
2.5 precision test
Get 2.2 following Hesperidin reference substance solution, continuous sample introduction 6 times, each 5 μ L, the record peak area calculates peak area RSD=0.44%, shows that this method precision is good.(n＝6)。
2.6 replica test
By 2.3 following methods, 6 parts of preparation sample solutions, the difference sample introduction, sample size 15 μ L record peak area, calculate RSD=2.91% (n=6), show that this method repeatability is better.
2.7 stability test
The accurate absorption with a collection of need testing solution 5 μ L, every interval certain hour sample introduction is measured 6 times, as a result Hesperidin RSD=0.72% altogether.Show that need testing solution is stable in 24h.
2.8 recovery test
Get 6 parts of the self-control capsule samples of known content, precision is weighed, and adds Hesperidin reference substance solution 2ml respectively; Put in the apparatus,Soxhlet's, add an amount of methanol, putting refluxes in the water-bath is extracted into extracting liquid colourless; Put coldly, filter, filtrating is concentrated near doing; Place 10mL volumetric flask bottle, methanol constant volume is to 10mL.Sample thief liquid 10 μ
L injects high performance liquid chromatograph, measures by the assay method of sample.The average average recovery of Hesperidin is 100.8616% as a result, RSD=1.12% (n=6).
3. Determination of Hesperidin Content is measured
Precision takes by weighing three batches of capsule samples by the preparation of embodiment 1 method, by 2.3 preparation samples, carries out assay.See table 17.
Table 17 three lot sample article Determination of Hesperidin Content
Three lot sample article Hesperidin average contents are 0.68mg/g, RSD=2.8%.
Description of drawings
Fig. 1: drug regimen object detecting method linear relationship of the present invention is investigated canonical plotting, y=1625.5x+45.087, R2=0.9997.
The specific embodiment
Embodiment 1: the detection method of capsule
Rhizoma valerianae latifoliae 12g Cortex Albiziae 9g
Semen Ziziphi Spinosae 9g Medulla Junci 1g
Get Rhizoma valerianae latifoliae and be ground into coarse powder, with 35% ethanol that is equivalent to Rhizoma valerianae latifoliae medical material 10 times of weight, reflux, extract, 3 times, each 1 hour; Semen Ziziphi Spinosae and Cortex Albiziae are ground into coarse powder, add the water of 10 times of weight, reflux, extract, 3 times, each 2 hours; Medulla Junci is ground into coarse powder, with 95% ethanol of 50 times of weight, reflux, extract, 3 times, each 1 hour; Above extracting solution is reclaimed solvent respectively, be condensed into dried cream, the dried cream of each composition adds the dextrin of the dried cream amount of 0.5 times of weight by the prescription mixed; Mixing, 90% ethanol is granulated as wetting agent; Cross 20 mesh sieves, granule should be kept at critical relative humidity below 80%, promptly gets.
Chromatograph and testing conditions: Agilent C18 chromatographic column (4.6mm * 250mm, 5 μ m); With 20: 80 acetonitriles-0.3% phosphoric acid solution is mobile phase; 35 ℃ of column temperatures; Flow velocity 1mL/min detects wavelength 280nm.
The preparation of reference substance solution: precision takes by weighing Hesperidin 9.8mg, with dissolve with methanol and be settled to the 25mL volumetric flask, shakes up, and promptly gets.
The preparation of sample solution: get this medicament composition capsule preparation and shell, take by weighing the 5.00g granule, put in the apparatus,Soxhlet's, add an amount of methanol; Putting in the water-bath refluxes is extracted into extracting liquid colourless, puts coldly, filters; Filtrating is concentrated near doing, and places 100mL volumetric flask bottle, and methanol constant volume is to 100mL.
Algoscopy: accurate respectively reference substance solution 5 μ l and the sample solution 15 μ l of drawing, inject chromatograph of liquid, measure, promptly get.
Content of hesperidin is 0.68mg/g in the medicament composition capsule preparation of the present invention.
Embodiment 2: the detection method of capsule
Rhizoma valerianae latifoliae 8g Cortex Albiziae 12g
Semen Ziziphi Spinosae (parched) 14g Medulla Junci 0.8g
Get Rhizoma valerianae latifoliae and be ground into coarse powder, with 35% ethanol that is equivalent to Rhizoma valerianae latifoliae medical material 6 times of weight, reflux, extract, 4 times, each 1.2 hours; Semen Ziziphi Spinosae and Cortex Albiziae are ground into coarse powder, add the water of 8 times of weight, reflux, extract, 2 times, each 2.5 hours; Medulla Junci is ground into coarse powder, with 95% ethanol of 56 times of weight, reflux, extract, 2 times, each 0.8 hour; Above extracting solution is reclaimed solvent respectively, be condensed into dried cream, the dried cream of each composition adds the dextrin of the dried cream amount of 0.6 times of weight by the prescription mixed; Mixing, 95% ethanol is granulated as wetting agent; Cross 20 mesh sieves, granule should be kept at critical relative humidity below 75%, promptly gets.
Chromatograph and testing conditions: Agilent C18 chromatographic column (4.6mm * 250mm, 5 μ m); With 15: 86 acetonitriles-0.3% phosphoric acid solution is mobile phase; 35 ℃ of column temperatures; Flow velocity 1mL/min detects wavelength 280nm.
The preparation of reference substance solution: precision takes by weighing Hesperidin 9.8mg, with dissolve with methanol and be settled to the 25mL volumetric flask, shakes up, and promptly gets.
The preparation of sample solution: get medicament composition capsule preparation of the present invention and shell, take by weighing the 6.5g granule, put in the apparatus,Soxhlet's, add an amount of methanol; Putting in the water-bath refluxes is extracted into extracting liquid colourless, puts coldly, filters; Filtrating is concentrated near doing, and places 100mL volumetric flask bottle, and methanol constant volume is to 100mL.
Algoscopy: accurate respectively reference substance solution 3 μ l and the sample solution 12 μ l of drawing, inject chromatograph of liquid, measure, promptly get.
Content of hesperidin is 0.52mg/g in the medicament composition capsule preparation of the present invention.
Embodiment 3: the detection method of capsule
Rhizoma valerianae latifoliae 10g Cortex Albiziae 14g
Semen Ziziphi Spinosae (parched) 12g Medulla Junci 0.6g
Get the above-mentioned raw materials medicine, add conventional adjuvant,, process capsule according to common process.
Chromatograph and testing conditions: Agilent C18 chromatographic column (4.6mm * 250mm, 5 μ m); With 26: 72 acetonitriles-0.3% phosphoric acid solution is mobile phase; 35 ℃ of column temperatures; Flow velocity 1mL/min detects wavelength 280nm.
The preparation of reference substance solution: precision takes by weighing Hesperidin 9.8mg, with dissolve with methanol and be settled to the 25mL volumetric flask, shakes up, and promptly gets.
The preparation of sample solution: get medicament composition capsule preparation of the present invention and shell, take by weighing the 5g granule, put in the apparatus,Soxhlet's, add an amount of methanol; Putting in the water-bath refluxes is extracted into extracting liquid colourless, puts coldly, filters; Filtrating is concentrated near doing, and places 100mL volumetric flask bottle, and methanol constant volume is to 100mL.
Algoscopy: accurate respectively reference substance solution 7.5 μ l and the sample solution 18 μ l of drawing, inject chromatograph of liquid, measure, promptly get.
Content of hesperidin is 0.75mg/g in the medicament composition capsule preparation of the present invention.
Embodiment 4: slow releasing agent
Rhizoma valerianae latifoliae 12g Cortex Albiziae 9g
Semen Ziziphi Spinosae 9g Medulla Junci 1g
Get Rhizoma valerianae latifoliae and be ground into coarse powder, with 35% ethanol that is equivalent to Rhizoma valerianae latifoliae medical material 10 times of weight, reflux, extract, 3 times, each 1 hour; Semen Ziziphi Spinosae and Cortex Albiziae are ground into coarse powder, add the water of 10 times of weight, reflux, extract, 3 times, each 2 hours; Medulla Junci is ground into coarse powder, with 95% ethanol of 50 times of weight, reflux, extract, 3 times, each 1 hour; Above extracting solution is reclaimed solvent respectively, be condensed into dried cream, process slow releasing agent through conventional technology.
Chromatograph and testing conditions: Agilent C18 chromatographic column (4.6mm * 250mm, 5 μ m); With 20: 80 acetonitriles-0.3% phosphoric acid solution is mobile phase; 35 ℃ of column temperatures; Flow velocity 1mL/min detects wavelength 280nm.
The preparation of reference substance solution: precision takes by weighing Hesperidin 9.8mg, with dissolve with methanol and be settled to the 25mL volumetric flask, shakes up, and promptly gets.
The preparation of sample solution: get pharmaceutical composition slow releasing agent of the present invention, pulverize, take by weighing 5g, put in the apparatus,Soxhlet's; Add an amount of methanol, putting in the water-bath refluxes is extracted into extracting liquid colourless, puts coldly, filters; Filtrating is concentrated near doing, and places 100mL volumetric flask bottle, and methanol constant volume is to 100mL.
Algoscopy: accurate respectively reference substance solution 5 μ l and the sample solution 15 μ l of drawing, inject chromatograph of liquid, measure, promptly get.
Content of hesperidin is 0.68mg/g in the pharmaceutical composition slow releasing agent of the present invention.
Embodiment 7: granule
Rhizoma valerianae latifoliae 8g Cortex Albiziae 12g
Semen Ziziphi Spinosae (parched) 14g Medulla Junci 0.8g
Get Rhizoma valerianae latifoliae and be ground into coarse powder, with 35% ethanol that is equivalent to Rhizoma valerianae latifoliae medical material 6 times of weight, reflux, extract, 4 times, each 1.2 hours; Semen Ziziphi Spinosae and Cortex Albiziae are ground into coarse powder, add the water of 8 times of weight, reflux, extract, 2 times, each 2.5 hours; Medulla Junci is ground into coarse powder, with 95% ethanol of 56 times of weight, reflux, extract, 2 times, each 0.8 hour; Above extracting solution is reclaimed solvent respectively, be condensed into dried cream, the dried cream of each composition adds the dextrin of the dried cream amount of 0.6 times of weight by the prescription mixed, and mixing, 95% ethanol are processed granule as wetting agent through conventional technology.
Chromatograph and testing conditions: Agilent C18 chromatographic column (4.6mm * 250mm, 5 μ m); With 16: 82 acetonitriles-0.3% phosphoric acid solution is mobile phase; 35 ℃ of column temperatures; Flow velocity 1mL/min detects wavelength 280nm.
The preparation of reference substance solution: precision takes by weighing Hesperidin 9.8mg, with dissolve with methanol and be settled to the 25mL volumetric flask, shakes up, and promptly gets.
The preparation of sample solution: get medicament composition granule agent of the present invention, take by weighing 7.5g, put in the apparatus,Soxhlet's, add an amount of methanol; Putting in the water-bath refluxes is extracted into extracting liquid colourless, puts coldly, filters; Filtrating is concentrated near doing, and places 100mL volumetric flask bottle, and methanol constant volume is to 100mL.
Algoscopy: accurate respectively reference substance solution 2.5 μ l and the sample solution 13 μ l of drawing, inject chromatograph of liquid, measure, promptly get.
Content of hesperidin is 0.48mg/g in the medicament composition granule agent of the present invention.
Embodiment 8: tablet
Rhizoma valerianae latifoliae 18g Cortex Albiziae 6g
Semen Ziziphi Spinosae (parched) 8g Medulla Junci 1.2g
Get Rhizoma valerianae latifoliae and be ground into coarse powder, with 35% ethanol that is equivalent to Rhizoma valerianae latifoliae medical material 12 times of weight, reflux, extract, 2 times, each 0.8 hour; Semen Ziziphi Spinosae and Cortex Albiziae are ground into coarse powder, add the water of 14 times of weight, reflux, extract, 4 times, each 1.5 hours; Medulla Junci is ground into coarse powder, with 95% ethanol of 46 times of weight, reflux, extract, 4 times, each 1.4 hours; Above extracting solution is reclaimed solvent respectively, be condensed into dried cream, process tablet through conventional technology.
Chromatograph and testing conditions: Agilent C18 chromatographic column (4.6mm * 250mm, 5 μ m); With 27: 72 acetonitriles-0.3% phosphoric acid solution is mobile phase; 35 ℃ of column temperatures; Flow velocity 1mL/min detects wavelength 280nm.
The preparation of reference substance solution: precision takes by weighing Hesperidin 9.8mg, with dissolve with methanol and be settled to the 25mL volumetric flask, shakes up, and promptly gets.
The preparation of sample solution: get pharmaceutical composition tablet of the present invention, pulverize, take by weighing 4.5g, put in the apparatus,Soxhlet's; Add an amount of methanol, putting in the water-bath refluxes is extracted into extracting liquid colourless, puts coldly, filters; Filtrating is concentrated near doing, and places 100mL volumetric flask bottle, and methanol constant volume is to 100mL.
Algoscopy: accurate respectively reference substance solution 6 μ l and the sample solution 17 μ l of drawing, inject chromatograph of liquid, measure, promptly get.
Content of hesperidin is 0.74mg/g in the pharmaceutical composition tablet of the present invention.
Claims (10)
1. detection method with drug combination preparation of angst resistance effect is characterized in that this method is:
This method adopts HPLC to detect;
Chromatograph and testing conditions: Agilent C18 chromatographic column; Acetonitrile-0.3% phosphoric acid solution with 10-30: 70-90 is a mobile phase; Detect wavelength 280nm; Be dissolved in organic solvent with Hesperidin and prepare reference substance solution; The said composition preparation prepares sample solution by organic solvent extraction; Draw reference substance solution and sample solution respectively and inject chromatograph of liquid, measure;
The crude drug of wherein said drug combination preparation consists of:
Rhizoma valerianae latifoliae 5-20 weight portion Cortex Albiziae 5-15 weight portion
Semen Ziziphi Spinosae 5-15 weight portion Medulla Junci 0.5-4 weight portion.
2. the method for claim 1 is characterized in that the crude drug of said drug combination preparation consists of:
3. method as claimed in claim 2 is characterized in that the method for preparing of drug combination preparation is:
Choose the aforementioned proportion crude drug, the Rhizoma valerianae latifoliae medical material is with 15-55% alcohol reflux 2-5 time, each 0.5-1.5 hour; Semen Ziziphi Spinosae and Cortex Albiziae add the water of 5-15 times of weight, reflux, extract, 2-5 time, each 1-3 hour; Medulla Junci was with the 60-99% alcohol reflux of 40-60 times of weight 2-5 time, each 0.5-1.5 hour; Above extracting solution is reclaimed solvent respectively, be condensed into dried cream, promptly get.
4. the method for claim 1 is characterized in that this method is:
HPLC chromatograph and testing conditions: Agilent C18 chromatographic column; Acetonitrile-0.3% phosphoric acid solution with 10-30: 70-90 is a mobile phase; 35 ℃ of column temperatures; Flow velocity 1mL/min detects wavelength 280nm;
The preparation of reference substance solution: get Hesperidin and use dissolve with methanol;
The preparation of sample solution: get this drug combination preparation, pulverize, make with the methanol eddy extraction;
Algoscopy: accurate respectively reference substance solution and the sample solution injection chromatograph of liquid drawn, measure, promptly get.
5. method as claimed in claim 4 is characterized in that in this method:
The method for preparing of reference substance solution is: precision takes by weighing Hesperidin 9.8mg, with dissolve with methanol and be settled to the 25mL volumetric flask, shakes up, and promptly gets;
The method for preparing of sample solution is: get drug combination preparation of the present invention, pulverize, take by weighing 2-8g, put in the apparatus,Soxhlet's; Add an amount of methanol, putting in the water-bath refluxes is extracted into extracting liquid colourless, puts coldly, filters; Filtrating is concentrated near doing, and places 100mL volumetric flask bottle, and methanol constant volume is to 100mL.
6. method as claimed in claim 4 is characterized in that this method is:
This method adopts HPLC to detect;
Chromatograph and testing conditions: Agilent C18 chromatographic column (4.6mm * 250mm, 5 μ m); With 20: 80 acetonitriles-0.3% phosphoric acid solution is mobile phase; 35 ℃ of column temperatures; Flow velocity 1mL/min detects wavelength 280nm;
The preparation of reference substance solution: precision takes by weighing Hesperidin 9.8mg, with dissolve with methanol and be settled to the 25mL volumetric flask, shakes up, and promptly gets;
The preparation of sample solution: get drug combination preparation of the present invention, pulverize, take by weighing 5 weight portions, put in the apparatus,Soxhlet's; Add an amount of methanol, putting in the water-bath refluxes is extracted into extracting liquid colourless, puts coldly, filters; Filtrating is concentrated near doing, and places 100mL volumetric flask bottle, and methanol constant volume is to 100mL;
Algoscopy: accurate respectively reference substance solution 5 μ l and the sample solution 15 μ l of drawing, inject chromatograph of liquid, measure, promptly get;
Content of hesperidin is 0.68mg/g in the pharmaceutical composition of the present invention.
7. method as claimed in claim 5 is characterized in that acetonitrile-0.3% phosphoric acid solution with 20: 80 is a mobile phase in chromatograph and the testing conditions.
8. method as claimed in claim 4 is characterized in that the capsule preparations of pharmaceutical composition described in this method is prepared by following method:
Get Rhizoma valerianae latifoliae and be ground into coarse powder, with 35% ethanol that is equivalent to Rhizoma valerianae latifoliae medical material 5-15 times of weight, reflux, extract, 2-5 time, each 0.5-1.5 hour; Semen Ziziphi Spinosae and Cortex Albiziae are ground into coarse powder, add the water of 5-15 times of weight, reflux, extract, 2-5 time, each 1-3 hour; Medulla Junci is ground into coarse powder, with 95% ethanol of 40-60 times of weight, reflux, extract, 2-5 time, each 0.5-1.5 hour; Above extracting solution is reclaimed solvent respectively, be condensed into dried cream, the dried cream of each composition adds the dextrin of the dried cream amount of 0.2-0.8 times of weight by the prescription mixed; Mixing, 80-95% ethanol is granulated as wetting agent; Cross 20 mesh sieves, granule should be kept at below the critical relative humidity 70-90%, promptly gets.
9. method as claimed in claim 6 is characterized in that the capsule preparations of this pharmaceutical composition is prepared by following method:
Get Rhizoma valerianae latifoliae and be ground into coarse powder, with 35% ethanol that is equivalent to Rhizoma valerianae latifoliae medical material 5-15 times of weight, reflux, extract, 2-5 time, each 0.5-1.5 hour; Semen Ziziphi Spinosae and Cortex Albiziae are ground into coarse powder, add the water of 5-15 times of weight, reflux, extract, 2-5 time, each 1-3 hour; Medulla Junci is ground into coarse powder, with 95% ethanol of 40-60 times of weight, reflux, extract, 2-5 time, each 0.5-1.5 hour; Above extracting solution is reclaimed solvent respectively, be condensed into dried cream, the dried cream of each composition adds the dextrin of the dried cream amount of 0.2-0.8 times of weight by the prescription mixed; Mixing, 80-95% ethanol is granulated as wetting agent; Cross 20 mesh sieves, granule should be kept at below the critical relative humidity 70-90%, promptly gets.
10. method as claimed in claim 9 is characterized in that the capsule preparations of pharmaceutical composition is prepared by following method:
Get Rhizoma valerianae latifoliae and be ground into coarse powder, with 35% ethanol that is equivalent to Rhizoma valerianae latifoliae medical material 10 times of weight, reflux, extract, 3 times, each 1 hour; Semen Ziziphi Spinosae and Cortex Albiziae are ground into coarse powder, add the water of 10 times of weight, reflux, extract, 3 times, each 2 hours; Medulla Junci is ground into coarse powder, with 95% ethanol of 50 times of weight, reflux, extract, 3 times, each 1 hour; Above extracting solution is reclaimed solvent respectively, be condensed into dried cream, the dried cream of each composition adds the dextrin of the dried cream amount of 0.5 times of weight by the prescription mixed; Mixing, 90% ethanol is granulated as wetting agent; Cross 20 mesh sieves, granule should be kept at critical relative humidity below 80%, promptly gets.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103091437A (en) * | 2012-11-13 | 2013-05-08 | 江苏艾兰得营养品有限公司 | Determination method of hesperidin content |
| CN112773849A (en) * | 2020-12-31 | 2021-05-11 | 武汉中博绿亚生物科技有限公司 | Pharmaceutical composition for preventing pet anxiety and application thereof |
-
2011
- 2011-03-31 CN CN201110080067.7A patent/CN102716275B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| 程静 等: "野生蜘蛛香与家种蜘蛛香中陈皮苷含量对比", 《中国现代药物应用》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103091437A (en) * | 2012-11-13 | 2013-05-08 | 江苏艾兰得营养品有限公司 | Determination method of hesperidin content |
| CN103091437B (en) * | 2012-11-13 | 2014-12-24 | 江苏艾兰得营养品有限公司 | Determination method of hesperidin content |
| CN112773849A (en) * | 2020-12-31 | 2021-05-11 | 武汉中博绿亚生物科技有限公司 | Pharmaceutical composition for preventing pet anxiety and application thereof |
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