CN102397333A

CN102397333A - Preparation and quality detection methods of honeysuckle flower-baikal skullcap root injection

Info

Publication number: CN102397333A
Application number: CN2010102844548A
Authority: CN
Inventors: 江厚生; 王秀敏; 侯晓礁; 李学良
Original assignee: BEIJING CENTRE BIOLOGY Co Ltd
Current assignee: BEIJING CENTRE BIOLOGY Co Ltd
Priority date: 2010-09-17
Filing date: 2010-09-17
Publication date: 2012-04-04

Abstract

The invention discloses preparation and quality detection methods of a honeysuckle flower-baikal skullcap root injection. The preparation method comprises the following steps of: 1, adding an appropriate amount of water into a baikal skullcap root extract for dissolving; 2, regulating the pH to 6-10 with a 2-20 percent sodium hydroxide solution, and filtering; 3, combining the filtrate with a honeysuckle flower extract, and regulating the pH to 6.0-7.5 with a 2-20 percent sodium hydroxide solution; 4, boiling the solution for one hour, filtering, and adding 1-10 grams of sodium sulfite; 5, adding water to full dose, stirring uniformly, and regulating the pH to 6.0-7.5 with a 2-20 percent sodium hydroxide solution; 6, adding water to 1,000 ml and filtering; and 7, filling and sterilizing to obtain the honeysuckle flower-baikal skullcap root injection. The injection has the advantages of good curative effect, convenience for administration, easiness for transportation and storage, easiness for accepting by a large quantity of raisers and dealers, small toxic and side effects, high meat quality, contribution to the health of human beings, remarkable social benefit and the like.

Description

A kind of preparation of YINHUANG ZHUSHEYE and quality determining method

Technical field

The present invention relates to a kind of YINHUANG ZHUSHEYE, and the preparation of YINHUANG ZHUSHEYE and quality determining method.

Background technology

Flos Lonicerae and Radix Scutellariae all are good clear pathogen medicine of a warm nature things that Chinese veterinarian uses always, through being usually used in various febrile illnesses.

Flos Lonicerae is the dry flower that Caprifoliaceae Lonicera Caprifoliaceae plant Radix Ophiopogonis Lonicera L.japonicaThunb. belongs to various plants together, is traditional Chinese medical science medicine commonly used, has the function of heat-clearing and toxic substances removing, cool breeze heat radiation.It is a kind of heat-clearing and detoxifying herb the most frequently used in the veterinary clinic.Be mainly used in epidemic febrile disease heating, anemopyretic cold, carbuncle furuncle poison, sore throat, erysipelas, toxic-heat and blood stasis, anal fistula, disease such as have blood in stool.Be used to treat multiple diseases such as epidemic febrile disease heating, anemopyretic cold, laryngopharynx swelling and pain, pneumonia, erysipelas, septicemia, enteritis and infectious intestinal disease more clinically, the title of " penicillin among the Chinese medicine " is arranged.In the prescriptions of Chinese medicine of SARS prevention, the Flos Lonicerae frequency of occurrences is maximum.Have broad-spectrum antiseptic, antiviral, antitumor, enhance immunity and antipyretic and anti-inflammatory, function of gallbladder promoting, protect the liver, multiple pharmacological effect such as blood fat reducing, antifertility, hemostasis, antiulcer; The multiple disease that escherichia coli, streptococcus pneumoniae, pasteurellosis bacillus, streptococcus etc. are caused all has good curing and preventive effect; Its determined curative effect, do not see toxic side effect and drug resistance, can use yet with multiple antibiotics commonly used is collaborative.Medicine fit applications such as normal and Radix Scutellariae, Rhizoma Coptidis, Fructus Forsythiae in clinical.

The chemical constitution study of Flos Lonicerae shows, mainly contains volatile oil, flavonoid, organic acid, triterpenes and inorganic elements during it is spent.Furl according to " modernization of cmm and application " the 3rd and to carry, Flos Lonicerae has effects such as resisting pathogenic microbes, antiinflammatory, analgesic, immunomodulating.Flos Lonicerae also has effects such as function of gallbladder promoting protects the liver, antioxidation, blood fat reducing, central excitation in addition.

Radix Scutellariae is the dry root of labiate Radix Scutellariae Scutellaria baicalensis Georgi, is to use wider Chinese medicine in the traditional Chinese veterinary medicine, press the traditional Chinese veterinary medicine theory, and Radix Scutellariae has heat clearing and damp drying, eliminating fire and detoxication, hemostasis, effect such as antiabortive.Also be a kind of Chinese medicine the most frequently used in the veterinary clinic.Be mainly used in anti-inflammation and infection, cure mainly hyperpyrexia excessive thirst, cough due to lung-heat, damp-heat dysentery, jaundice, pyretic stranguria, conjunctival congestion and swelling pain, carbuncle furuncle etc.Radix Scutellariae has antibiotic, antiviral, inflammation-inhibiting reaction, hepatic cholagogic, diuresis, removing free radical and antioxidation, effects such as anticoagulation and antithrombotic formation.The multiple disease that escherichia coli, streptococcus pneumoniae, pasteurellosis bacillus, staphylococcus aureus etc. are caused all has good preventive and therapeutic effect.

Radix Scutellariae contains five kinds of main flavone components: baicalin (C ₂₁H ₁₈O ₁₁) be Radix Scutellariae-T-glucuronide, wogonoside (C ₂₂H ₂₀O ₁₁) be wogonin-7-glucuronide, wogonin (C ₁₆H ₁₂O ₈), also contain baicalinase in the root and can promote baicalin and wogonoside hydrolysis, generate baicalein and Wogonin, still contain cupreol, benzoic acid, Radix Scutellariae fecula in addition.Radix Scutellariae has antiinflammatory, immunomodulating and antioxidative effect.Radix Scutellariae also has antitumor action, cardiovascular system is had protective effect in addition, and tumor necrosis factor and the inductive hepatocellular apoptosis of D actinomycin D are had inhibitory action.

Silver yellow share and can obviously heighten the effect of a treatment.Clinically, Flos Lonicerae, Radix Scutellariae Chang Peiwu share.For further improving its curative effect, enlarge scope of medication and make things convenient for medication, modern Chinese medicine is clinical to have begun to extract its effective ingredient with Chinese veterinarian is clinical, with the use law of Chinese medicine, forms " silver yellow preparation " and uses by a certain percentage.A large amount of pharmaceutical researches are verified; The silver yellow combination formulations is used than the single drug use; Have effects such as more powerful heat-clearing and toxic substances removing, anti-inflammation, antiviral; Produce the obvious synergistic effect, for upper respiratory tract and respiratory tract infection, acute tonsillitis, pharyngitis, mumps, intestinal tract disease etc. curative effect is preferably arranged all, and good synergism is arranged with penicillin, lincomycin etc.; Can also antiviral, show the dual anti-infective effect that can not be ignored.

Only silver yellow soup is a kind of for dosage form the earliest; Continuous development along with dosage form research; The people doctor has developed tens kinds of dosage forms such as oral liquid, tablet (comprising buccal tablet), injection, electuary, pill, powder, enema, collutory, spray, aerosol at present, and is widely used in clinical according to the characteristics of different dosage form.But in veterinary applications, available silver yellow preparation is still few, further develops and more very imitates related preparations easily, comprises the silver yellow acid injection, with the present situation of preventing and treating of obviously improving its relevant disease, promotes animal husbandry development.Simultaneously, reduce application of antibiotics, ensure human health.

Summary of the invention

The preparation and the quality determining method that the purpose of this invention is to provide a kind of YINHUANG ZHUSHEYE.

One of purpose provides a kind of method for preparing of YINHUANG ZHUSHEYE, comprises the steps:

1, Radix Scutellariae extract is added water and make its dissolving in right amount;

2, regulate pH to 6-10 with the 2-20% sodium hydroxide solution, filter;

3, filtrating and Flos Lonicerae extract merge, and regulate pH to 6.0-7.5 with the 2-20% sodium hydroxide solution;

4, solution was boiled 1 hour, filter, add sodium sulfite 1-10g;

5, add water to nearly full dose, stir, regulate pH to 6.0-7.5 with the 2-20% sodium hydroxide solution;

6, add water to 1000ml, filter;

7, embedding, sterilization promptly gets YINHUANG ZHUSHEYE.

Chinese medicine belongs to the novel form of herbal medicine, and this patent is processed the injection drug administration by injection through fully extracting the effective ingredient in the crude drug, has improved bioavailability of medicament greatly, has strengthened curative effect;

Medication is convenient, and transportation, preservation simply are prone to accepted by numerous raisers and distributor;

Simultaneously because the toxic and side effects of Chinese medicine own is little, widely popularize the silver yellow acid injection after, can obviously reduce the application of antibiotic and chemicals, improve meat quality, guarantee human health, obvious social benefit;

Therefore, the silver yellow acid injection has good market prospect.

Two of purpose provides a kind of YINHUANG ZHUSHEYE quality determining method, and especially a kind of content assaying method comprises the steps:

Assay: adopt chlorogenic acid and content of baicalin in the hplc simultaneous determination YINHUANG ZHUSHEYE.

(1) chromatographic condition and system suitability test are filler with the octadecylsilane chemically bonded silica; With methanol-0.5% potassium dihydrogen phosphate is mobile phase, methanol in the time of 0 minute-0.5% potassium dihydrogen phosphate (20: 80), and methanol in the time of 11 minutes-0.5% potassium dihydrogen phosphate (80: 20), methanol in the time of 11.01 minutes-0.5% potassium dihydrogen phosphate (20: 80) stopped in the time of 20 minutes; The detection wavelength is 316nm;

(2) it is quantitative to sighting article that the preparation of reference substance solution, precision take by weighing baicalin, places volumetric flask, adds methanol to scale, and ultrasonic dissolution 15 minutes is processed the solution that concentration is 100 μ g/ml, filters subsequent use; It is an amount of to get the chlorogenic acid reference substance, and accurate the title decides, and puts in the brown bottle, adds the solution that methanol is processed 20 μ g/ml, filters subsequent use;

(3) preparation of need testing solution, accurate absorption YINHUANG ZHUSHEYE is quantitative, places volumetric flask, adds methanol to scale, and ultrasonic dissolution 15 minutes is cool to room temperature, filters, and it is quantitative that precision is measured subsequent filtrate, places volumetric flask, adds methanol to scale, and mixing is subsequent use;

(4) algoscopy, each is quantitative for accurate respectively absorption reference substance solution and need testing solution, injects chromatograph of liquid, measures, and promptly gets.

The every 1ml of these article contains Flos Lonicerae with chlorogenic acid (C ₁₆H ₁₈O ₉) meter, must not be less than 0.6mg, contain Radix Scutellariae with baicalin (C ₂₁H ₁₈O ₁₁) meter, must not be less than 6.0mg.

The specific embodiment

Through embodiment the present invention is done further elaboration below:

The preparation technology of YINHUANG ZHUSHEYE of the present invention:

Embodiment 1:

Radix Scutellariae extract 28.4g is added water make its dissolving in right amount, regulate pH to 8 with 2% sodium hydroxide solution, filter, filtrating merges with Flos Lonicerae extract 80g; Regulate pH to 7.2 with 10% sodium hydroxide solution, boiled 1 hour, filter, add sodium sulfite 1g; Add water to nearly full dose, stir, regulate pH to 7.2, add water to 1000ml with 2% sodium hydroxide solution; Filter, embedding, sterilization promptly gets YINHUANG ZHUSHEYE 1000ml.

Embodiment 2:

Radix Scutellariae extract 40g is added water make its dissolving in right amount, regulate pH to 10 with 8% sodium hydroxide solution, filter, filtrating merges with Flos Lonicerae extract 78.6g; Regulate pH to 6.0 with 20% sodium hydroxide solution, boiled 1 hour, filter, add sodium sulfite 2g; Add water to nearly full dose, stir, regulate pH to 6.0, add water to 1000ml with 5% sodium hydroxide solution; Filter, embedding, sterilization promptly gets YINHUANG ZHUSHEYE 1000ml.

Embodiment 3:

Radix Scutellariae extract 32.1g is added water make its dissolving in right amount, regulate pH to 6 with 20% sodium hydroxide solution, filter, filtrating merges with Flos Lonicerae extract 86g; Regulate pH to 7.5 with 2% sodium hydroxide solution, boiled 1 hour, filter, add sodium sulfite 10g; Add water to nearly full dose, stir, regulate pH to 7.5, add water to 1000ml with 20% sodium hydroxide solution; Filter, embedding, sterilization promptly gets YINHUANG ZHUSHEYE 1000ml.

Embodiment 4: about the preparation of YINHUANG ZHUSHEYE and the correlational study and experiment YINHUANG ZHUSHEYE Research on Process of quality determining method

YINHUANG ZHUSHEYE of the present invention is the compound preparation by Flos Lonicerae and Radix Scutellariae configuration after extracting processing.Have broad-spectrum antiseptic and antivirus action, can stop toxin to absorb, raise immunity alleviates or eliminates stress, improves capillary resistance, protects mucosal epithelium and hepatocyte function.It is one of time-honored conventional medicament of China.Chinese medicine is the emphasis and the difficult point of Chinese medicine preparation research and development in recent years.This test is on Flos Lonicerae and Radix Scutellariae Study on extraction basis, the production technology of YINHUANG ZHUSHEYE have been done detailed research.

1 test apparatus and reagent

1.1 instrument SHIMADZU L VP C-10AT high performance liquid chromatograph; SPD-10A VP UV, visible light detector; Chromatographic column: SHIMADZU ODSC18 post (150 * 4.6mm, 5 μ m); Thunder magnetic PHS-25 type digital display pH meter.

1.2 reagent baicalin standard substance, the chlorogenic acid standard substance, Nat'l Pharmaceutical & Biological Products Control Institute provides; Ethanol, phosphoric acid (analyzing alcohol) potassium dihydrogen phosphate (analyzing alcohol), methanol (chromatograph alcohol) are purchased the chemical reagents corporation in Beijing.

2 test methods

2.1 the variation of medicinal liquid pH value and content before and after the sterilization

2.1.1 the configuration of medicinal liquid

Medicinal liquid dispensing according to configuration 500ml.Add Flos Lonicerae extract (being equivalent to the 0.4g chlorogenic acid) and Radix Scutellariae extract (being equivalent to the 4g baicalin) in the 500ml water for injection, add the 1g sodium sulfite, mix homogeneously; Regulate pH value to 6.0 with sodium hydroxide, it is dissolved fully, filter; Be divided into 10 parts; Except that portion progressively increases as all the other 9 parts amounts with the NaOH 0.5ml of 0.1mol/ml the blank, measure pH value and the content of effective of respectively organizing medicinal liquid, the amount embedding of propping up by 10ml/ then; 100 ℃ of sterilization 30min cool the back and are measuring pH value and the content of respectively organizing medicinal liquid respectively.

2.1.2pH the mensuration of value

Standard operating procedure according to PHS-25 type digital display pH meter is directly measured.

2.1.3 Determination on content

2.1.3.1 chromatographic condition

Chromatographic column: ODSC18 post (150 * 4.6mm, 5 μ m); Mobile phase: it is 2.6 that methanol-0.5% potassium dihydrogen phosphate, 0.5% potassium dihydrogen phosphate use phosphoric acid to regulate pH value, detects wavelength: 316nm; Column temperature: 40 ℃; Flow velocity: 1ml/min.

2.1.3.2 the preparation of standard solution

Precision takes by weighing baicalin standard substance 2.5mg, places the volumetric flask of 25ml; Add methanol to scale, ultrasonic dissolution 15 minutes is processed the solution that concentration is 100 μ g/ml.

Precision takes by weighing chlorogenic acid standard substance 2.5mg, places the volumetric flask of 50ml, adds methanol to scale, and ultrasonic dissolution 15 minutes is processed the solution that concentration is 50 μ g/ml.

With two kinds of standard solutions by subsequent use after 1: 1 the mixed.

2.1.3.3 the preparation of sample solution

Precision is drawn 1ml, places the volumetric flask of 25ml, adds methanol to scale, and ultrasonic dissolution 15 minutes filters, and gets and considers liquid 1ml, places the 10ml volumetric flask, adds methanol to scale, and mixing is subsequent use.

2.1.2.4 the mensuration of sample

Accurate respectively mixed standard solution and each 10 μ l injecting chromatograph of sample solution drawn, mensuration promptly gets.

2.2 best sterilization time and sterilising temp

2.2.1 the configuration of medicinal liquid

Medicinal liquid dispensing according to configuration 500ml.Add Flos Lonicerae extract (being equivalent to the 0.08g chlorogenic acid) and Radix Scutellariae extract (being equivalent to the 0.8g baicalin) in the 100ml water for injection, add the 1g sodium sulfite behind the mix homogeneously, regulate pH value to 7.0 with sodium hydroxide; It is dissolved fully, filter, the amount embedding of propping up by 10ml/; 80 ℃, 90 ℃, 100 ℃; Under 110 ℃ of conditions, sterilization 10min, 20min, 30min, 40min carry out steriling test after cooling.

2.2.2 steriling test

2.2.2.1 the configuration of contrast bacterium

Nutrient agar inclined-plane fresh cultured thing 1 oese of depletion Staphylococcus aureus [CMCC (B) 26003]; Be seeded in the nutrient broth medium; 30～35 ℃ cultivate 16～18 hours after, be diluted to 0.9% aseptic sodium chloride solution and contain 10～100 bacterium among every 1ml, subsequent use.

2.2.2.2 the cultivation of fungus, mycete

Adopt test tube that membrane-filter procedure will be equipped with THIOGLYCOLLIC ACID salt fluid culture medium to be put in 30～35 ℃ the incubator and cultivated 2 days, the test tube that fungi culture medium is housed is put into mold incubator and 20～25 ℃ of cultivations 3 days.

2.2.2.3 the result judges

When the positive control pipe shows muddy and truly has bacteria growing; When the negative control pipe is negative; The result who gets according to observation post judges: as be clarification in THIOGLYCOLLIC ACID salt fluid culture medium and the fungal culture parent tube or show muddy but verified be not that bacteria growing is arranged, the judgement test sample is qualified; If any pipe shows muddy and proves conclusively in two kinds of cultivation parent tubes bacteria growing is arranged, then get the doubling dose test sample again, reexamine, except that the positive control pipe, other each Guan Jun do not have bacterial growth, judge that test sample is qualified.

3 result of the tests

3.1 the variation of medicinal liquid pH value and content before and after the sterilization.(seeing table 1)

Medicinal liquid pH value and content (mg/ml) table before and after table 1 sterilization

3.2 best sterilization time (min) and sterilising temp (℃) see table 2

The different sterilising temps of the different sterilization times of table 2 are to the influence of bacterial growth situation

Annotate: "+" is for there being bacteria growing; "-" is asepsis growth

4 discuss

4.1 can find out that from table 1 result sterilization back pH value reduces; And content of baicalin increases to some extent; Chlorogenic acid contents o'clock decreases in pH value＜7; But change not obvious, sterilization is described before and after baicalin, chlorogenic acid content stable, it is little to the influence of baicalin chlorogenic acid in regulation pH value scope that hence one can see that; When pH value greater than 7.5 the time, chlorogenic acid contents reduces, sterilization back pH value fall is bigger; When pH value 6.0～7.5 the time, the variation of sterilization back pH is less, so pH value transfers to 6.0～7.5 ideals comparatively.

4.2 in the experimentation, pH value changes before and after sterilization, fall increases with the increase of pH value.Prompting should arouse attention when transferring pH in the preparation process, and sterilization back pH value should be in prescribed limit.

4.3 the data in the table 2 show, 90 ℃ of 30min, 40min; 100 ℃ of 20min, 30min, 40min; There is not bacterial growth fully in 110 ℃ of 20min, 30min, the 40min sample.But consider cost and needs of production, select 100 ℃ of sterilizations 20 minutes.

4.4 whole process of production is to accomplish in the GMP workshop, before embedding, through twice filtration of 0.45 and 0.22 μ m, can antibacterial and thermal source be filtered basically, so only need 100 ℃ of 20min to get final product.

5 conclusions

The pH value of solution transfers to 6.0～7.5 before the sterilization, and the pH of sterilization back solution is in the scope of regulation; But consider cost and needs of production, select 100 ℃ of sterilizations 20 minutes.

The research of chlorogenic acid and content of baicalin assay method in the YINHUANG ZHUSHEYE

The main component of YINHUANG ZHUSHEYE is baicalin, chlorogenic acid, is pure Chinese medicinal preparation, is mainly used in poultry pneumonia, asthma and the treatment of the mixed infection that caused by various bacteria, has the function of heat-clearing and toxic substances removing, anti-inflammation.Main bioactive ingredients in the Radix Scutellariae is a baicalin; Its topmost pharmacological action is an antiinflammatory; Baicalin can significantly suppress the biosynthesis of leukotriene B4, leukotriene C in the cell; Also can significantly suppress the interior Ca2+ rising of leukocyte that artificial tripeptides (fMLP) excites, and promote that the cAMP level improves in the cell, show the leukocytic multiple function of baicalin appreciable impact and disclosed its antiinflammatory action mechanism.Main bioactive ingredients in the Flos Lonicerae is a chlorogenic acid; Flos Lonicerae all has certain inhibitory action to various pathogens; Comprise staphylococcus aureus, Hemolytic streptococcus, escherichia coli, dysentery bacterium, vibrio cholera, Bacillus typhi, Salmonella paratyphi; Also effective to streptococcus pneumoniae, meningococcus, bacillus pyocyaneus, tubercule bacillus, Flos Lonicerae also has the obvious antiinflammatory refrigeration function that gets simultaneously.Therefore, research has been carried out assay as index components to YINHUANG ZHUSHEYE with baicalin, chlorogenic acid.

1 instrument and reagent

1.1 instrument

SHIMADZU LC-10ATVP high performance liquid chromatograph; SPD-10AVP UV, visible light detector; Chromatographic column: SHIMADZU ODSC18 post (150 * 4.6mm, 5 μ m)

1.2 reagent

YINHUANG ZHUSHEYE (our factory); Baicalin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute); Chlorogenic acid reference substance (Nat'l Pharmaceutical & Biological Products Control Institute); Potassium dihydrogen phosphate (analytical pure); Phosphoric acid (analytical pure); Methanol (chromatographically pure); The mobile phase water is a distilled water; All the other reagent are analytical pure.

2 test methods and result

2.1 chromatographic condition

Chromatographic column: ODSC18 post (150 * 4.6mm, 5 μ m); Mobile phase: methanol-0.5% potassium dihydrogen phosphate; It is 2.6 that 0.5% potassium dihydrogen phosphate uses phosphoric acid to regulate pH value; 0.01 minute the time 0.5% potassium dihydrogen phosphate-methanol (80: 20); 0.5% potassium dihydrogen phosphate in the time of 11 minutes-methanol (20: 80), 0.5% potassium dihydrogen phosphate in the time of 11.01 minutes-methanol (80: 20) stopped in the time of 20 minutes; Detect wavelength: 316nm; Column temperature: 40 ℃; Flow velocity: 1ml/min.

2.2 the preparation of need testing solution

2.2.1 the preparation of standard solution

Precision takes by weighing baicalin standard substance 2.5mg, places the volumetric flask of 25ml, adds methanol to scale, and ultrasonic dissolution 15 minutes is processed the solution that concentration is 100 μ g/ml, filters subsequent use.

Precision takes by weighing chlorogenic acid standard substance 2.5mg, places the volumetric flask of 50ml, adds methanol to scale, and ultrasonic dissolution 15 minutes is processed the solution that concentration is 50 μ g/ml, filters subsequent use.

2.2.2 the preparation of sample solution

Precision is drawn YINHUANG ZHUSHEYE 1ml, places the volumetric flask of 25ml, adds methanol to scale, and ultrasonic dissolution 15 minutes is cool to room temperature, filters, and gets filtrating 1ml, places the volumetric flask of 10ml, adds methanol to scale, and mixing is subsequent use.

2.2.3 the preparation of sample negative control solution

After removing Flos Lonicerae in the prescription, Radix Scutellariae respectively, prepare the negative control solution of YINHUANG ZHUSHEYE, prepare negative controls by the method for " 2.2.2 " according to production technology.

2.3 linear relationship is investigated

Accurate mixing reference substance (1: 1) solution 4,6,8,10, the 12 μ l that draw chlorogenic acid and baicalin; Measure the peak area integrated value by above-mentioned chromatographic condition; With the peak area is vertical coordinate, and the time is abscissa, gets regression equation through linear regression treatment; The regression equation of baicalin is Y=5.085819e-005+3.61012 (r=0.9935750), shows that the sample size of baicalin is good linear relationship in 20～60 μ g scopes; The regression equation of chlorogenic acid is Y=3.665887e-005X+2.31978 (r=0.9990650), shows that the sample size of chlorogenic acid is good linear relationship in 10～30 μ g scopes.

2.4 precision test

Precision is drawn mixing reference substance (1: 1) the solution 10 μ l of chlorogenic acid and baicalin, repeats sample introduction 5 times, measures peak area by above-mentioned chromatographic condition, and according to peak area value, the average peak area that gets baicalin is 1037524.02, RSD=1.42%; The average peak area of chlorogenic acid is 257640.48, RSD=2.05%.

2.5 repeatability test

Get 5 of same batch of YINHUANG ZHUSHEYEs, prepare, and under above-mentioned chromatographic condition, measure peak area according to the method for preparing of above-mentioned sample solution, according to peak area value, the baicalin average peak area be 720291.3, RSD=2.55%; The average peak area of chlorogenic acid is 21428.12RSD=2.14%.

2.6 average recovery test

Get 5 of the YINHUANG ZHUSHEYEs of known content respectively, whenever draw 5ml, add baicalin, chlorogenic acid reference substance respectively, by above-mentioned sample preparation methods and chromatographic condition mensuration peak area, the average recovery rate of baicalin is 99.04%, RSD=1.61% (n=5).The average recovery rate of chlorogenic acid is 99.99%, RSD=2.02% (n=5).

Table 1 YINHUANG ZHUSHEYE determination of recovery rates result (n=5)

2.7 sample determination

Get the YINHUANG ZHUSHEYE of different lot numbers respectively, the accurate absorption measured peak area by above-mentioned sample solution method for preparing and chromatographic condition, each batch replication three times, and the result sees table 2.

Table 2 YINHUANG ZHUSHEYE assay result (n=5)

3 discuss

We once adopted the acetonitrile-0.5% potassium dihydrogen phosphate system of different proportion, the acetonitrile-water system of different proportion, the methanol-water system of different proportion; The methanol of different proportion-0.5% potassium dihydrogen phosphate system, the result shows: when being set at gradient elution, i.e. methanol-0.5% potassium dihydrogen phosphate; Potassium dihydrogen phosphate is regulated pH to 2.6,0.5% potassium dihydrogen phosphate in the time of 0.01 minute-methanol (80: 20), 0.5% potassium dihydrogen phosphate in the time of 11 minutes-methanol (20: 80) with phosphoric acid; 11.01 minute the time 0.5% potassium dihydrogen phosphate-methanol (80: 20), stop the peak shape of chlorogenic acid, baicalin symmetry in the time of 20 minutes; Separate fully, negative control does not disturb.

4 conclusions

The HPLC specificity is strong; Can measure chlorogenic acid and content of baicalin in the YINHUANG ZHUSHEYE fast and accurately; When chlorogenic acid and baicalin are measured in to the YINHUANG ZHUSHEYE of different lot numbers; Find that its content difference is not remarkable, therefore, can be used as the quality control standard of YINHUANG ZHUSHEYE.

The stability study of YINHUANG ZHUSHEYE

YINHUANG ZHUSHEYE is that Flos Lonicerae, Radix Scutellariae are through extracting the refining sterile water solution that forms.Have broad-spectrum antiseptic and antivirus action, can stop toxin to absorb, raise immunity alleviates or eliminates stress, improves capillary resistance, protects mucosal epithelium and hepatocyte function.Cure mainly the pig lung plague that pasteurellosis bacillus causes, the diseases such as pig lung plague that Mycoplasma hyopneumoniae causes.Can eliminate disease pig fever caused by exogenous pathogens rapidly, not eat, dysentery, cough and asthma, fervescence, mucosa cyanosis, symptoms such as the basal part of the ear, veutro, the hemorrhage speckle of extremity inside skin.

This paper is intended to study the stability of YINHUANG ZHUSHEYE, for clinical application provides foundation.

1 instrument and medicine

1.1 instrument

SHIMADZU LC-10ATVP high performance liquid chromatograph; SPD-10AVP UV, visible light detector; Chromatographic column: SHIMADZU ODSC18 post (150 * 4.6mm, 5 μ m); Thunder magnetic PHS-25 type digital display pH meter (Shanghai Precision Scientific Apparatus Co., Ltd).

1.2 reagent

Baicalin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute); Chlorogenic acid reference substance (Nat'l Pharmaceutical & Biological Products Control Institute); Potassium dihydrogen phosphate (analytical pure); Phosphoric acid (analytical pure); Methanol (chromatographically pure); The mobile phase water is a distilled water; Sodium hydroxide (analytical pure); Aluminum nitrate (analytical pure); Ferric chloride (analytical pure); Chile saltpeter (analytical pure); YINHUANG ZHUSHEYE of the present invention is provided by Beijing Centre Biology Co., Ltd..

2 experimental techniques

2.1 accelerated tests

Accelerated test by the pharmacopeia regulation; Three lot sample article are at 40 ± 2 ℃; The condition held of relative humidity 75 ± 5% six months, the every month of sampling are regularly once measured chlorogenic acid and content of baicalin in the YINHUANG ZHUSHEYE, pH value then; And observe outward appearance, color and luster (character), according to prediction of result effect duration.

2.2 illumination accelerated tests

Get and supply three batches of test agents, place the light kitchen, the condition held of illumination 4500 ± 500Lx 10 days; The regularly sampling in the 5th, 10 day; Measure chlorogenic acid and content of baicalin in the YINHUANG ZHUSHEYE, pH value then, and observe outward appearance, clarity (character), according to prediction of result effect duration.

2.3 reserved sample observing method

2.3.1 three batches of test samples are pressed commercially available back, normal temperature condition held 12 months.Sampling in per 3 months once, respectively at 0,3,6,9,12 month, to chlorogenic acid in the YINHUANG ZHUSHEYE and content of baicalin, pH value, outward appearance, clarity (character) etc. detected then.

2.3.212 detected respectively at sampling in 18,24 months with the continued observation in individual month.With result and comparison in 0 month, confirm the effect duration of medicine.Because the dispersibility of measured data carries out statistical analysis by 95% fiducial limit, draws rational effect duration.Less like three batches of statistic analysis result difference, then getting its meansigma methods is expiry date; If difference is bigger, then gets it and the shortest be effect duration.Data show very stable medicine, and analysis does not take statistics.

2.4 identification experiment

These article of getting 1ml adds 75% ethanol 9ml, shakes up, as need testing solution.Other gets baicalin reference substance and chlorogenic acid reference substance, adds methanol respectively and processes the solution that every 1ml contains 1mg, 0.3mg, as reference substance solution.According to thin layer chromatography (Chinese Pharmacopoeia version appendix in 2000) test, draw each 2 μ l of above-mentioned three kinds of solution, put respectively on same polyamide film; With acetic acid is developing solvent, launches, and takes out; Dry, put under the uviol lamp (365nm) and inspect, whether identically observe with the speckle of reference substance chromatograph relevant position.

3 experimental results

3.1 YINHUANG ZHUSHEYE accelerated test result

YINHUANG ZHUSHEYE is at 40 ± 2 ℃; The condition held of relative humidity 75 ± 5% six months, its appearance luster and clarity do not have significant change, and pH value changes not remarkable; Baicalin and chlorogenic acid contents variation difference is not remarkable in the YINHUANG ZHUSHEYE, can confirm tentatively that its stability is 2 years.The result sees table 1.

3.2 YINHUANG ZHUSHEYE acceleration by light result of the test

YINHUANG ZHUSHEYE is the condition held of illumination 4500 ± 500Lx 10 days, the regularly sampling in the 5th, 10 day, and its appearance character changes not obvious, and pH changes little, and baicalin in the YINHUANG ZHUSHEYE, that chlorogenic acid contents changes difference is not remarkable.The result sees table 2.

3.3 reserved sample observing result

YINHUANG ZHUSHEYE was kept somewhere 24 months under approaching actual storage requirement, and its outward appearance and color and luster do not have significant change, and the variation difference of baicalin, chlorogenic acid content is not remarkable, and the quality index difference is very little, can confirm that effect duration is 2 years.The result sees table 3.

Table 1 different batches YINHUANG ZHUSHEYE accelerated test result

Annotate: chlorogenic acid content is green contain (mg/ml) content of baicalin Huang contain (mg/ml) rufous supernatant liquid+

Table 2 different batches YINHUANG ZHUSHEYE acceleration by light result of the test

Table 3 different batches YINHUANG ZHUSHEYE reserved sample observing result

3.4 when doing discrimination test, in the test sample chromatograph, with the corresponding position of chlorogenic acid reference substance chromatograph on, show the fluorescence speckle of same color.With the corresponding position of baicalin reference substance chromatograph on, show an identical dirty-green speckle.

4 conclusions

Result of study shows that YINHUANG ZHUSHEYE character under temperature accelerated test and acceleration by light condition changes not obvious; Chlorogenic acid and content of baicalin are stable, can confirm tentatively that its stability is 2 years.Through the stability of reserved sample observing method investigation YINHUANG ZHUSHEYE, to place 2 years, each index changes very little, so it has good stability.

Person skilled in the art should be familiar with; The above embodiment is used for explaining the object of the invention; And be not with opposing qualification of the present invention; As long as in essential scope of the present invention, variation, modification (like the similar adjustment that time, temperature, concentration etc. are done) that the foregoing description is done all will drop in the claim protection domain of the present invention.

Claims

1. the preparation of a YINHUANG ZHUSHEYE and quality determining method comprise the steps: that Radix Scutellariae extract is added water makes its dissolving in right amount; Regulate pH to 6-10 with the 2-20% sodium hydroxide solution, filter; Filtrating merges with Flos Lonicerae extract, regulates pH to 6.0-7.5 with the 2-20% sodium hydroxide solution; Solution was boiled 1 hour, filter, add sodium sulfite 1-10g; Add water to nearly full dose, stir, regulate pH to 6.0-7.5 with the 2-20% sodium hydroxide solution; Add water to 1000ml, filter; Embedding, sterilization promptly gets YINHUANG ZHUSHEYE.It is characterized in that: adopt chlorogenic acid and content of baicalin in the hplc simultaneous determination YINHUANG ZHUSHEYE.

2. the preparation of a kind of YINHUANG ZHUSHEYE according to claim 1 and quality determining method, it is characterized in that: every 10ml injection is equivalent to crude drug in whole 2-12g.