CN1813859A - Particle for treating cardio-cerebrovascular disease, and tis preparing and active constituent content measuring method - Google Patents

Abstract

The present invention relates to a persimmon leaf extract granules preparation, its preparation and effective component content determination method. Its name is Naoxinqing granules preparation, and is an oral medicine preparation for curing the diseases of coronary heart disease, angina pectoris, cerebral arteriosclerosis and ischemic cerebrovascular disease, etc.

Description

The clear granule of the brain heart and the preparation and the active constituent content measuring method of treatment cardiovascular and cerebrovascular disease

Technical field

The present invention relates to technical field of Chinese medicines.Relate to a kind of clear granule of the brain heart and preparation and active constituent content measuring method that is used for cardiovascular and cerebrovascular disease, being particularly related to contain Chinese medicine Folium Kaki extraction of active ingredients thing is raw material, a kind of drug composition oral preparation that is prepared from suitable pharmaceutically useful adjuvant.

Background technology

NAOXINQING PIAN is to be raw material through extracting a kind of oral formulations that processing is made with the Chinese medicine Folium Kaki, and its standard derives from the Sanitation Ministry medicine standard WS ₃-B-3974-98.Have the arteria coronaria of increasing and cerebral artery blood flow amount, improve the blood supply oxygen supply state of the heart, cerebral tissue, increase the effect of erythrocyte electrophoresis rate, be used for the treatment of angina pectoris, cerebral arteriosclerosis, ischemic cerebrovascular, clinical verification shows, determined curative effect, untoward reaction is less, is clinical commonly used drug.

But since technology of preparing itself, dosage forms such as existing NAOXINQING PIAN agent, capsule, and because it is longer to exist dissolve scattered time limit, dissolution is lower, influences indexs such as its absorption and bioavailability, thereby influences the performance of drug effect, even therapeutic effect.

Summary of the invention:

Purpose of the present invention, be to replenish the existing angina pectoris that is used for the treatment of, cerebral arteriosclerosis, the deficiency of the oral drug preparation of ischemic cerebrovascular particularly is different from dosage forms such as existing NAOXINQING PIAN agent, capsule, and a kind of dispersion height is provided, bioavailability is good, and have quick release, fast Absorption and the fast clear granule of the drug composition oral preparation brain heart of imitating produce effects comprise sugar-free and sugar (containing low sugar) type granule are arranged.

The clear granule of the brain heart involved in the present invention is a raw material with the Chinese medicine Folium Kaki, is prepared from the pharmaceutically acceptable additive as adjuvant.The every gram granule of its finished product is equivalent to medical material Folium Kaki dry extract 25～500mg, and contained ethyl acetate extractum is 50mg～220mg.Simultaneously, carried out method of quality control research, the result is that the contained Folium Kaki of every gram granule is with Quercetin (C ₁₅H ₁₀O ₇) meter, must not be less than 0.50mg.

The present invention has adopted following technical scheme, has studied clear granule preparing process scheme of the brain heart and Quality Control Technology thereof.

1, technological process (seeing Figure of description 1)

2, Study on Preparation

(1) design of extraction process route

This product is a granule, is to change agent by tablet to form, and does not change in order to make its pharmacodynamics that changes after the agent, when technical study, its medicinal material extract method is the same with tablet, is decoction and alcohol sedimentation technique, gained extractum is dissolved in water, filter, filtrate is used ethyl acetate extraction four times, merges ethyl acetate liquid, reclaim ethyl acetate, because former technology determines technological factors such as solvent loads, therefore, this time during technical study to technological factor research experiment such as solvent load, baking temperatures.

(2) pre-treatment of medical material

Medical material is made decoction pieces after cleaning.

(3) Folium Kaki water is proposed the condition investigation

(3.1) water absorption rate is investigated

Take by weighing three batches of Folium Kaki, every crowd of 500g adds 10 times of water gagings respectively, is dipped to the heart, filters, and recording average water absorption rate is 405%.

(3.2) Folium Kaki water is proposed the condition investigation:

With extraction time, add the water multiple, extraction time is the investigation factor, carries out L ₉(3 ⁴) orthogonal test, and with receive the cream rate, quercetin content (%) be an evaluation index, screening optimum process condition (receipts cream rate, quercetin content weight coefficient are respectively 0.6,0.4).

Experimental technique: get 9 parts of Folium Kaki medical materials, every part of each 500g, water is carried, filters, merging filtrate, filtrate decompression is concentrated into the clear paste that relative density is 1.12 (60 ℃).Survey and receive cream rate and quercetin content.

Receive the cream rate: take by weighing Folium Kaki by prescription, water is carried, filter, and merging filtrate, filtrate decompression is concentrated into the clear paste that relative density is 1.12 (60 ℃).Calculate the percentage ratio of the clear paste and the medical material weight of carrying.

The assay of Quercetin (HPLC): use high effective liquid chromatography for measuring

Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; With ethanol (30～95%)-0.1% phosphoric acid (1: 20～1: 50) is mobile phase, detects wavelength 270nm, and number of theoretical plate calculates by the Quercetin peak should be not less than 4000.

It is an amount of that the Quercetin reference substance is got in the preparation of reference substance solution, accurate claims surely, adds ethanol (30%～90%) and dissolve and be diluted to the solution that every 1ml contains the about 40 μ g of Quercetin.

The about 2g of this product is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug flask, adds ethanol 20ml, and 25% sulphuric acid 5ml claims to decide weight.Water-bath backflow 30min, the cooling back claims to decide weight, supplies the weight that subtracts mistake with ethanol, and mixing filters, the reuse membrane filtration, promptly.

Accurate respectively above-mentioned two kinds of reference substance solution and each the 10 μ l of need testing solution of drawing of sample determination method inject chromatograph of liquid, according to high effective liquid chromatography for measuring, calculate quercetin content (%).The results are shown in Table 1-2.

Table 1 factor level table

Table 2 orthogonal test table

Tested number	A	B	C	D (blank)	Evaluation index
								Receive cream rate (%)	Quercetin content	Comprehensive grading (%)
					1 2 3 4 5 6 7 8 9 K 1 K 2 K 3 R	1 2 3 1 2 3 1 2 3 215.4 271.4 281.1 21.9	1 2 3 2 3 1 3 1 2 230.4 265.7 271.8 13.8				1 2 3 3 1 2 2 3 1 247.8 262.4 257.7 3.3	1 1 1 2 2 2 3 3 3 257.3 266.2 254.4 3.93	17.7 31.4 32.8 22.5 29.8 28.6 23.2 24.5 30.6	0.0186 0.0220 0.0226 0.0195 0.0218 0.0198 0.0205 0.0210 0.0213	61.0 96.3 100 75.7 93.1 87.4 78.7 82.0 93.7

Annotate: following receipts cream rate, quercetin content (C ₁₅H ₁₀O ₇) two evaluation indexes, all adopt the comprehensive grading method to carry out data analysis, receive cream rate, quercetin content (C ₁₅H ₁₀O ₇) weight coefficient is respectively 0.6,0.4.

Receive cream rate scoring=clear paste amount/maximum clear paste amount * 0.6*100%

Quercetin content scoring=quercetin content/maximum quercetin content * 0.4*100%

Comprehensive grading=receipts cream rate scoring+quercetin content scoring

As shown in Table 2, (K in the A factor ₁, K ₂) result differs bigger, (K in the A factor ₂, K ₃) result is more or less the same, and considers that big production saves factors such as cost, determines A ₂(extracting 2 times) is optimum condition; The C factor influences not quite the result, after taking all factors into consideration, determines that optimum process condition is A ₂B ₃C ₃, because the C factor influences not quite the result,, can consider that extraction time is 1 hour for the second time in order to save combustion energy and reduction of erection time, be: Folium Kaki decocts with water secondary, and 2 hours for the first time, add 12 times of water gagings, 1 hour for the second time, add 12 times of water gagings.

(3) alcohol precipitation process condition examination

The alcohol amount that contains with added concentration of alcohol, quiescent time, medicinal liquid.Be the examination factor, respectively get three levels, adopt L ₉(3 ⁴) orthogonal test, and be evaluation index with the quercetin content, screening precipitate with ethanol condition.

Test method: get 9 parts of Folium Kaki medical materials, every part of each 500g, according to the extraction process by water that top quadrature filters out, it is 1.12～1.15 (60 ℃) that filtrate is concentrated into relative density, add ethanol and reach 85% to containing the alcohol amount, standing over night, the leaching supernatant is standby; Precipitate with 65% washing with alcohol twice, collect cleaning mixture, leave standstill, draw supernatant and preceding supernatant and merge, reclaim ethanol, get extractum (ρ=1.35).The quercetin content assay method is the same, calculates quercetin content (mg/g).The results are shown in Table 3-4.

Table 3 factor level table

Table 4 orthogonal test table

Tested number	1 A	2 B	3 C	Quercetin content (mg/g)
					1 2 3 4 5 6 7 8	1 1 1 2 2 2 3 3	1 2 3 1 2 3 1 2	1 2 3 2 3 1 3 1	0.82 0.85 0.88 1.00 1.06 1.06 1.20 1.25

9 K 1 K 2 K 3 R

3 2.55 3.12 3.71 0.39

3 3.02 3.16 3.20 0.06

2 3.13 3.11 3.14 0.01

1.26 ∑＝9.38

As shown in Table 4, precipitate with ethanol factor affecting size is followed successively by A＞B＞C, because B, C factor are less, considers the big practical situation of producing, and optimum process condition is A ₃B ₂C ₃

(4) ethyl acetate extraction process conditions examination

With the density of ethyl acetate extraction number of times, medicinal liquid, the temperature of medicinal liquid is the examination factor, respectively gets three levels, adopts L ₉(3 ⁴) orthogonal test, and be evaluation index with extract (dried cream), screening ethyl acetate extraction process conditions.

Test method: get 9 parts of Folium Kaki medical materials, every part of each 500g, the aqueous extraction-alcohol precipitation technology that filters out according to top quadrature, gained extractum is dissolved in water, filters, filtrate is used ethyl acetate extraction, merge ethyl acetate liquid, reclaim ethyl acetate, the thick paste cold drying becomes dry extract.With the dry extract is evaluation index.

The results are shown in Table 5-6.

Table 5 factor level table

Table 6 orthogonal test table

Tested number	1 A	2 B	3 C	Dry extract weight (g)
					1 2 3 4 5 6 7 8 9	1 1 1 2 2 2 3 3 3	1 2 3 1 2 3 1 2 3	1 2 3 2 3 1 3 1 2	10.2 9.6 9.8 13.2 10.8 9.6 15.6 12.4 10.6

K 1 K 2 K 3 R

23.6 27.6 32.6 3.0

39.0 32.8 30.0 3.0

32.2 33.4 37.8 1.8

∑＝101.8

As shown in Table 6, ethyl acetate extraction influences size and is followed successively by A=B＞C, and optimum process condition is A ₃B ₁C ₃

(5) extracting solution concentrates

In Chinese medicine was produced, the concentrating under reduced pressure required time was short, can prevent that again effective ingredient is subjected to heat damage for a long time, in conjunction with factory's actual production conditions, adopted triple effect concentrating under reduced pressure equipment to concentrate.Extracting solution is condensed into thick paste, and recording its relative density is 1.12 (60 ℃).

(6) optimised process repeated authentication test:

In order to verify the accuracy of The above results,, arrange 3 batches of demonstration tests by above-mentioned fixed process conditions to guarantee the rationally feasible of extraction process, get dried Folium Kaki 1000g for every batch, decoct with water secondary, 2 hours for the first time, 1 hour for the second time, collecting decoction, filter, it is 1.12 (60 ℃) that filtrate is concentrated into relative density, adds ethanol and reaches 85% to containing the alcohol amount, standing over night, the leaching supernatant is standby; Precipitation is with twice of 65% washing with alcohol, collect cleaning mixture, leave standstill, draw supernatant and preceding supernatant and merge, reclaim ethanol, gained extractum is dissolved in water, and filters, and filtrate is used ethyl acetate extraction four times, merge ethyl acetate liquid, reclaim ethyl acetate, the thick paste cold drying becomes dry extract, and result of the test sees Table 7.

Table 7 Folium Kaki extract powder extraction process repeated experiments result

Numbering	Crude drug amount (g)	Dried cream amount (g)	Quercetin content in the dried cream (mg)	Paste-forming rate (%)
					1 2 3	1000 1000 1000	15.2 14.6 15.5	175 172 184	1.52 1.46 1.55

From above-mentioned result of the test as can be seen: the technology that is screened is rationally feasible, and is reliable and stable, has operability and repeatability.

(7) calculating of the quercetin content rate of transform in the Folium Kaki

Get 3 parts of Folium Kaki medical materials, every part of 1000g measures quercetin content in its medical material (mg) by top survey quercetin content method, and the optimised process that filters out by quadrature extracts then, measure quercetin content (mg) in its dried cream amount and the dried cream, the rate of transform of quercetin content is as follows in the Folium Kaki:

The rate of transform of table 8 Folium Kaki

Numbering	Medical material amount (g)	Quercetin content in the medical material (mg)	Dried cream amount (g)	Quercetin content in the dried cream (mg)	The rate of transform of Quercetin (%)	Mean transferred rate (%)
							1 2 3	1000 1000 1000	390 390 390	15.2 14.6 15.5	17.5 172 184	44.87 44.10 47.17	45.38

3, granular preparation technical study

This product is a granule, changes agent by Chinese medicine NAOXINQING PIAN promulgated by the ministries or commissions of the Central Government and forms.Therefore, we are reference to the medication amount (dried cream amount) of taking at every turn with the primary standard, and the determining of the adjuvant amount supplementary product kind that need add when going out to make granule in prediction on such basis

Adjuvant of granulating commonly used has dextrin, starch, sucrose etc.This product is to change agent according to Chinese medicine NAOXINQING PIAN promulgated by the ministries or commissions of the Central Government to form, and it is adjuvant that primary standard is selected starch for use.After my company changed tablet agent and becomes granule, the situation of taking according to particulate character and patient had changed adjuvant into sucrose and dextrin.

Supplementary product consumption

By the aforementioned calculation method as can be known: the extractum amount of at every turn taking is 0.1g, press the design of 3g/ bag, the dry extract amount that contains in every bag is 0.1g, adjuvant is 2.9g, dry extract: adjuvant=1: 29, because extract is that ethyl acetate extraction comes out, for ethyl acetate is fully volatilized, with its extractum bone dry, still use starch slurry as binding agent, its ratio is

Dry extract: sucrose: dextrin=1: 25: 4.

Granulate

Raw material: aforementioned 12.5.1. joint " semi-finished product Study on Preparation " prepared dried cream.

Operation: take by weighing dried cream 0.1kg, get sucrose and dextrin and be respectively 2.5kg, 0.4kg and put in barrel formula premix machine, add starch slurry 0.1kg mixing gradually, make the soft material of degree of tightness appropriateness, cross 14 mesh sieves and granulate.It is easy than 16 mesh sieves in operation with 14 mesh sieves,, granularity is also comparatively even, therefore adopts 14 mesh sieve wet granulations.Dry extract in the granulation: sucrose: the investigation of dextrin mixed proportion

Table 9

Concentration of alcohol %	Dry extract: sucrose: dextrin
						1∶25∶4	1∶25∶4	1∶25∶4	1∶25∶4	1∶25∶4
	15％	The group of being	The group of being	The group of being	The group of being						Powder
25％						The group of being	The group of being	The group of being	The group of being	Powder
	35％	The group of being	The group of being	The group of being	Be grain						Powder
45％						The group of being	The group of being	Powder	Powder	Powder

By table 9 as seen, dry extract: sucrose: the ratio of dextrin 1: 25: 4, granulating efficiency is best during concentration of alcohol 35%.

Particulate drying and granulate

This product in drying course, is used cold drying after granulating, and initial temperature is set at 40 ℃, is slowly rising to 60 ℃ within an hour then, is dried to particulate moisture and is lower than at 2.0% o'clock, and drying finishes.

Granulate

For making even particle size, guarantee dosage, be convenient to packing, need carry out granulate, this product is carried out granulate with 14 mesh sieves, 1 good sieve and No. 5 sieve screenings.

Qualified particulate yield is about 94.2% behind the granulate.

Particulate physical property

Character this product is faint yellow to brown granular; It is sweet to distinguish the flavor of, little hardship.

Bulk density is got granule 15g, places the 50ml graduated cylinder, measures particle volume, calculates bulk density.Measurement result is as follows:

The density measurement of table 10 granulation mass

Tested number	Granule heavy (g)	Volume (ml)	Bulk density (g/ml)	On average
					1 2 3	15.118 15.201 15.157	35.16 31.02 33.68	0.43 0.49 0.45	0.46

This product bulk density is 0.46g/ml.

Mobile

Adopt the funnel method to measure particulate flowability, calculate angle of repose, measurement result is as follows:

The particulate fluidity determining of table 11

Tested number	1	2	3	On average
					Angle of repose	29.5	29.1	29.3	29.3

Be 29.3 ° the angle of repose of this product, and particulate flowability is better, is easy to particulate packing.

The mensuration of critical relative humidity

This product is a granule, and granule is moisture absorption easily in the process of packing, influences the quality of product.Therefore must investigate granule is subjected to influence, the especially humidity of environment in minute process of assembling influence, carry out wettability test, measure critical relative humidity.Get 5 parts of granules, put in the weighing botle, accurate claim surely, put into relative humidity respectively and be 33%, 42.8%, 59.7%, 75.3%, 92.5% environment, in 25 ℃ of constant incubators, placed 48 hours, take out and measure its moisture.With the relative humidity is abscissa, and moisture is vertical coordinate, draws moisture equilibrium at dry side curve (seeing figure), and result of the test is as follows:

Table 12 critical relative humidity measurement result

Tested number	1	2	3	4	5
						Relative humidity (%) moisture (%)	33 3.7	42.8 4.2	59.7 7.0	75.3 13.3	92.5 42.2

The clear granule sucting wet curve of brain heart figure sees Figure of description 2.

By measurement result as can be known, the clear granule of the brain heart in the environment of different relative humiditys, its hygroscopic capacity difference, relative humidity less than the environment below 60% in particulate hygroscopicity not strong, packing does not influence the moisture of this product in this environment.Therefore, relative humidity should be controlled at below 60% during this product packing.

This product loading amount

From the above: the 3g/ bag, take the 1-2 bag, 3 times on the 1st at every turn.

Determining of packaging material

Because the critical relative humidity of this product is lower, therefore moisture absorption easily should select trapping, every moist strong compound membrane bag, as the packaging material of this product.

4, the research of quality control and HPLC method

Adopt thin layer chromatography, furancarboxylic acid, protocatechuic acid, the succinic acid reference substance that contains in the prescription carried out qualitative identification research, the preparation of reference substance solution and need testing solution is as follows according to the proper mass standard fabrication:

Get the about 8g of this product, add ethyl acetate 50ml, reflux 1 hour filters, and filtrate is concentrated into about 2ml, as need testing solution.Other gets furancarboxylic acid, protocatechuic acid, each 1mg of succinic acid reference substance, with ethyl acetate 1ml dissolving, makes reference substance solution respectively.

Detect: according to thin layer chromatography (appendix VIB) test, draw each 6 μ l of above-mentioned four kinds of solution, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid (5: 4: 1) is developing solvent, launches, and takes out, dry, 105 ℃ the heating half an hour, put cold, the spray with 0.05% bromophenol blue alcoholic solution.Discovery is in the test sample chromatograph, not obvious with speckle on the reference substance chromatograph relevant position, so consider it is that concrete test sample is prepared as follows because therefore this product reason that the adjuvant ratio increases in changing the agent process has increased the ethyl acetate extraction process again in the test sample preparation process:

Get the about 8g of this product, add ethyl acetate 50ml, reflux 1 hour filters, and filtrate evaporate to dryness, residue add the water ultrasonic dissolution, filter, and filtrate is used ethyl acetate extraction, gets acetic acid ethyl fluid and is concentrated into about 2ml, as need testing solution.

According to the thin layer chromatography test, draw each 6 μ l of need testing solution and each contrast solution, put respectively on same silica gel g thin-layer plate, be developing solvent with toluene-ethyl acetate-formic acid (5: 4: 1), launch, take out, dry, 105 ℃ the heating half an hour, put cold, the spray with 0.05% bromophenol blue alcoholic solution.In the test sample chromatograph, respectively with each corresponding position of reference substance chromatograph on, show the speckle of same color.

All the other conditions are constant, are developing solvent with toluene-ethyl acetate-acetic acid (2: 2: 1), launch, and also can obtain separating effect preferably.Negative noiseless.

Arsenic salt inspection (an appendix IX of Pharmacopoeia of the People's Republic of China version in 2005 F Yi Fa Gucaishi method) is in accordance with the law checked three batches of lab scale samples.

The preparation of standard arsenic solution: take by weighing arsenic trioxide 0.132g, put in the 1000ml measuring bottle, add 20% sodium hydroxide solution 5ml dissolving after, neutralize with an amount of dilute sulfuric acid, add dilute sulfuric acid 10ml again, be diluted with water to scale, shake up, as stock solution.

Before facing usefulness, precision is measured stock solution 10ml, puts in the 1000ml measuring bottle, adds dilute sulfuric acid 10ml, is diluted with water to scale, shakes up, and promptly gets (As that every 1ml is equivalent to 1 μ g).

Apparatus: with the instrument of pharmacopeia regulation, the lead acetate Cotton Gossypii 60mg that packs in airway C (tubulature highly is 70mm) is put a slice mercuric bromide reagent paper again on the plane, top of cock D, covers cock lid E and screws, promptly.

The preparation of standard arsenic speckle: precision is measured standard arsenic solution 2ml, put in the A bottle, add hydrochloric acid 5ml and water 21ml, add 5 of the inferior stannum test solutions of potassium iodide test solution 5ml and acid chlorization again, after room temperature is placed 10 minutes, add zinc granule 2g, to adorn immediately that appropriate airway C is close to be filled on the A bottle, and the A bottle will be put in 30 ℃ of water-baths, react 45 minutes, take out the mercuric bromide reagent paper, promptly.

Check: take by weighing three crowdes of each 1g of test sample (two parts every crowd), put in the ancient Cai Shi bottle, respectively add water 20ml and make dissolving, the preparation of the accurate arsenic speckle of sighting target, from " adding potassium iodide test solution 5ml again ", operation in accordance with the law.With 6 arsenic speckles and standard arsenic speckle (2ppm) comparison of three batch samples that generate, color is more shallow as a result, illustrates that arsenic content is all less than 2ppm in three batches of test samples.By pertinent regulations, the text standard is not listed in this inspection.

Microbial limit

According to microbial limit test (an appendix XIII of Chinese Pharmacopoeia version in 2005 C) test, sample is all up to specification.

Table granule general rule check result table

Project	Lot number
				050301	050302	050303
	Content uniformity granularity moisture melting arsenic salt (ppm) heavy metal (ppm) bacterial population (individual/g) fungi count is (individual/g) Escherichia coli	Qualified up to specification 4.1% up to specification＜2＜10＜10/g＜10/g does not detect	Qualified up to specification 2.9% up to specification＜2＜10＜10/g＜10/g does not detect				Qualified up to specification 3.2% up to specification＜2＜10＜10/g＜10/g does not detect

Conclusion: laboratory sample rules of preparations check result is all qualified.

The ethyl acetate extractum is checked

Getting each about 10g of three batches of lab scale samples of this product (accurately to 0.01g), is solvent with ethyl acetate, measures according to the ethanol-soluble extractives algoscopy.

Detected object selects not have the assay item in the former granule quality standard, has increased the assay item newly in this research.

This product is the granule of being made through extraction by Folium Kaki, according to the pharmacology documents and materials of Folium Kaki as can be known, Folium Kaki flavone has bigger pharmacological action aspect cardio-cerebrovascular, so we have paid the utmost attention to the Folium Kaki flavone constituents when revising and enlarging the assay item, and contain Quercetin in the Folium Kaki flavone constituents, its content assaying method research is a lot, usefulness be high performance liquid chromatography the most widely, this detection method accuracy height, favorable reproducibility are methods commonly used during modern medicines detect.Therefore, this product adopts the content of its Quercetin of high effective liquid chromatography for measuring.

Content assaying method learn this research of research with reference to contain the Quercetin composition in other preparations contain survey method (high performance liquid chromatography), draft the method for measuring quercetin content in this product, and on the quercetin content basis of measuring pilot product, tentatively determined the content limit of Quercetin in this product.

Detect wavelength determination

It is an amount of to get the Quercetin reference substance, adds methanol and makes the solution that every 1ml contains 0.5mg, with ultraviolet device scanning, found that Quercetin has absorption maximum at the 270nm place in 200～400nm scope, is decided to be 270nm so will detect wavelength.

Chromatographic condition and system suitability test

The selection of mobile phase: with reference to the mobile phase that the quercetin content of other preparations is measured, carried out the screening test of mobile phase, selected at last mobile phase is: methanol-0.1% phosphoric acid (1: 20～1: 50); Column temperature: 40 ℃, flow velocity 1.0ml/min, blank adjuvant experimental results show that simultaneously: negative sample is noiseless (seeing Fig. 3～5).

The screening of chromatographic column: this research is screened chromatographic column, successively respectively with KromasilC ₁₈Post (4.6 * 250mm, 5 μ m), Dikma diamonds C ₁₈(4.6 * 250mm, 5 μ m) analyze for analytical column, through overtesting, find with Kromasil C ₁₈The separating effect that post (4.6 * 250mm, 5 μ m) is analyzed is best, the theoretical cam curve height.

The preparation of reference substance solution

Precision takes by weighing Quercetin reference substance 9.5mg, puts in the 25ml measuring bottle, with dissolve with methanol and be diluted to scale, shakes up, as stock solution (0.38mg/ml).The accurate stock solution 1ml that draws is transferred in the 10ml measuring bottle, is diluted to scale with methanol, shakes up, and promptly gets the reference substance solution that every 1ml contains Quercetin 0.038mg.

The investigation of extraction conditions

Measure the preparation method of need testing solution down with reference to other formulation contents that contain the Quercetin composition, we select hydrochloric acid with methanol and 25% as the extraction solvent, and have carried out extracting mode and extraction time is investigated.

The investigation of extracting mode

Get each two parts of this product, every part of about 2g, the accurate title, decide, and all puts in the tool plug flask, adds methanol 20ml, and 25% hydrochloric acid 5ml claims to decide weight.A water-bath backflow 30min, the cooling back claims to decide weight, supplies the weight that subtracts mistake with methanol, and mixing filters, and the membrane filtration of reuse 0.45 μ m promptly gets test sample sample 1.

Another part is used supersound extraction 30min, and the cooling back claims to decide weight, supplies the weight that subtracts mistake with methanol, and mixing filters, and the membrane filtration of reuse 0.45 μ m promptly gets test sample sample 2.

The accurate reference substance solution 10 μ l that draw, need testing solution 1,2 each 10 μ l inject high performance liquid chromatograph, measure, and result of calculation, the result of reflux, extract, and supersound extraction relatively see Table 3 (Fig. 6～8).

Quercetin content in the table 3 Different Extraction Method sample

Extracting method	Reflux extraction	Ultrasonic extraction
			Quercetin content (mg/g)	1.0445	0.7729

Two kinds of methods are extracted sample, and result difference is bigger, and the reflux, extract, effect is better, so select reflux extraction for use.

The investigation of extraction time

Get three parts of this product, every part of about 2g, the accurate title, decide, and all puts in the tool plug flask, adds methanol 20ml, and 25% hydrochloric acid 5ml claims to decide weight.A water-bath refluxed 1 hour, a backflow 1.5 hours, and a the backflow 2 hours, the cooling back claims to decide weight, supplies the weight that subtracts mistake with methanol, and mixing filters, and the membrane filtration of reuse 0.45 μ m promptly gets test sample sample 3,4,5.

The accurate reference substance solution 10 μ l that draw, need testing solution 3,4,5 each 10 μ l inject high performance liquid chromatograph, measure, result of calculation, extraction time is investigated the result and relatively sees Table 4 (Fig. 9～12).

The investigation test of table 4 extraction time

Time (h)	0.5	1	1.5	2
					Quercetin content (mg/g)	1.0445	1.0411	1.0466	1.0509

It is basicly stable to extract after 0.5 hour content, so the selective extraction time is 0.5 hour.

Linear relationship investigation and the accurate absorption of regression equation Quercetin reference substance stock solution (0.38mg/ml) 0.5ml, 2ml, 4ml, 5ml put respectively in the 10ml measuring bottle, be diluted to scale with methanol, shake up, concentration is 0.019mg/ml, 0.076mg/ml, 0.152mg/ml, 0.19mg/ml.Accurate each 10 μ l of reference substance solution that draw above-mentioned variable concentrations measure peak area by above-mentioned chromatography condition, calculate regression equation with peak area and sample size, the results are shown in Table 5.

Table 5 Quercetin sample size and peak area linear relationship

Reference substance concentration (mg/ml)	0.019	0.038	0.076	0.152	0.19
						Sample size (mg * 10 -6) peak area	0.19 652744	0.38 1202746	0.76 2408936	1.52 4975327	1.9 6388031
Regression equation	Y＝3E+06X-60942			r＝0.9991

The Quercetin linear relationship chart is seen Figure of description 3.

Quercetin has good linear relationship (seeing last figure) in 0.19～1.9 μ g scope.

Accurate above-mentioned reference substance solution (0.038mg/ml) the 10 μ l that draw of precision experiment repeat sample introduction 5 times, and RSD＜2% of Quercetin reference substance peak area the results are shown in Table 6.

Table 6 Precision test result

The sample introduction number of times	1	2	3	4	5	RSD(％)
							Peak area	1202746	1148729	1176245	1184628	1189656	1.71

Replica test is got parallel 6 tests of this product, carries out assay by the text method, and the quercetin content meansigma methods is 1.0474mg/g as a result, and RSD is 1.02%, sees table 7 for details.

Table 7 replica test result

Sample number	1	2	3	4	5	6	Meansigma methods	RSD(％)
									Quercetin content (mg/g)	1.0451	1.0615	1.0315	1.0413	1.0558	1.0493	1.0474	1.02

The sample solution stability test is got above-mentioned need testing solution 1, respectively at the 0th, 2,4,6,8 hour sample introduction, and each 10 μ l, record peak area integrated value, RSD is 1.77%, shows in the sample solution that Quercetin measured in 8 hours good stability is arranged.

Table 8 sample solution stability test result

Time (h)	0	2	4	6	8	RSD( ％)
							The peak area integrated value	2335911	2267842	2314870	2285446	2371522	1.77

Recovery test

Adopt the average recovery method, precision takes by weighing 9 parts of each about 1g of known content this product, and accurate the title decides, put in the tool plug flask, (precision takes by weighing Quercetin reference substance 17.4mg, puts in the 100ml measuring bottle to add a certain amount of reference substance respectively, add dissolve with methanol and be diluted to scale, shake up, concentration is 0.174mg/ml, adds different amounts respectively, see Table 9), add methanol 20ml, 25% hydrochloric acid 5ml claims to decide weight.Water-bath backflow 30min, the cooling back claims to decide weight, supplies the weight that subtracts mistake with methanol, and mixing filters, the membrane filtration of reuse 0.45 μ m, promptly.

Accurate reference substance solution and each 10 μ l injection high performance liquid chromatograph of each need testing solution drawn measured, and result of calculation with the following formula calculate recovery rate, the results are shown in Table 9:

Table 9 determination of recovery rates result

Number of times	Sample size (g)	Quercetin content in the sample (mg)	Add Quercetin amount (mg)	Measure Quercetin amount (mg)	The response rate (%)	Meansigma methods (%)	RSD (％)
								1 2 3 4 5 6 7 8 9	0.9172 0.9103 0.8612 0.9027 0.9129 0.8794 0.8863 0.9042 0.9148	0.9607 0.9534 0.9020 0.9455 0.9562 0.9211 0.9283 0.9471 0.9582	1.392 1.392 1.392 1.74 1.74 1.74 2.088 2.088 2.088	2.3378 2.2782 2.2833 2.6506 2.6992 2.6355 2.9433 3.0297 3.0014	98.93 95.17 99.23 97.99 100.17 98.53 96.50 99.75 97.86	98.24	1.62

The result shows that this law response rate is better.

Description of drawings

Fig. 1: technological process

Fig. 2: the clear granule sucting wet curve of brain heart figure

Fig. 3: Quercetin linear relationship chart

The specific embodiment

Embodiment 1:

Get dried Folium Kaki, decoct with water secondary, 2 hours for the first time, 1 hour for the second time, add 12 times of water gagings at every turn, collecting decoction filters, and it is 1.12～1.15 (60 ℃) that filtrate is concentrated into relative density, add ethanol and reach 85% to containing the alcohol amount, standing over night, the leaching supernatant is standby; Precipitation is collected cleaning mixture with 65% washing with alcohol twice, leaves standstill, draw supernatant and preceding supernatant and merge, reclaim ethanol, gained extractum is dissolved in water, filter, filtrate is used ethyl acetate extraction four times, merges ethyl acetate liquid, reclaim ethyl acetate, the thick paste cold drying becomes dry extract, and every 33.4g dry extract is with sucrose and dextrin 970g (sucrose: dextrin=25: 4), mixing, an amount of with 35% ethanol, make granule, 1000g is made in cold drying, promptly.

Embodiment 2:

As above the obtained extractive of persimmon leaves ethyl acetate dry extract of method 150g adds dextrin 2000g and an amount of stevioside's correctives, and mixing is an amount of with 30% ethanol, granulate, and cold drying, granulate promptly gets the clear sugar type granules of the brain heart.

Claims (4)

1, a kind of clear granule of the brain heart and preparation and active constituent content measuring method that is used for cardiovascular and cerebrovascular disease.It is characterized by the granule dosage form, comprise Sugarless type and sugared type (containing low sugar) type is arranged.All adjuvants are amylodextrin and correctives etc.

2, the clear granule of the brain heart as claimed in claim 1, the ethyl acetate extract that it is characterized by by Folium Kaki is the granule that active effective ingredient makes, the contained ethyl acetate extract of every gram granule (or dry extract) is 25mg～220mg.

3, the clear granule of the brain heart as claimed in claim 1 is characterized in that the mixed proportion of described drug extract and used adjuvant can be 1: 0.5～1: 9.5.

4, the clear granular preparation content of effective of the brain heart as claimed in claim 1 assay method is characterized in that adopting the HPLC method to carry out, and institute's measurement result is that the contained Folium Kaki of every gram granule is with Quercetin (C ₁₅H ₁₀O ₇) meter, must not be less than 0.50mg.

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