CN1810272A - Prepn process of red sage prepn and its quality control method - Google Patents

Abstract

The present invention discloses the preparation process of one kind of red sage preparation. The preparation process includes the steps of: crushing red sage as the medicine material, extracting with 70-95 % concentration alcohol solution, filtering, merging filtrate, decompression concentrating to obtain extract while recovering alcohol, washing with hot water at 70-90 deg.c to eliminate water soluble component, drying the residual extract, crushing to obtain the extract and further adding proper supplementary material for preparing into required preparation form. The process is simple and practical, and the product has the functions of the medicinal material maintained. Furthermore, the present invention also discloses the quality control method.

Description

A kind of preparation method of red sage formulation and method of quality control

Technical field

The present invention relates to a kind of extract of red rooted salvia and the preparation method and the method for quality control of preparation thereof, belong to the field of Chinese medicines.

Background technology

Radix Salviae Miltiorrhizae is the dry root and rhizome of labiate Radix Salviae Miltiorrhizae Salviamiltiorrhiza Bge..Spring, Qiu Erji excavate, and remove silt, drying.Liposoluble constituent in the Radix Salviae Miltiorrhizae has the effect of anti-inflammation, is used for osteomyelitis, acne, tonsillitis, otitis externa, furuncle, carbuncle, trauma infection contamination, burn infection, mastitis, cellulitis etc.If the extracts active ingredients in the red rooted salvia is made with extra care out, make suitable dosage form, will greatly make things convenient for clinical practice, have good market prospect.

Summary of the invention

The technical problem to be solved in the present invention is, to the mainly containing after effective constituent furthers investigate of salviamiltiorrhizabung, sets up the preparation method of effective ingredient in the Radix Salviae Miltiorrhizae, and make preparation be applied to clinical in.For this reason, the inventor has entered deep research.

According to simple prerun, the inventor thinks that following technology is more reasonable: red rooted salvia is pulverized, and the alcoholic solution with 70～95% extracts, and filters, merging filtrate, decompression recycling ethanol also is condensed into thick paste, removes the hydrotrope with 70～90 ℃ of hot water wash, remaining extract dry is pulverized, and gets extract.In view of the above, carry out further Study on thinning:

(1) extraction process

The investigation that 1 solvent is doubly measured

1.1 the mensuration of index components

Radix Salviae Miltiorrhizae medical material simply only in the prescription, effective ingredient has TANSHINONES, cryptotanshinone, Tanshinone I I A etc. in the Radix Salviae Miltiorrhizae.Consider that finished product carries out assay to cryptotanshinone,, carry out craft screening so be index components with the cryptotanshinone in the test.

Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Acetonitrile-water (68: 32) is a mobile phase, and flow velocity is 1.0ml/min, and column temperature is 40 ℃, and the detection wavelength is 270nm, and number of theoretical plate calculates by the cryptotanshinone peak should be not less than 3000.

The cryptotanshinone reference substance is got in the preparation of reference substance solution, and accurate the title decides, and uses dissolve with methanol, makes the solution that every 1ml contains 50 μ g, promptly.

It is an amount of that the preparation precision of need testing solution takes by weighing this product, and the accurate methanol 25ml that adds claims decide weight, and (100w 50kHz), is put coldly, supplies the weight of loss, shakes up, with microporous filter membrane (0.45 μ m) filtration, promptly for supersound process 30 minutes.

Accurate respectively reference substance and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.

Trial test: take by weighing one times of recipe quantity medical material, add 8 times of amount 90% ethanol, refluxed 2 hours, filter, by the volume of filtrate, the liquid absorption that calculates medical material is 2.1 times.

By the trial test result, testing program is doubly measured in the design alcohol extraction: take by weighing 1/100 times of recipe quantity medical material in the prescription ratio, totally 3 parts, add 90% alcohol reflux three times respectively, each 1 hour, every part added 4,6,8 times of amount 90% ethanol respectively and carries out reflux, extract, test, merge extractive liquid,, to contain the cryptotanshinone total amount is that index is carried out optimal process, concrete data and the results are shown in Table 1.

Table 1 solvent is doubly measured investigation

Extracting solvent doubly measures	Cryptotanshinone total amount (mg)
		4 times of amounts	140
6 times of amounts	172
		8 times of amounts	173

Result of the test shows, it is lower to add 4 times of amounts of 90% ethanol cryptotanshinone content, adds 6 times of amounts and to add 8 times of amount results close, is to save solvent, and energy savings selects to add 6 times of amount 90% alcohol reflux 3 times.

The investigation of 2 reflux extracting times

Take by weighing 1/100 times of recipe quantity medical material in the prescription ratio, totally 3 parts, add 90% alcohol reflux three times, add 6 times of amounts, decocting time was respectively 1 hour, 1.5 hours, 2 hours, merge extractive liquid, at every turn, to contain the cryptotanshinone total amount is that index is carried out optimal process, concrete data and the results are shown in Table 2.

The investigation of table 2 decocting time

Extraction time	Cryptotanshinone total amount (mg)
		1.0	168
1.5	181
		2.0	172

Result of the test shows that the cryptotanshinone content that reflux, extract, obtained in 1 hour, 2 hours is lower, and the cryptotanshinone content that reflux, extract, obtained in 1.5 hours is higher, is 1.5 hours so select reflux extracting time.

3 best reflux, extract, technological designs

In the actual production, because the medical material liquid absorption is bigger, so it is big for the first time to extract solvent adding amount during reflux, extract,, extraction time is long, and two, three times the used solvent of reflux, extract, is doubly measured and all corresponding for the first time minimizing of extraction time, this process choice adds 6 times of amount 90% ethanol for the first time, reflux, extract, 1.5 hours, two, three times reflux, extract, selects to add 4 times of amount 90% ethanol, reflux, extract, 1 hour, get final product, can reduce production costs.By test, select two parts of 1/100 times of recipe quantity medical materials, be index with the cryptotanshinone total amount, determine optimum extraction process.Result of the test sees Table 3.

Table 3 optimum extraction process is investigated

Extracting method	Cryptotanshinone total amount (mg)
		Reflux, extract, 3 times adds 6 times of amounts at every turn, refluxes 1.5 hours	184
For the first time add 6 times of amounts, refluxed 1.5 hours; Two, add 4 times of amounts for three times, refluxed 1 hour.	179

Result of the test shows that two kinds of extraction process cryptotanshinone content are more or less the same, and shows that designed extraction process is comparatively reasonable.

(2) reclaim the research of ethanol and drying process

1 reclaims the ethanol condition selects

By test, investigated of the influence of different recovery ethanol conditions to cryptotanshinone content in the sample, serve as to investigate index with the content of cryptotanshinone and recovery time, preferably reclaim the ethanol condition, concrete test seeing Table 4.

Test method: get this product alcohol extract, be divided into three parts, reclaim with different condition.

Table 4 concentration technology is investigated

Reclaim the ethanol condition	Recovery time (h)	Cryptotanshinone content (mg)
			40～50℃，-0.08Mpa	7.2	21
50～60℃，-0.08Mpa	5.5	36
			60～70℃，-0.08Mpa	4.3	25

Result of the test shows, reclaim the ethanol condition select (50～60 ℃ ,-0.08Mpa), during recovery the used time short, the effective component content height is so select this condition recovery ethanol.

The investigation of 2 thick paste density

In the former technology, the water liquid that reclaims behind the ethanol is condensed into thick paste, adds the hot water wash soluble constituent that anhydrates.By test, thick paste density is investigated, remain with effective constituent as far as possible, remove water-soluble components.

Test method: fetch receive ethanol (50～60 ℃ ,-0.08Mpa) water liquid is divided into three parts, is condensed into the thick paste of different relative densities, washes remove impurity by technology, is index with the content of cryptotanshinone, investigates, result of the test sees Table 5.

Table 5 thick paste density is investigated

Thick paste density (50 ℃)	The rate of extract (%)	Latent red ketone total amount (mg)
			1.25	3.9	10.3
1.30	3.4	9.8
			1.35	2.8	7.6

Result of the test shows that when thick paste density was 1.30 (50 ℃), loss of effective components was less in the sample, and the rate of extract is lower, is convenient to the molding of preparation.

3 drying meanss

Extract, concentrate by preferably good extraction process, get three parts of equivalent thick pastes, carry out the drying means screening.Adopt constant pressure and dry respectively, drying under reduced pressure (55～60 ℃ ,-0.08MPa), drying under reduced pressure (65～70 ℃ ,-0.08MPa) carry out drying, and be index with the total content of cryptotanshinone in the dry thing, investigate experiment, the results are shown in Table 6.

Table 6 drying means is investigated

Drying means	Drying time	Latent red ketone content (mg)
			Constant pressure and dry	13.2	16.2
Drying under reduced pressure (55～60 ℃ ,-0.08MPa)	4.1	30.5
			Drying under reduced pressure (65～70 ℃ ,-0.08MPa)	3.6	25.3

Learn that by test constant pressure and dry is than the drying under reduced pressure length that expends time in, loss of effective components is bigger, does not generally adopt constant pressure and dry during therefore big production; Drying under reduced pressure (55～60 ℃ ,-0.08MPa) than drying under reduced pressure (65～70 ℃ ,-0.08MPa) drying time short, but the composition temperature influence is bigger in the sample, thus drying condition selection drying under reduced pressure (55～60 ℃ ,-0.08MPa).

So far, the research that the inventor has extracted part before having finished all obtains following result: red rooted salvia is ground into coarse powder, add 90% alcohol reflux three times, add 6 times of amounts, reflux, extract, 1.5 hours for the first time, second and third time adds 4 times of amounts respectively, and reflux, extract, 1 hour filters, merging filtrate, 50～60 ℃ of following decompression recycling ethanols and when being condensed into 50 ℃ relative density be 1.28～1.32 extractum, remove the hydrotrope with 80 ℃ of hot water wash, dry under 55～60 ℃ of conditions, pulverize, get extract.

Then, inventor's design is made various dosage forms with this extract, and its tablet is preferred.Because the active ingredient of this prescription is fat-soluble, sample viscosity is bigger, by test, selects to add an amount of filler and disintegrating agent, to guarantee the qualified and good stability of product.Result by test optimizes the best disintegrating agent and the addition of filler: add microcrystalline Cellulose 40g, and carboxymethylstach sodium 30g, the alcohol granulation with 80% is crossed 20 mesh sieves, drying, granulate.Behind the granulate, add magnesium stearate 1g again, can improve particulate flowability and unilateral smoothness, add an amount of carboxymethylstach sodium to total amount at last and reach 420g, mixing, tabletting gets final product.The disintegrating agent of adding at last can effectively improve the disintegrate effect of tablet, makes the rapid disintegrate of tablet, improves bioavailability.

To a little, the inventor has finished the research contents of whole Radix Salviae Miltiorrhizae extract preparations.

But in above research process; quality control process is very crucial, by the quality of control intermediate and final product, could guarantee the meaning of each technological parameter; could finally make clinical effective product, so the inventor also lists method of quality control in protection domain.

This method of quality control comprises qualitative identification and assay two parts, below respectively the narration:

Qualitative identification: it is an amount of to get extract of the present invention or preparation, adds methanol 10ml, and supersound extraction 10 minutes can fully be extracted it, filters, and gets subsequent filtrate as need testing solution.Other gets Radix Salviae Miltiorrhizae IIA and cryptotanshinone reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution.According to the thin layer chromatography test, draw need testing solution and each 5ul of reference substance solution, put respectively on same silica gel g thin-layer plate, be developing solvent with 19: 1 toluene-ethyl acetates, launch, take out, dry.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

Content assaying method:

Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; 74: 26 methanol-water is as mobile phase; The detection wavelength is 270nm.Number of theoretical plate calculates by the cryptotanshinone peak should be not less than 3000.

The preparation precision of reference substance solution takes by weighing cryptotanshinone and each 2.5mg of Tanshinone I I A reference substance, puts in the 100ml measuring bottle, adds methanol and is diluted to scale, shakes up, promptly.

Preparation 0.1g of the present invention is got in the preparation of need testing solution, and accurate the title decides, and precision adds methanol 25ml, claim to decide weight, supersound process 20 minutes claims to decide weight again, replenish the weight that subtracts mistake with methanol, shake up, filter, the accurate suction measured subsequent filtrate 1ml, adding methanol is diluted in the 5ml measuring bottle, shake up, with the microporous filter membrane filtration of 0.45um, promptly.

Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid respectively, measure, promptly.

The specific embodiment:

The preparation that makes with the inventive method is the effect with anti-inflammation, and following experiment conclusion can prove.

1, chronic bronchitis

Matched group: 40 routine patients, select quiet of cephalosporin 4g or ofloxacin 0.2g for use, a twice-daily.Application is simultaneously eliminated the phlegm, cough medicine; Cooperate the aerosol therapy in case of necessity, oxygen uptake and Supporting Therapy, and regulate water, electrolyte balance.The treatment group: 35 routine patients, add on this basis and to use tablet of the present invention, oral, 3 slices/time, three times on the one, 2 weeks of logotype.Two groups equal 7 days be 1 course of treatment.Treat 2 the course of treatment result: the treatment group is cured 13 examples, produce effects 15 examples, 5 examples that take a turn for the better, and total effective rate 94.29%, matched group are followed successively by 7,12,15,87,50%, two groups of relatively P＜0.01.

2, urticaria perstans

40 examples are organized in treatment, 4 slices/time in oral tablet of the present invention, and three times on the one, matched group 39 routine oral cetirizine 10mg, once-a-day, equal 7 days was 1 course of treatment, took for 2 courses of treatment continuously.With examining 1 time, continued to follow up a case by regular visits to 2 weeks after finishing the course of treatment weekly.The result: treatment group 32 examples (80%) of fully recovering are better than matched group 21 examples (53 85%), two groups of P＜0.05 relatively.

3 psoriasises

Patient's 329 examples are divided into 3 groups at random.Treatment group: 4 slices/time in oral tablet of the present invention, three times on the one; WEIANZHI JIAONANG 25mg, three times on the one; Contrast I group: WEIANZHI JIAONANG 25mg, three times on the one.Contrast II group: placebo vitamin E three times on the 0.1st,, equal two week checks 1 time, in totally 8 weeks, treatment finishes back 4 weeks of tracing study.The result: treatment is organized and is cured 110 examples, produce effects 75 examples, effective 28 examples, total effective rate 93.01% in 229 examples.Contrast I organizes 60 examples, respectively is 0,7,12,3167%.Contrast II organizes 40 examples, respectively is 0,1,3,100%.Treatment group and two matched groups are P＜0.01 relatively, and this disease of tablet in treatment of the present invention has the effect of very strong antibiotic, antiinflammatory and microcirculation improvement, especially to the G+ coccus and there is tangible immunoregulation effect relevant.Tretinoin medicines has antiinflammatory, inhibition cell proliferation, promotes the effect of cell differentiation that the two associating has remedied deficiency separately, makes therapeutic effect desirable more.

Further specify technical scheme of the present invention by the following examples:

Embodiment one

[method for making] gets Radix Salviae Miltiorrhizae 10kg, is ground into coarse powder, adds 90% alcohol reflux three times, adds 6 times of amounts for the first time, reflux, extract, 1.5 hours, and second and third time adds 4 times of amounts respectively, reflux, extract, 1 hour, merge extractive liquid, filters (50 ℃～60 ℃ of decompression recycling ethanols;-0.08Mpa) and be condensed into the thick extractum of 1.30 (50 ℃), add 4 times of water gagings (80 ℃), stir evenly, filter, get precipitation, reduce pressure (55 ℃～60 ℃;-0.08Mpa) drying is pulverized, and gets extract fine powder 280g, adds microcrystalline Cellulose 40g, carboxymethylstach sodium 20g, use 80% alcohol granulation, dry (45～55 ℃), granulate adds magnesium stearate 1g, carboxymethylstach sodium is in right amount to total amount 350g, and tabletting wraps film-coat, promptly.

1 of this product is got in [discriminating] (1), removes film-coat, and porphyrize adds methanol 10ml, and supersound extraction 10 minutes filters, and gets subsequent filtrate as need testing solution.Other gets Radix Salviae Miltiorrhizae IIA and cryptotanshinone reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution.According to the thin layer chromatography test, draw need testing solution and each 5ul of reference substance solution, put respectively on same silica gel g thin-layer plate, be developing solvent with toluene-ethyl acetate (19: 1), launch, take out, dry.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

[assay] is according to high effective liquid chromatography for measuring.

Chromatographic condition and system suitability test are filler (φ * L=4.6 * 250mm, granularity 5 μ m) with octadecylsilane chemically bonded silica; Methanol-water (74: 26) is a mobile phase; The detection wavelength is 270nm.Number of theoretical plate calculates by the cryptotanshinone peak should be not less than 3000.

The preparation precision of reference substance solution takes by weighing cryptotanshinone and each 2.5mg of tanshinone reference substance, puts in the 100ml measuring bottle, adds methanol and is diluted to scale, shakes up, and promptly gets (every 1ml cryptotanshinone and each 25ug of tanshinone).

5 of this product are got in the preparation of need testing solution, remove film-coat, porphyrize, get 0.1g, the accurate title, decide, and precision adds methanol 25ml, claim to decide weight, supersound process (250W, 40Hz) 20 minutes, claim to decide weight again, replenish the weight that subtracts mistake, shake up with methanol, filter, the accurate suction measured subsequent filtrate 1ml, adds methanol and is diluted in the 5ml measuring bottle, shake up, filter with microporous filter membrane (0.45um), promptly.

Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid respectively, measure, promptly.

Every of this product contains Radix Salviae Miltiorrhizae with cryptotanshinone (C ₁₉H ₂₀O ₃) and tanshinone (C ₁₉H ₁₈O ₃) meter, all must not be less than 10mg.

Embodiment two

[method for making] gets Radix Salviae Miltiorrhizae, is ground into coarse powder, adds 90% alcohol reflux three times, adds 6 times of amounts for the first time, reflux, extract, 1.5 hours, and second and third time adds 4 times of amounts respectively, reflux, extract, 1 hour, merge extractive liquid, filters (50 ℃～60 ℃ of decompression recycling ethanols;-0.08Mpa) and be condensed into the thick extractum of 1.30 (50 ℃), add 4 times of water gagings (80 ℃), stir evenly, filter, get precipitation, reduce pressure (55 ℃～60 ℃;-0.08Mpa) drying is pulverized, and gets extract fine powder 280g, and the amount of PEG400 is 300g, propylene glycol 20g, and to total amount 600g, the compacting soft capsule, promptly.

1 of this product is got in [discriminating] (1), adds methanol 20ml, and supersound extraction 10 minutes filters, and gets subsequent filtrate 10ml, evaporate to dryness, residue add water 20ml makes dissolving, uses ether extraction 3 times, each 20ml, merge ether solution, evaporate to dryness, residue add methanol 1ml dissolving, as need testing solution.Other gets Radix Salviae Miltiorrhizae IIA and cryptotanshinone reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution.According to the thin layer chromatography test, draw need testing solution and each 5ul of reference substance solution, put respectively on same silica gel g thin-layer plate, be developing solvent with toluene-ethyl acetate (19: 1), launch, take out, dry.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

[assay] is according to high effective liquid chromatography for measuring.

Chromatographic condition and system suitability test are filler (φ * L=4.6 * 250mm, granularity 5 μ m) with octadecylsilane chemically bonded silica; Methanol-water (74: 26) is a mobile phase; The detection wavelength is 270nm.Number of theoretical plate calculates by the cryptotanshinone peak should be not less than 3000.

The preparation precision of reference substance solution takes by weighing cryptotanshinone and each 2.5mg of tanshinone reference substance, puts in the 100ml measuring bottle, adds methanol and is diluted to scale, shakes up, and promptly gets (every 1ml cryptotanshinone and each 25ug of tanshinone).

5 of this product are got in the preparation of need testing solution, get content, mixing, get 0.1g, the accurate title, decide, and precision adds methanol 25ml, claim to decide weight, supersound process (250W, 40Hz) 20 minutes, claim to decide weight again, replenish the weight that subtracts mistake, shake up with methanol, filter, the accurate suction measured subsequent filtrate 1ml, adds methanol and is diluted in the 5ml measuring bottle, shake up, filter with microporous filter membrane (0.45um), promptly.

Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid respectively, measure, promptly.

Every of this product contains Radix Salviae Miltiorrhizae with cryptotanshinone (C ₁₉H ₂₀O ₃) and tanshinone (C ₁₉H ₁₈O ₃) meter, all must not be less than 10mg.

Claims (8)

1. each method of the system of a salviamiltiorrhizabung preparation, it is characterized in that this method comprises following processing step: red rooted salvia is pulverized, alcoholic solution with 70～95% extracts, filter, merging filtrate, decompression recycling ethanol also is condensed into thick paste, remove the hydrotrope with 70～90 ℃ of hot water wash, remaining extract dry is pulverized, and gets extract.

2. as the preparation method of claim 1 described red sage formulation, it is characterized in that red rooted salvia is ground into coarse powder, add 90% alcohol reflux three times, for the first time add 6 times of amounts, reflux, extract, 1.5 hours, second and third time adds 4 times of amounts respectively, reflux, extract, 1 hour, filter, merging filtrate, 50～60 ℃ of following decompression recycling ethanols and when being condensed into 50 ℃ relative density be 1.28～1.32 extractum, remove the hydrotrope with 80 ℃ of hot water wash, dry under 55～60 ℃ of conditions, pulverize, get extract.

3. the preparation method of the red sage formulation described in claim 2 is characterized in that the extract that will obtain at last adds the conventional adjuvant of pharmacy, makes clinical required dosage form, comprises tablet, granule, soft capsule, drop pill.

4. the preparation method of red sage formulation as claimed in claim 3 is characterized in that adjuvant used in the preparation can be one or more the compositions in starch, microcrystalline Cellulose, carboxymethylstach sodium, magnesium stearate and the Polyethylene Glycol.

5. the preparation method of red sage formulation as claimed in claim 4, it is characterized in that the extract that 1000 weight portion red rooted salvias make is added microcrystalline Cellulose 4 weight portions, carboxymethylstach sodium 3 weight portions, use 80% alcohol granulation, drying, granulate, add magnesium stearate 0.1 weight portion, mixing, compacting is in blocks, promptly.

6. one kind is used to monitor the method for quality that makes product as preparation method as described in the claim 3, it is characterized in that this method contains following qualitative identification method: it is an amount of to get extract of the present invention or preparation, add methanol 10ml, supersound extraction can fully be mixed in 10 minutes, mixture is filtered, get subsequent filtrate as need testing solution; Other gets Radix Salviae Miltiorrhizae IIA and cryptotanshinone reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution; According to the thin layer chromatography test, draw need testing solution and each 5ul of reference substance solution, put respectively on same silica gel g thin-layer plate, be developing solvent with 19: 1 toluene-ethyl acetates, launch, take out, dry; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

7. monitoring product method for quality as claimed in claim 6 is characterized in that this method also comprises following content assaying method:

Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; 68: 32 acetonitrile-water is a mobile phase; The detection wavelength is 270nm.Number of theoretical plate calculates by the cryptotanshinone peak should be not less than 3000;

The preparation precision of reference substance solution takes by weighing cryptotanshinone and each 2.5mg of tanshinone reference substance, puts in the 100ml measuring bottle, adds methanol and is diluted to scale, shakes up, promptly;

0.1g preparation of the present invention is got in the preparation of need testing solution, and accurate the title decides, and precision adds methanol 25ml, claim to decide weight, supersound process 30 minutes claims to decide weight again, replenish the weight that subtracts mistake with methanol, shake up, filter, the accurate suction measured subsequent filtrate 1ml, adding methanol is diluted in the 10ml measuring bottle, shake up, with the microporous filter membrane filtration of 0.45um, promptly;

Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid respectively, measure, promptly.

8. the red rooted salvia extract that makes by claim 1 or 2 described methods.