CN107132285A

CN107132285A - A kind of detection method of the Chinese medicine composition for the treatment of cancer

Info

Publication number: CN107132285A
Application number: CN201710302545.1A
Authority: CN
Inventors: 杨胜玉; 陈勇; 汤美健
Original assignee: CHENGDU DIAO GROUP TIANFU MEDICINE Co Ltd
Current assignee: CHENGDU DIAO GROUP TIANFU MEDICINE Co Ltd
Priority date: 2013-10-29
Filing date: 2013-10-29
Publication date: 2017-09-05
Also published as: CN104569169A

Abstract

The invention provides a kind of detection method of the Chinese medicine composition for the treatment of cancer, the traditional Chinese medicinal composition raw materials composition includes the taste Chinese medicine of motherwort, safflower, Chinese prickly ash, leech etc. 34.Detection method includes：Chromatographic condition：Using methanol-water as mobile phase, isocratic elution, the 320nm of Detection wavelength 250；It is prepared by reference substance solution：Eugenol reference substance is taken to be prepared into reference substance solution；It is prepared by need testing solution：Chinese medicine composition is taken, need testing solution is prepared into after being extracted with organic solvent.The inventive method specificity is strong, and precision, stability, repeatability are good.

Description

A kind of detection method of the Chinese medicine composition for the treatment of cancer

It is on October 29th, 2013, Application No. 201310520036.8, entitled " one kind the applying date that the application, which is, The divisional application of the application for a patent for invention of the detection method of the Chinese medicine composition for the treatment of cancer ".

Technical field

The present invention relates to a kind of detection method of Chinese medicine composition, and in particular to a kind of HPLC detection sides of Chinese medicine composition Method, belongs to technical field of Chinese medicine.

Background technology

Eugenol, molecular formula is C₁₀H₁₂O₂, colourless or pale yellow liquid has strong cloves fragrance, water insoluble.Pharmacology Research shows that eugenol has inhibitory action in 1: 8000~1: 16000 concentration to pathogenic fungus；1: 2000~1: During 8000 concentration, there is suppression to make the bacillus such as staphylococcus aureus and pneumonia, dysentery (will is congratulated low), large intestine, deformation, tuberculosis With；Rabbit vein injection can produce anesthesia, reduce blood pressure, respiration inhibition is acted on anticonvulsion etc.；Show through muscle specimen experiment There is strong antihistaminicum effect.In addition eugenol can also be used for perfume fragrance and various cosmetic essences and fragrance for detergents formula In, it can be also used for the allotment of flavoring essence.

Prior art to using HPLC methods the assay of eugenol more, the progress by mobile phase of the acid solution of organic solvent Elution, such as multitude meets [right《Chinese Pharmacopoeia》The discussion of eugenol content assaying method, new Chinese medicine in version cloves medicinal material in 2010 With clinical pharmacology, 2012,23 (5)：579-582] with the acetic acid of methanol -1% water (55: 45) be mobile phase to cloves in cloves medicinal material The content assaying method of phenol is improved.

The present invention provides a kind of content assaying method of eugenol in Chinese medicine composition of the treating cancer of specific composition, existing There is technology to have no report to this.

The content of the invention

It is an object of the invention to provide a kind of detection method of the Chinese medicine composition for the treatment of cancer.

The purpose of the present invention is achieved by the following technical solution：

A kind of detection method of the Chinese medicine composition for the treatment of cancer, this method includes：

Chromatographic condition：Using methanol-water as mobile phase, isocratic elution, Detection wavelength 250-320nm；

It is prepared by reference substance solution：Eugenol reference substance is taken to be prepared into reference substance solution；

It is prepared by need testing solution：Chinese medicine composition is taken, need testing solution is prepared into after being extracted with organic solvent；

Determine：Reference substance and need testing solution is taken to be injected separately into liquid chromatograph, according to high effective liquid chromatography for measuring.

Further, the mobile phase volume percentage is methanol: water=(50: 50)~(70: 30)；Further, methanol: Water=(55: 55)~(65: 45)；Further, methanol: water=60: 40.

Further, during prepared by reference substance solution, reference substance solution concentration is per 1ml reference substance solutions 20-80 containing eugenol μ g；Further, reference substance solution concentration is per the 1ml μ of 40-60 containing eugenol g；

Further, during prepared by need testing solution, the organic solvent is dichloromethane, chloroform, ethyl acetate, third The mixing that any one or a few in ketone, ether, n-butanol, methanol, 50% methanol aqueous solution, ethanol, Diluted Alcohol solution is constituted Solvent；Further, the organic solvent is methanol；Further, need testing solution concentration is suitable per 1ml need testing solutions In crude drug 0.01-0.1g；Further, need testing solution concentration is equivalent to crude drug 0.04g per 1ml.

Traditional Chinese medicinal composition raw materials of the present invention are constituted：

Further, the bulk drug composition of the Chinese medicine composition is：

Wherein, the Chinese prickly ash is that charcoal Chinese prickly ash, leech are that leech processed, Rhizoma Sparganii are that vinegar prepared RHIZOMA SPARGANII with vine-gar, rhizoma cyperi are vinegar prepared RHIZOMA CYPERI, myrrh Processed for vinegar, semen armeniacae amarae is that to fry semen armeniacae amarae, fennel seeds be that salt fennel(parched), excrementum pteropi are that to process excrementum pteropi, corydalis tuber be that vinegar is processed and prolonged to vinegar Hu Suo, cattail pollen be cattail pollen charcoal, frankincense be vinegar process, dried lacquer be forge dried lacquer, evodia rutaecarpa be liquorice beverage process evodia rutaecarpa, folium artemisiae argyi is processs Chinese mugwort Leaf.

" Chinese medicine composition " can be the composition that each bulk drug is obtained after crushed described in detection method, also may be used To be the extract obtained after each bulk drug is extracted through conventional method, can also be each bulk drug extracted through conventional method after again by The composite preparation that conventional formulation technique is prepared from, such as oral liquid, tablet, capsule, granule.

After the invention described above traditional Chinese medicinal composition raw materials are extracted, add following auxiliary materials and be prepared into oral liquid；The auxiliary material Including：Flavouring, sweetener, stabilization agent, thickener, dissolving adjuvant, pH adjusting agent, colouring agent, essence；The flavouring One or more in following：PVP, menthol, dipotassium glycyrrhizinate, malic acid, natrium malicum, tartaric acid, amber Acid, sodium succinate, acetic acid, glutamic acid, citric acid, sodium citrate, glucolactone, sodium chloride.The sweetener is selected from down One or more in stating：Sucrose, fructose, glucose, lactose, D-sorbite, maltitol, erythrose, Sucralose, honeybee Honey, saccharin, steviol glycoside extract, Abbas's sweet tea, acesulfame potassium, sweet protein, radix glycyrrhizae, honey element.Stabilizer may be selected from as follows It is one or more of：PVP, glycerine, arabo-ascorbic acid and its salt, edetic acid(EDTA), metaphosphoric acid etc..Thickener may be selected from following one kind Or it is several：Sodium carboxymethylcellulose, agar, PVP, polyvinyl alcohol, xanthans.Dissolving adjuvant may be selected from it is following a kind of or It is several：Polysorbate, polyethylene glycol, polyoxyethylene hardened castor oil, PVP, sodium hydroxide, polyoxyethylene sorbitan monoleate.PH is adjusted Agent is selected from following one or more of：Citric acid, sodium citrate, lactic acid, sodium hydroxide, hydrochloric acid, malic acid, natrium malicum, winestone Acid, butanedioic acid, acetic acid, gluconic acid, phosphoric acid etc..The auxiliary material of oral liquid is most preferably：1-5 parts by weight polyoxyethylene sorbitan monoleate, 1-5 weights Measure part honey element.

The oral liquor of the Chinese medicine composition comprises the following steps：

Step 1：Turtle shell is added water to cook 2-4 times, each 2-4 hours, merges extract solution, and filtration, filtrate concentration is decocted for turtle shell Liquid；

Step 2：Radix Angelicae Sinensis, Ligusticum wallichii, turmeric, galangal, Chinese cassia tree, cloves, asafoetide, dalbergia wood steam distillation are extracted 5-7 hours, Volatile oil and distillate are collected,

Step 3：Step 2 steam distillation is extracted the remaining dregs of a decoction and added water to cook 2-4 times with remaining crude drug, each 1-3 Hour, collecting decoction, filtration, filtrate is concentrated into clear cream 1,

Step 4：The clear cream 1 plus ethanol of step 3 to alcohol content reach 60-80%, and regulation pH value is 7.5~8.0, is stood, filter Cross, filtrate recycling ethanol, add water to obtain clear cream 2,

Step 5：The turtle shell decocting liquid of the step 1 of clear cream 2 in step 4, boils, lets cool, plus step 2 volatile oil and distillate And the customary adjuvant of oral liquid, add water and be stirred to dissolve, stand, oral liquid is made through common process in filtration, filtrate；

The step 2 and/or 4 standing are：0~5 DEG C of standing；

The auxiliary material of addition oral liquid is in the step 5：1-5 parts by weight polyoxyethylene sorbitan monoleate, 1-5 parts by weight honey elements.

Brief description of the drawings

Fig. 1 eugenol contrast solution chromatograms

Fig. 2 need testing solution chromatograms

Fig. 3 negative control solution chromatograms

Fig. 4 mobile phases investigate experimental result, and chromatographic peak A is eugenol chromatographic peak

A, the chromatogram of mobile phase 1；B, the chromatogram of mobile phase 2；C, the chromatogram of mobile phase 3；D, the chromatogram of mobile phase 4.

Fig. 5 Extraction solvents investigate experimental result, and chromatographic peak A is eugenol chromatographic peak

A, using methanol as the HPLC chromatogram of Extraction solvent；B, using 50% methanol aqueous solution as the HPLC chromatogram of Extraction solvent Figure；C, using ethanol as the HPLC chromatogram of Extraction solvent；D, using Diluted Alcohol solution as the HPLC chromatogram of Extraction solvent.

Experimental example 1

1 instrument and reagent

1.1 instrument：SSI1500 high performance liquid chromatographs (UVIS1000 detectors, CSchromPlus chromatographies system System)；The a ten thousandth electronic balances of Sai Duolisi CPA225D ten, Water purifirer ultrapure water machines, TU-1810 UV, visible lights Spectrophotometer.

1.2 reagent：Methanol is chromatographically pure, and remaining reagent is that analysis is pure, and water is ultra-pure water.

1.3 reference substance：Eugenol provides (lot number by Nat'l Pharmaceutical ＆ Biological Products Control Institute：110725-201112, for containing Measure fixed use).

1.4 sample：Embodiment 1 prepares oral liquid, lot number 130801,130802,130803, by the Chengdu Di Ao groups self-sufficient and strategically located region Medicine company limited company provides.

2 methods and result

2.1 method

2.1.1 chromatographic condition and system suitability test

Chromatographic column：Diamonsil-C18 (5 μm, 250 × 4.6mm)；

Mobile phase：Methanol-water (60: 40) isocratic elution；

Detection wavelength：280nm；

Flow velocity：1.0ml·min-1；

Column temperature：Room temperature；

2.1.2 prepared by reference substance solution：Precision weighs eugenol reference substance 50.46mg, puts in 100ml measuring bottles, plus methanol Scale is diluted to, is shaken up, precision measures 10ml, is put in 100ml measuring bottles, plus methanol is to scale, shakes up, and produces (50.46 μ g ml^-1)。

2.1.3 prepared by need testing solution：Precision measures sample 1ml, puts in 25ml volumetric flasks, plus methanol is to scale, shakes up, Filtration, takes subsequent filtrate, produces.

2.1.4 prepared by negative control solution：The negative sample of scarce eugenol is taken, is prepared by 2.1.3 lower need testing solutions Method prepares negative solution.

Under these conditions, other component chromatographic peaks can be kept completely separate in eugenol and sample, eugenol color adjacent thereto The separating degree of spectral peak meets the requirements, and number of theoretical plate (n) is calculated as more than 3000 by eugenol peak, and tailing factor (T) meets the requirements, And negative control solution in identical retention time without chromatographic peak.See Fig. 1-3.

2.2 method validation

2.2.1 mobile phase is investigated

Take sample (lot number 130801) by need testing solution is prepared under 2.1.3, eluted respectively with mobile phase 1-4, As a result Fig. 4 is seen.

Mobile phase 1：The glacial acetic acid solution of methanol -1% (55: 45)；

Mobile phase 2：Acetonitrile-water (35: 65)；

Mobile phase 3：Methanol-water (60: 40)；

Mobile phase 4：Methanol-water (70: 30).

From fig. 4, it can be seen that with methanol-water (60: 40) for mobile phase, the peak area of index components eugenol is maximum, and at this Under the conditions of in eugenol and sample other component chromatographic peak separating effects it is preferable.

2.2.2 Extraction solvent is investigated

Sample is extracted with methanol, 50% methanol solution, ethanol, four kinds of solvents of Diluted Alcohol solution respectively, extract solution Dissolved after being evaporated with methanol, be measured by 2.1.1 lower chromatographic conditions, as a result see Fig. 5.

As seen from Figure 5, using methanol as Extraction solvent, the peak area of index components eugenol is maximum, and fourth under this condition Other component chromatographic peak separating effects are preferable in fragrant phenol and sample.

2.2.3 the range of linearity is investigated

Precision weighs eugenol reference substance 15.21mg, puts in 100ml measuring bottles, plus methanol dissolves and is diluted to scale, shakes It is even, obtain reference substance solution (0.1581mgml^-1).Precision measures above-mentioned solution 1,2,3,4,5,6ml, and 10ml measuring bottles are put respectively In, plus methanol dilution is to scale, shakes up, the μ l of sample introduction 20, injection liquid chromatograph record chromatogram, determine its peak area respectively, And using peak area as ordinate, mapped by abscissa of reference substance sample size, standard curve is drawn, and calculate regression equation and be：Y =792244X+691, r=0.9998.

As a result show, in 0.3162~1.8972 μ g ranges, linear relationship is good, is shown in Table 1.

The linear relationship of table 1 investigates result

Sample size (μ g)	0.3162	0.6324	0.9486	1.2648	1.5810	1.8972
							Peak area	252298	498657	756586	1008757	1234758	1513758

2.2.4 precision test

Precision draws reference substance solution (50.46 μ gml-1) 20 μ l, repeats sample introduction 5 times, determines eugenol peak area, knot Fruit is shown in Table 2.

Test result indicates that：The precision of instrument is preferable.

The Precision test result of table 2

2.2.5 replica test

Same batch of six parts of sample (lot number 130801) is taken, by need testing solution is respectively prepared under 2.1.3, by 2.1.1 Lower chromatographic condition determines eugenol content, the results are shown in Table 3.

Test result indicates that：This method repeatability is good.

The replica test of table 3

2.2.6 stability test

Take need testing solution (lot number 130801) respectively at 0,2,4,6,8 hours, the μ l of sample introduction 20, eugenol in determination sample Peak area, the results are shown in Table 4.

Test result indicates that：Need testing solution is basicly stable in 8 hours.

The stability test of table 4

2.2.7 average recovery is tested

Sample (lot number 130801, content 0.7432mgml-1) about 0.5ml of six points of known contents is taken, precision is measured, It is accurate respectively to add 0.7388mgml^-1Reference substance storing solution 0.5ml, by the preparation method system of 2.1.3 lower need testing solutions Standby sample-adding reclaims test sample liquid, and sample introduction determines its content under 2.1.1 chromatographic conditions, calculates the rate of recovery, the results are shown in Table 5.

The average recovery result of the test of table 5

Test result indicates that：The average recovery measurement result of six experiments is between 95~105%, and its average value is 99.32%, RSD are 1.80%.

2.2.8 sample is determined

Different sample lots are taken, by preparing need testing solution under 2.1.3 and carry out contents by 2.1.1 lower chromatographic conditions Determine, the results are shown in Table 6.

The sample size of table 6 determines (n=2)

Lot number	Eugenol content (mgml-1)
		130801	0.74
120101	0.36
		130101	0.54

Embodiment

The oral liquid of embodiment 1

Bulk drug is constituted：

Preparation method：

The four traditional Chinese medicine material of the above 30, turtle shell is added water to cook 3 times (plus 9 times of water), 3 hours every time, collecting decoction, is filtered, filter Liquid is concentrated into (about 180ml) in right amount；Radix Angelicae Sinensis, Ligusticum wallichii, turmeric, galangal, Chinese cassia tree, cloves, asafoetide, dalbergia wood steam distillation are extracted 6 hours (plus 12 times of water), collect volatile oil and distillate is appropriate (about 50ml), and the dregs of a decoction and remaining 25-component add water to cook three Secondary, 2 hours (adding water 12 times), second and third time each 1.5 hours (adding water respectively 10 times, 8 times), collecting decoction, are filtered for the first time, Filtrate is concentrated into the clear cream that relative density is 1.15 (50~60 DEG C), plus ethanol makes alcohol content up to 70%, and regulation pH value is 7.5~ 8.0, (0~5 DEG C) 24 hours are stood, are filtered, filtrate recycling ethanol, it is the clear of 1.06 (50~60 DEG C) to add water to relative density Cream, adds turtle shell decocting liquid, boils 20 minutes, lets cool, plus volatile oil and distillate and polyoxyethylene sorbitan monoleate, honey element, adds water and stirs Mixing makes dissolving, stands (0~5 DEG C) 24~48 hours, filtration, and filtrate adds water to ormal weight (1000ml), embedding, sterilizing (105 DEG C, 60min), produce.Every 10ml.

Detection method：

Chromatographic condition：Using octadecylsilane chemically bonded silica as filler；Using 60: 40 methanol-water as flowing equality Elution, Detection wavelength 280nm, flow velocity 1.0ml/min, detection temperature is room temperature；

It is prepared by need testing solution：Oral liquid 1ml is taken, is placed in 25ml measuring bottles, plus methanol dilution is to scale, shakes up, with micro- (0.45 μm) filtration of hole filter membrane, takes subsequent filtrate, produces；

It is prepared by reference substance solution：Precision weighs eugenol reference substance 50.46mg, puts in 100ml measuring bottles, plus methanol dilution is extremely Scale, shakes up, and precision measures 10ml, puts in 100ml measuring bottles, plus methanol is to scale, shakes up, and produces (50.46 μ gml-1)；

Determine：Reference substance and each 20 μ l injections high performance liquid chromatograph of need testing solution are drawn respectively, according to high-efficient liquid phase color Spectrometry is determined, and is produced.

Embodiment 2

Bulk drug is constituted：

Preparation method：

The taste of the above 34, turtle shell is added water to cook 3 times, and 3 hours every time, collecting decoction, filtration, filtrate was concentrated into right amount； Radix Angelicae Sinensis, Ligusticum wallichii, turmeric, galangal, Chinese cassia tree, cloves, asafoetide, dalbergia wood steam distillation are extracted 6 hours, collect volatile oil and distillation Appropriate liquid, the dregs of a decoction and remaining 25-component are added water to cook three times, and 2 hours for the first time, second and third time each 1.5 hours, merges and decocts Liquid, filtration, filtrate is concentrated into the clear cream of relative density about 1.15 (50~60 DEG C), plus ethanol makes alcohol content up to 70%, adjusts pH It is worth for 7.5~8.0, stands 24 hours, filtration, filtrate recycling ethanol adds water to the clear of relative density about 1.06 (50~60 DEG C) Cream, adds turtle shell decocting liquid, boils 20 minutes, lets cool, plus volatile oil and distillate, and granule is made through common process.

Detection method：

Chromatographic condition：Using octadecylsilane chemically bonded silica as filler；Using 65: 35 methanol-water as flowing equality Elution, Detection wavelength 280nm, flow velocity 1.0ml/min, detection temperature is room temperature；

It is prepared by need testing solution：Granule is taken equivalent to crude drug 2g, plus methanol 20ml ultrasonic extraction 30min, extract solution Stand, filtering takes subsequent filtrate 1ml to be placed in 25ml measuring bottles, plus methanol dilution is to scale, shakes up, with miillpore filter (0.45 μm) Filtration, takes subsequent filtrate, produces；

Reference substance solution is prepared and assay method be the same as Example 1.

Claims

1. a kind of detection method of the Chinese medicine composition for the treatment of cancer, it is characterised in that this method includes：

It is prepared by reference substance solution：Eugenol is taken to be prepared into reference substance solution；

Determine：Reference substance and need testing solution is taken to be injected separately into liquid chromatograph, according to high effective liquid chromatography for measuring；

Wherein, the traditional Chinese medicinal composition raw materials composition is：

2. detection method as claimed in claim 1, it is characterised in that the bulk drug of the Chinese medicine composition, which is constituted, is：

3. detection method as claimed in claim 1, it is characterised in that the Chinese prickly ash is that charcoal Chinese prickly ash, leech are leech processed, Rhizoma Sparganii It is that vinegar prepared RHIZOMA CYPERI, myrrh are that vinegar is processed, semen armeniacae amarae is that to fry semen armeniacae amarae, fennel seeds be salt fennel(parched), five spirits for vinegar prepared RHIZOMA SPARGANII with vine-gar, rhizoma cyperi Fat is that to process excrementum pteropi, corydalis tuber be that to process corydalis tuber, cattail pollen be that cattail pollen charcoal, frankincense are that vinegar is processed, dried lacquer is forges dried lacquer, evodia rutaecarpa to vinegar to vinegar Evodia rutaecarpa, the folium artemisiae argyi processed for liquorice beverage are to process folium artemisiae argyi.

4. detection method as claimed in claim 1, it is characterised in that the mobile phase volume percentage is methanol: water=(50 : 50)~(70: 30).

5. detection method as claimed in claim 4, it is characterised in that the mobile phase volume percentage is methanol: water=(55 : 55)~(65: 45).

6. detection method as claimed in claim 5, it is characterised in that the mobile phase volume percentage is methanol: water=60: 40。

7. detection method as claimed in claim 1, it is characterised in that in prepared by reference substance solution, reference substance solution concentration is Per the 1ml reference substance solutions μ of 20-80 containing eugenol g.

8. detection method as claimed in claim 1, it is characterised in that in prepared by need testing solution, the organic solvent is two Chloromethanes, chloroform, ethyl acetate, acetone, ether, n-butanol, methanol, 50% methanol aqueous solution, ethanol, Diluted Alcohol solution In any one or a few constitute mixed solvent.

9. detection method as claimed in claim 8, it is characterised in that in prepared by need testing solution, the organic solvent is first Alcohol.

10. detection method as claimed in claim 8, it is characterised in that need testing solution concentration is per 1ml need testing solution phases When in crude drug 0.01-0.1g.