CN105663719A

CN105663719A - Application of cough and asthma treating Bulbophyllum calodictyon Schlecht tablets in preparation of medicine for inhibiting proliferation of histocyte lymphoma cells U937

Info

Publication number: CN105663719A
Application number: CN201610088736.8A
Authority: CN
Inventors: 张静
Original assignee: Jinan Xinshidai Medicine Science and Technology Co Ltd
Current assignee: Jinan Xinshidai Medicine Science and Technology Co Ltd
Priority date: 2016-02-17
Filing date: 2016-02-17
Publication date: 2016-06-15

Abstract

The invention belongs to the technical field of traditional Chinese medicines, in particular to application of cough and asthma treating Bulbophyllum calodictyon Schlecht tablets in preparation of a medicine for inhibiting the proliferation of histocyte lymphoma cells U937 and a preparation method of the cough and asthma treating Bulbophyllum calodictyon Schlecht tablets. The cough and asthma treating Bulbophyllum calodictyon Schlecht tablets are prepared from medicinal raw materials including 200 g of Bulbophyllum calodictyon Schlecht, 200 g of a Chinese fevervine herb, 100 g of Chinese mahonia stems, 100 g of marsdeniae tenacissimae caulis, 100 g of rhizoma bletillae, 100 g of giant knotweeds, 100 g of garden balsam stems and 100 g of many-flower solomonseal rhizomes and are prepared by adopting supercritical extraction, so that the rheum emodin content is greatly improved.

Description

Green and cough with asthma sheet suppresses the application in histocytic lymphoma's cell U937 cell proliferation in preparation

Technical field

The invention belongs to technical field of Chinese medicines, be specifically related to a kind of green and cough with asthma sheet is in preparing suppression histocytic lymphoma cell U937 cell proliferation application and green and cough with asthma sheet preparation method.

Background technology

Green and cough with asthma particulate level WS-10616(ZD-0616)-2002, it is recorded in country's standard for traditional Chinese medicines compilation internal medicine lung system (two) fascicle. It is made up as crude drug of little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, there is effect of nourishing YIN and moistening the lung, heat-clearing and toxic substances removing, removing stasis to stop bleeding, for the cough that hot dry criminal's lung causes, hectic fever, the disease such as night sweat.

In prior art, not yet there is the green and cough with asthma sheet report in the application suppressed in human tissue cell's lymphoma cell U937 cell proliferation in preparation, also extract preparation aspect there are no green and cough with asthma sheet and adopt the report of supercritical extraction, and traditional water decoction is boiled, the method for alcohol reflux, technique is coarse, backward, and impurity is many, causes that patient's consumption is excessive, be inconvenient to take, had a strong impact on this product and applied clinically.

Summary of the invention

Goal of the invention: it is an object of the invention to provide a kind of green and that cough with asthma sheet is in preparing suppression histocytic lymphoma cell U937 cell proliferation application.

Further object is that the preparation method that a kind of green and cough with asthma sheet are provided.

It is an object of the invention to by following scheme realization:

Green and cough with asthma sheet suppresses the application in histocytic lymphoma's cell U937 cell proliferation in preparation, described green and cough with asthma sheet is made up as crude drug of little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, described green and cough with asthma sheet preparation method comprises the steps: to take little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, joins CO₂In supercritical extraction device, ethanol is as entrainer, and it is 4-6%, extracting pressure 15-30MPa that entrainer accounts for the percent by volume of total extractant, temperature 30-50 DEG C, CO₂Flow l-3ml/g crude drug min, extraction time 700-800min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, dry, and tabletting makes 200, every tablet weight 0.50g.

Preferably, above-mentioned green and cough with asthma sheet suppresses the application in histocytic lymphoma's cell U937 cell proliferation in preparation, described green and cough with asthma sheet preparation method comprises the steps: to take little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, joins CO₂In supercritical extraction device, ethanol is as entrainer, and it is 5% that entrainer accounts for the percent by volume of total extractant, extracting pressure 25MPa, temperature 40 DEG C, CO₂Flow 2ml/g crude drug min, extraction time 750min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, dry, and tabletting makes 200, every tablet weight 0.50g.

In prior art, green and cough with asthma particulate oral, a 10g, 3-4 time on the one. Green and cough with asthma granule dosage is big. Green and the every tablet weight 0.50g of cough with asthma sheet adopting the inventive method to prepare, only needs 2 every time, within 1st, takes 3-4 time. Dose is greatly reduced when having more active component. This conclusion can pass through following it have been experienced that.

The comparison of emodin content in green and cough with asthma sheet prepared by test one, distinct methods

L, instrument and the reagent present invention be green and cough with asthma sheet: prepares by embodiment 1 method, uses 1000g crude drug, extracted makes 200, every tablet weight 0.50g. Former green and cough with asthma granule, according to WS-10616(ZD-0616) prepared by-2002 standard methods. Agilent1200 high performance liquid chromatograph; METTLERAE240 electronic analytical balance; Rheum emodin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).

2, method

Measure according to high performance liquid chromatography (Chinese Pharmacopoeia the 4th note on the use 15 of version in 2015).

Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; Methanol-0.1% phosphoric acid solution (80: 20) is mobile phase; Detection wavelength is 437nm. Number of theoretical plate calculates by rheum emodin peak should be not less than 4000.

It is appropriate that the preparation precision of reference substance solution weighs rheum emodin reference substance, adds methanol and makes every 1ml solution containing 10 μ g, to obtain final product.

The preparation of need testing solution takes that the present invention is green and cough with asthma sheet 10, finely ground, mixing, taking 0.1g, accurately weighed, the 20ml that adds water makes dissolving, add hydrochloric acid 2ml, shake up, be heated to reflux 30 minutes, cooling, adds chloroform 30ml, supersound process 10 minutes, proceed in separatory funnel, dividing and take chloroform solution, water liquid extracts 2 times with chloroform jolting again, each 15ml, combined chloroform liquid, being evaporated in 80 DEG C of water-baths, residue methanol dissolves and is transferred in 10ml measuring bottle, adds methanol dilution to scale, shake up, filter with microporous filter membrane (0.45 μm), take filtrate, to obtain final product.

The preparation of reference product need testing solution takes the green of comparison and cough with asthma granule 20g, finely ground, mixing, take 1g, accurately weighed, the 20ml that adds water makes dissolving, add hydrochloric acid 2ml, shake up, it is heated to reflux 30 minutes, cooling, add chloroform 30ml, supersound process 10 minutes, proceed in separatory funnel, divide and take chloroform solution, water liquid extracts 2 times with chloroform jolting again, each 15ml, combined chloroform liquid, 80 DEG C of water-baths are evaporated, residue methanol dissolves and is transferred in 10ml measuring bottle, add methanol dilution to scale, shake up, filter with microporous filter membrane (0.45 μm), take filtrate, obtain.

Algoscopy precision respectively draws reference substance solution and need testing solution, each 20 μ l of reference product need testing solution, injects chromatograph of liquid, measures, to obtain final product.

3, result

It is shown that the present invention is green and in cough with asthma sheet, the content of rheum emodin is 1.65-3.28mg/ sheet; And the content of rheum emodin is 1.62mg/ bag (every bag containing granule 10g) in former green and cough with asthma granule, each serving 2-4 times that emodin content is former granule 10g content of consumption 2, when dose reduces, emodin content improves a lot.

The studies above shows, adopts green and cough with asthma sheet prepared by preparation method of the present invention, and active constituent content is significantly larger than WS-10616(ZD-0616) the green and cough with asthma granule prepared of method recorded of-2002 standards.

Detailed description of the invention

Below in conjunction with specific embodiment, the present invention is further described, so that those skilled in the art knows more about the present invention, but this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to Examples below, and all technology realized based on foregoing of the present invention belong to the scope of the present invention.

Embodiment 1

Take little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, join CO₂In supercritical extraction device, ethanol is as entrainer, and it is 5% that entrainer accounts for the percent by volume of total extractant, extracting pressure 25MPa, temperature 40 DEG C, CO₂Flow 2ml/g crude drug min, extraction time 750min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, dry, and tabletting makes 200, every tablet weight 0.50g.

After testing, in finished product, the content of rheum emodin is 3.28mg/ sheet.

Embodiment 2

Take little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, join CO₂In supercritical extraction device, ethanol is as entrainer, and it is 4% that entrainer accounts for the percent by volume of total extractant, extracting pressure 15MPa, temperature 30 DEG C, CO₂Flow 1ml/g crude drug min, extraction time 900min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, dry, and tabletting makes 200, every tablet weight 0.50g.

After testing, in finished product, the content of rheum emodin is 1.65mg/ sheet.

Embodiment 3

Take little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, join CO₂In supercritical extraction device, ethanol is as entrainer, and it is 6% that entrainer accounts for the percent by volume of total extractant, extracting pressure 30MPa, temperature 60 C, CO₂Flow 3ml/g crude drug min, extraction time 800min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, dry, and tabletting makes 200, every tablet weight 0.50g.

After testing, in finished product, the content of rheum emodin is 2.08mg/ sheet.

Embodiment 4: green and cough with asthma sheet suppresses the experimentation data of human tissue cell's lymphoma cell U937 cell proliferation

1. experiment material

1.1 experiment cell strains

Human tissue cell's lymphoma cell U937 cell, Shandong University's laboratory cell storehouse, DMEM+10%FBS cellar culture.

1.2 Experimental agents

Drugs: the present invention is green and cough with asthma sheet: prepare by embodiment 1 method.

Medicinal liquid liquid storage: weigh that 100mg is green and cough with asthma sheet, is dissolved in 5ml dehydrated alcohol, 0.2 μm of frit, 500 μ ldoff pipe subpackages ,-20 DEG C of storages, and 0.2 μm of frit dehydrated alcohol is in order to the use of matched group simultaneously.

1.3 experiment reagents

DMEM (GIBCO company Cat.No.12100-061Lot.No.758137); Hyclone (Tian Hang bio tech ltd, Zhejiang Lot.No.100419); NaHC0₃(Shanghai hundred million chemical reagent company limited Cat.No.11810-033Lot.No.1088387 of a specified duration); Trypsin(AMRESCO company); EDTA(AMRESCO company); PenicillinGSodiumSalt(AMRESCO company 1); StreptomycinSulfate (AMRESCO); Dehydrated alcohol (Zibo Ya Dulan Trade Co., Ltd.); MTT (Biosharp lot number: 0793): PBS(laboratory autogamy);

1.4 experiment equipments

Lycra inverted microscope (Germany Leica model: DMIL); Visible-ultraviolet light microwell plate detector (MD company of U.S. model: SPECTRAMAX190); C0₂Incubator (FORMA model: 3111); Super-clean bench (safe and sound company of Su Jing group manufactures model: SW-CJ-ZFD);Pure water instrument (Sprlng company of U.S. model: S/N020579); Accurate pipettor (Gilson Inc of France model: P2); Electronic balance (Sai Duolisi company limited of Germany model: BT323S); Full-automatic high-pressure autoclave (SANYO company of Japan model: MLS-3020); Table electrothermal air dry oven (Shanghai precision experimental facilities company model: DHG9123A); Refrigerator (Siemens Company's model: KG18V21TI); Liquid nitrogen container (CBS model: 2001); Low speed centrifuge (Anting Scientific Instrument Factory, Shanghai's model: KA-1000); 0.2 μm of filter (MILLIPORE model: SLGP033RB); 1cm culture dish (NEST company), 96 well culture plates (NEST company); Cell counting count board; Centrifuge tube, pipet, Tips are some.

2. experimental technique

1) U937 cell DMEM+10%FBS is in 37 DEG C, 5%C0₂Carry out cellar culture (10cm culture dish), when Growth of Cells to logarithmic (log) phase, collect cell, discard culture fluid, PBS fine laundering 3 times, add 3ml0.25% trypsin-0.04%EDTA, after 37 DEG C of digestion 2min, it is added thereto in 5ml complete medium and reaction, is proceeded in centrifuge tube after piping and druming cell, 1000rpm is centrifuged 5min, adjusts concentration of cell suspension 3 × 10⁴Individual/ml.

2) entering in 96 well culture plates by cell kind, every hole adds cell suspension 180 μ l, culture plate put in cell culture incubator (37 DEG C, 5%C0₂) cellar culture.

3) according to cell growth status, general long to 50%-70%, add green and cough with asthma sheet solution, continue to cultivate 24h.

4) add 20 μ lMTT solution (5mg/ml, i.e. 0.5%MTT) after 24h, continue to cultivate 4h.

5) after 4h, buckle method removes supernatant, pats dry gently with absorbent paper, and every hole adds 200 μ l dimethyl sulfoxide, puts low-speed oscillation 10min on shaking table, makes crystal fully dissolve. The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm place.

6) arranging background (being not added with cell, only add culture fluid), control wells (cell, the medicine dissolution medium of same concentrations, culture fluid, MTT, dimethyl sulfoxide), often group sets 6 multiple holes simultaneously.

7) suppression ratio of cell is represented by result with medicine: cell proliferation suppression ratio (%)=(control wells OD value-dosing holes OD value), control wells OD value × 100%. Experiment repeats 3 times.

3. statistical disposition

Adopting the correlation analysis in MicrosoftExcel2007 software and Studentt inspection, data represent with mean ± S.D..

4. experimental result

Statistical result showed after mtt assay experiment, compare with matched group, when dosage reaches 5mg/ml, to U937 cell inhibitory effect variant (P < 0.05), dosage this difference when 10mg/ml has significance (P < 0.01), has pole significant difference (P < 0.001) when dosage reaches 15-20mg/ml.

Table 1 is green and cough with asthma sheet is to U937 cell inhibitory effect influence research (X ± SD)

Group	Drug level (mg/ml)	Suppression ratio (%)
			Matched group	0	0
1	5	9.76±4.06
			2	10	18.25±5.69*
3	15	26.35±6.86**
			4	20	34.26±9.95**

Note: compare with matched group, * P < 0.01; * P < 0.001.

5. experiment conclusion

Green and the cough with asthma sheet of the present invention can suppress U937 cell proliferation, reduces the Growth of Cells number of U937 cell, and this effect is dose dependent.

Claims

1. green and cough with asthma sheet suppresses the application in histocytic lymphoma's cell U937 cell proliferation in preparation, described green and cough with asthma sheet is made up as crude drug of little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, it is characterized in that, described green and cough with asthma sheet preparation method comprises the steps: to take little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, joins CO₂In supercritical extraction device, ethanol is as entrainer, and it is 4-6%, extracting pressure 15-30MPa that entrainer accounts for the percent by volume of total extractant, temperature 30-50 DEG C, CO₂Flow l-3ml/g crude drug min, extraction time 700-800min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, dry, and tabletting makes 200, every tablet weight 0.50g.

2. the green and cough with asthma sheet according to claim l suppresses the application in histocytic lymphoma's cell U937 cell proliferation in preparation, it is characterized in that, described green and cough with asthma sheet preparation method comprises the steps: to take little green splendid achnatherum 200g, fevervine 200g, Caulis Mahoniae 100g, Caulis Marsdeniae Tenacissimae 100g, Pseudobulbus Bletillae (Rhizoma Bletillae) 100g, Rhizoma Polygoni Cuspidati 100g, Herba speranskiae tuberculatae 100g, Rhizoma Polygonati 100g, joins CO₂In supercritical extraction device, ethanol is as entrainer, and it is 5% that entrainer accounts for the percent by volume of total extractant, extracting pressure 25MPa, temperature 40 DEG C, CO₂Flow 2ml/g crude drug min, extraction time 750min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, dry, and tabletting makes 200, every tablet weight 0.50g.