CN105434487A - Application of chimonanthus nitens leaf tablets in preparing medicine for inhibiting cell proliferation of melanoma cell B16 - Google Patents

Application of chimonanthus nitens leaf tablets in preparing medicine for inhibiting cell proliferation of melanoma cell B16 Download PDF

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Publication number
CN105434487A
CN105434487A CN201510984152.4A CN201510984152A CN105434487A CN 105434487 A CN105434487 A CN 105434487A CN 201510984152 A CN201510984152 A CN 201510984152A CN 105434487 A CN105434487 A CN 105434487A
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Prior art keywords
blade
chimonanthus nitens
chimonanthusn itens
itens oliv
cell proliferation
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李晶
杨志华
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Jinan Xinshidai Medicine Science and Technology Co Ltd
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Jinan Xinshidai Medicine Science and Technology Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2059—Starch, including chemically or physically modified derivatives; Amylose; Amylopectin; Dextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Biotechnology (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention belongs to the technical field of traditional Chinese medicines and particularly relates to an application of chimonanthus nitens leaf tablets in preparing a medicine for inhibiting cell proliferation of melanoma cell B16 and a preparation method of the chimonanthus nitens leaf tablets. The chimonanthus nitens leaf tablets are prepared by adopting 1500g of chimonanthus nitens leaves as an active pharmaceutical ingredient and through supercritical extraction, and the content of quercetin and kaempferol is greatly increased.

Description

Chimonanthusn itens Oliv. blade is preparing the application in check melanin oncocyte B16 cell proliferation
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to the preparation method of a kind of Chimonanthusn itens Oliv. blade at the application prepared in check melanin oncocyte B16 cell proliferation and Chimonanthusn itens Oliv. blade.
Background technology
Chimonanthusn itens Oliv. blade standard No. WS-11310 (ZD-1310)-2002, is recorded in national standard for traditional Chinese medicines compilation internal medicine internal medicine lung system (one) fascicle.Be made up as crude drug of leaf of Chimonanthus Nitens 1500g, there are relieving the exterior syndrome with drugs of pungent in flavor and cool in nature, effect of heat-clearing and toxic substances removing.For anemopyretic cold, heating, aversion to cold, pharyngalgia.
In prior art, not yet there is Chimonanthusn itens Oliv. blade at the report suppressing the application in B16 mouse melanoma cell line cell proliferation in preparation, also extract there are no Chimonanthusn itens Oliv. blade the report that preparation aspect adopts supercritical extraction, and the method for soak by water, extraction by steam distillation volatile oil, technique is coarse, backward, and impurity is many, causes patient's consumption excessive, be inconvenient to take, had a strong impact on this product and applied clinically.
Summary of the invention
Goal of the invention: the object of the present invention is to provide a kind of Chimonanthusn itens Oliv. blade preparing the application in check melanin oncocyte B16 cell proliferation.
Another object of the present invention is the preparation method providing a kind of Chimonanthusn itens Oliv. blade.
The object of the invention is by following scheme realize:
Chimonanthusn itens Oliv. blade is preparing the application in check melanin oncocyte B16 cell proliferation, and described Chimonanthusn itens Oliv. blade is made up as crude drug of leaf of Chimonanthus Nitens 1500g, and the preparation method of described Chimonanthusn itens Oliv. blade is made up of the following step: get leaf of Chimonanthus Nitens, join CO 2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 4-6%, extracting pressure 15-30MPa, temperature 30-50 DEG C, CO 2flow l-3ml/g crude drug min, extraction time 700-800min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol granule, and dry, tabletting, makes 500, the heavy 0.35g of every sheet.
Preferably, above-mentioned Chimonanthusn itens Oliv. blade is preparing the application in check melanin oncocyte B16 cell proliferation, and the preparation method of described Chimonanthusn itens Oliv. blade is made up of the following step: get leaf of Chimonanthus Nitens, join CO 2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 5%, extracting pressure 25MPa, temperature 40 DEG C, CO 2flow 2ml/g crude drug min, extraction time 750min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol granule, and dry, tabletting, makes 500, the heavy 0.35g of every sheet.
In prior art, Chimonanthusn itens Oliv. blade is oral, one time 6 ~ 7,3 times on the one.Chimonanthusn itens Oliv. blade dosage is large.The heavy 0.35g of the every sheet of the Chimonanthusn itens Oliv. blade adopting the inventive method to be prepared into, only needs 3 at every turn, within 1st, takes 3 times.Dose is greatly reduced under the condition with more active component.This conclusion can be proved by following test.
The comparison of Quercetin and kaempferol content in Chimonanthusn itens Oliv. blade prepared by test one, distinct methods
L, instrument and reagent Chimonanthusn itens Oliv. blade of the present invention: by the preparation of embodiment 1 method, use 1500g crude drug, makes 500 through extracting, the heavy 0.35g of every sheet.Former Chimonanthusn itens Oliv. blade, prepares according to WS-11310 (ZD-1310)-2002 standard method.Agilent1200 high performance liquid chromatograph; METTLERAE240 electronic analytical balance; Quercetin and kaempferol reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; Methanol-0.5% phosphoric acid solution (45: 55) is mobile phase; Determined wavelength is 365nm.Number of theoretical plate calculates all should be not less than 1500 by Quercetin, kaempferol peak respectively.
The preparation precision of reference substance solution takes Quercetin, kaempferol reference substance is appropriate, adds methanol respectively and makes the solution of every 1ml containing 6 μ g, 8 μ g, to obtain final product.
10, Chimonanthusn itens Oliv. blade of the present invention is got in the preparation of need testing solution, accurately weighed, porphyrize, get 0.60g, accurately weighed, add methanol reflux 2 times (50ml, 50ml), each 1 hour, filter, merging filtrate, residue adds methanol and washs in right amount, and filter, washing liquid is incorporated in filtrate, evaporate to dryness, the residue 30ml that adds water makes dissolving, with glacial acetic acid adjust ph to 3 ~ 4, adds ethyl acetate jolting and extracts 5 times, each 30ml, merge ethyl acetate liquid, evaporate to dryness, residue adds methanol makes dissolving in right amount, be transferred in 10ml measuring bottle, add methanol to scale, shake up, to obtain final product.
20, the Chimonanthusn itens Oliv. blade of contrast is got in the preparation of reference product need testing solution, accurately weighed, porphyrize, get 1.2g, accurately weighed, add methanol reflux 2 (50ml, 50ml), each 1 hour, filter, merging filtrate, residue adds methanol and washs in right amount, filter, washing liquid is incorporated in filtrate, evaporate to dryness, the residue 30ml that adds water makes dissolving, with glacial acetic acid adjust ph to 3 ~ 4, add ethyl acetate jolting and extract 5 times, each 30ml, merge ethyl acetate liquid, evaporate to dryness, residue adds methanol makes dissolving in right amount, be transferred in 10ml measuring bottle, add methanol to scale, shake up, obtain.
Algoscopy is accurate respectively draws reference substance solution and need testing solution, each 5 μ l of reference product need testing solution, injection liquid chromatography, measures, to obtain final product.
3, result
Result shows, in Chimonanthusn itens Oliv. blade of the present invention, the content of Quercetin and kaempferol is 220-362 μ g/ sheet; And the content of Quercetin and kaempferol is 36 μ g/ grains in former Chimonanthusn itens Oliv. blade, each serving the Quercetin of consumption 3 and kaempferol content be the 3-5 of former tablet 6 content doubly, when dose reduces, Quercetin and kaempferol content improve a lot.
Above-mentioned research shows, adopts Chimonanthusn itens Oliv. blade prepared by preparation method of the present invention, Chimonanthusn itens Oliv. blade prepared by the method that active constituent content is recorded higher than WS-11310 (ZD-1310)-2002 standard far away.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described, so that those skilled in the art more understands the present invention, but this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example, all technology realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1
Get leaf of Chimonanthus Nitens 1500g, join CO 2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 5%, extracting pressure 25MPa, temperature 40 DEG C, CO 2flow 2ml/g crude drug min, extraction time 750min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol granule, and dry, tabletting, makes 500, the heavy 0.35g of every sheet.
After testing, in finished product, the content of Quercetin and kaempferol is 362 μ g/ sheets.
Embodiment 2
Get leaf of Chimonanthus Nitens 1500g, join CO 2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 4%, extracting pressure 15MPa, temperature 30 DEG C, CO 2flow 1ml/g crude drug min, extraction time 800min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol granule, and dry, tabletting, makes 500, the heavy 0.35g of every sheet.
After testing, in finished product, the content of Quercetin and kaempferol is 220 μ g/ sheets.
Embodiment 3
Get leaf of Chimonanthus Nitens 1500g, join CO 2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 6%, extracting pressure 30MPa, temperature 60 C, CO 2flow 3ml/g crude drug min, extraction time 700min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol granule, and dry, tabletting, makes 500, the heavy 0.35g of every sheet.
After testing, in finished product, the content of Quercetin and kaempferol is 308 μ g/ sheets.
Embodiment 4: the experimentation data of Chimonanthusn itens Oliv. blade check melanin oncocyte B16 cell proliferation
1. experiment material
1.1 experiment cell strains
B16 mouse melanoma cell line cell, Shandong University's laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: Chimonanthusn itens Oliv. blade of the present invention: prepare by embodiment 1 method.
Medicinal liquid liquid storage: take 100mg Chimonanthusn itens Oliv. blade, is dissolved in 5ml dehydrated alcohol, 0.2 μm of frit, 500 μ ldoff pipe subpackages ,-20 DEG C of storages, and simultaneously 0.2 μm of frit dehydrated alcohol is in order to the use of matched group.
1.3 experiment reagent
DMEM (GIBCO company Cat.No.12100-061Lot.No.758137); Hyclone (Tian Hang bio tech ltd, Zhejiang Lot.No.100419); NaHC0 3(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 equipment
Lycra inverted microscope (German Leica model: DMIL); Visible-ultraviolet light microwell plate detector (MD company of U.S. model: SPECTRAMAX190); C0 2incubator (FORMA model: 3111); Super-clean bench (safe and sound Inc. of Su Jing group moulding number: SW-CJ-ZFD); Pure water instrument (Sprlng company of U.S. model: S/N020579); Accurate pipettor (French Gilson Inc model: P2); Electronic balance (German Sai Duolisi company limited model: BT323S); Full-automatic high-pressure autoclave (Japanese SANYO company model: MLS-3020); Table electrothermal air dry oven (the accurate experimental facilities company in Shanghai 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) B16 cell DMEM+10%FBS is in 37 DEG C, 5%C0 2carry out cellar culture (10cm culture dish), when Growth of Cells is to logarithmic (log) phase, collecting cell, discards culture fluid, PBS fine laundering 3 times, add 3ml0.25% trypsin-0.04%EDTA, after 37 DEG C of digestion 2min, add 5ml complete medium neutralization reaction wherein, proceeded in centrifuge tube after piping and druming cell, the centrifugal 5min of 1000rpm, adjustment concentration of cell suspension 3 × 10 4individual/ml.
2) enter in 96 well culture plates by cell kind, every hole adds cell suspension 180 μ l, culture plate put into cell culture incubator (37 DEG C, 5%C0 2) cellar culture.
3) according to cell growth status, generally grow to 50%-70%, add Chimonanthusn itens Oliv. blade 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, crystal is fully dissolved.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm place.
6) background (do not add cell, only add culture fluid) is set, control wells (the medicine dissolution medium of cell, same concentrations, culture fluid, MTT, dimethyl sulfoxide) simultaneously, often organizes the multiple hole of setting 6.
7) result represents with the suppression ratio of medicine to cell: cell proliferation suppression ratio (%)=(control wells OD value-dosing holes OD value), control wells OD value × 100%.Experiment repetition 3 times.
3. statistical disposition
Adopt 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 B16 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 Chimonanthusn itens Oliv. blade is to B16 cell inhibitory effect influence research (X ± SD)
Group Drug level (mg/ml) Suppression ratio (%)
Matched group 0 0
1 5 10.28±5.46
2 10 18.79±7.95*
3 15 29.41±12.75**
4 20 40.29±14.16**
Note: compare with matched group, * P<O.01; * P<0.001.
5. experiment conclusion
Chimonanthusn itens Oliv. blade of the present invention can suppress B16 cell proliferation, and reduce the Growth of Cells number of B16 cell, this effect is dose dependent.

Claims (2)

1. Chimonanthusn itens Oliv. blade is preparing the application in check melanin oncocyte B16 cell proliferation, described Chimonanthusn itens Oliv. blade is made up as crude drug of leaf of Chimonanthus Nitens 1500g, it is characterized in that, the preparation method of described Chimonanthusn itens Oliv. blade is made up of the following step: get leaf of Chimonanthus Nitens, join CO 2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 4-6%, extracting pressure 15-30MPa, temperature 30-50 DEG C, CO 2flow l-3ml/g crude drug min, extraction time 700-800min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol granule, and dry, tabletting, makes 500, the heavy 0.35g of every sheet.
2. the Chimonanthusn itens Oliv. blade according to claim l, preparing the application in check melanin oncocyte B16 cell proliferation, is characterized in that, the preparation method of described Chimonanthusn itens Oliv. blade is made up of the following step: get leaf of Chimonanthus Nitens, join CO 2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 5%, extracting pressure 25MPa, temperature 40 DEG C, CO 2flow 2ml/g crude drug min, extraction time 750min, obtains supercritical extract, and supercritical extract is added starch, 70% ethanol granule, and dry, tabletting, makes 500, the heavy 0.35g of every sheet.
CN201510984152.4A 2015-12-24 2015-12-24 Application of chimonanthus nitens leaf tablets in preparing medicine for inhibiting cell proliferation of melanoma cell B16 Withdrawn CN105434487A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103494878A (en) * 2013-10-08 2014-01-08 南京正亮医药科技有限公司 Preparation method and application of compound danshen tablets

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103494878A (en) * 2013-10-08 2014-01-08 南京正亮医药科技有限公司 Preparation method and application of compound danshen tablets

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
国家药品监督管理局: "《国家中成药标准汇编内科肺系（一）分册》", 1 December 2002 *
李莎莎 等: "山蜡梅化学成分及药理作用的研究进展", 《华西药学杂志》 *

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