CN108379309A - A kind of extraction purification technology for treating lung cancer active component - Google Patents
A kind of extraction purification technology for treating lung cancer active component Download PDFInfo
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- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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Abstract
The technical program discloses a kind of extraction purification technology for treating lung cancer active component, what the treatment lung cancer active component was prepared as follows：It using Samson St Johswort as raw material, extracts, separation, purifies to get the treatment lung cancer active component of Er Evil Sampsoniones A, Samson St Johswort element E, xylocarpus granatum pyridine A compositions.
Description
Technical field
The invention belongs to biomedicine fields, are related to a kind of extraction purification technology for treating lung cancer active component, specifically
It is related to treating the extraction purification technology of lung cancer active component in a kind of Chinese medicine Samson St Johswort.
Background technology
Chinese medicine is the important component of drug from plant, animal, mineral.Early in initial stage Shang dynasty, people are just from Chinese medicine
Middle extraction prepares active component, for preventing and curing diseases, as Chinese medicine decoction is for oral administration or external application；The Ming Dynasty《Compendium of Materia Medica》It describes in detail
The process of camphor is prepared, purified with sublimed method etc.；《White ape note》It describes and extracts isolated crystalline crow from fresh radix aconiti agrestis
The method of head alkali；《Elementary Medicine》Middle to have recorded the process for preparing gallic acid from Chinese gall with fermentation method, this is in the world
The record of organic acid is prepared earliest.These all illustrate Ancient Times in China pharmacy man Chemistry for Chinese Traditional Medicine field outstanding contributions.According to state
Outer document is recorded, and German pharmacist fills in the morphine base that Tours is extracted from opium within 1805, it is considered to be in Chinese medicine first it is natural
Active constituent；In the early 1950s, being found that antihypertensive compositions reserpine from India traditional herbal medicine devilpepper；50 years 20th century
For latter stage, anticancer component vinblastine has been obtained from catharanthus roseus；The appearance of especially taxol is known as the 1990s
One of anticancer three greatly achievement in the world, is a kind of very promising anti-cancer drugs.In recent decades, with advanced section
The continuous development of technology, method for separating and analyzing, the extensive use of various chromatographic techniques, the extracting and developing of chemical composition of Chinese materia medica
With purification technique there has also been great progress, hydrophilic composition, micro constitutent, similar structures complex mixture ingredient separation
Through no longer difficult；The appearance of infrared, nuclear magnetic resonance, the wave spectrums new technology such as mass spectrographic simultaneously, make Structural Identification work tend to it is micro,
Quickly and accurate, the period of Study of Traditional Chinese Medicine chemical composition greatly shortens.
According to the literature, there are more than 8000 kinds of Chinese herbal medicine resource, more than 3000 kinds of ethnic group's medication, wherein big absolutely in China at present
Part is autonomic drug, and minority is animal drugs and mineral drug, and so abundant resource provides for further exploitation effective component of chinese medicine
Rich material base.But before the founding of the state, by the conditions such as the economic strength of entire country and scientific and technical integrated countermeasures system
Limitation, Chemistry for Chinese Traditional Medicine research substantially without what break through, more do not set up Chemistry for Chinese Traditional Medicine pharmaceuticals industry.Since the establishment of the nation, especially
Its is over nearly one, 20 year, and as all other cause of science, Chemistry for Chinese Traditional Medicine has welcome the booming new era.Ephedrine,
The industrial production of ten several tcm products such as rutin, cedilanid has carried out for many years, raw material --- the potato of steroid hormone class drug
The industrial production of taro sapogenin and its development of resources research more obtain huge achievement, not only ensure domestic demands, also largely
Outlet.Document report, at present in being related to more than 500 kinds of medicinal material, it was found that 8000 multiple compounds.Except original China
Shanghai institute of materia medica of the academy of sciences, Kunming plant institute, institute of Materia Medica,Chinese Academy of Medical Sciences and resources of medicinal plant develop institute
Outside, medical college in all parts of the country, hygiene department almost also all generally set up the research institution for being engaged in Chemistry for Chinese Traditional Medicine.Chinese medicine
In the process of the modernization of Chinese medicine play unprecedented effect, and as the compulsory course of Medical university.Nearly ten
Nian Lai has greatly pushed scientific circles of China with external with academic exchange in the ranks with carrying out for open policy
And personnel's contacts, Chemistry for Chinese Traditional Medicine are then to associate the most frequent, academic exchange the most with external personnel in pharmacy and chemical field
An active subject.This promotes the growth of studying team to play important function to improving China's research level.Currently, I
The paces of state's Chemistry for Chinese Traditional Medicine research work have been greatly speeded up, and research level is greatly enhanced, in addition the resource that China is abundant,
It is believed that the contribution of bigger is surely made to the healthy cause of the mankind in 21 century one.
Primary bronchogenic carcinoma of lung (abbreviation lung cancer) is to endanger one of principal disease of human health, and the incidence of lung cancer is close
In rising trend in China over year, when making a definite diagnosis, about 87% patient has belonged to late period, and most of patients has lost operative chance when finding
Or be reluctant to perform the operation, therefore treatment more limits to and curative effect is poor.Although new drug (gemcitabine, Japanese yew class, vinorelbine) and platinum
The third generation chemotherapy regimen curative effect that class constitutes jointly improves, and the survival rate of patient is still limited, although chemotherapy is being killed
It goes out, reduce tool in terms of tumour and have certain effect, but there is also the drawbacks such as damage human normal function, uncertain therapeutic efficacy be fixed.Chinese medicine
Medicine and Lung Cancer with Integrated Traditonal Chinese Westren Medicine, be intended to it is dialectical combined with the differentiation of disease, righting is combined with eliminating evil, improve symptom, improve existence
Quality stablizes lesion, extends life cycle and improves survival rate, and operation, chemicotherapy can be coordinated to reduce toxicity and raising
Clinical efficacy.The treatment work of lung cancer is extremely paid attention in countries in the world, with the fast development of 20th century science and technology, prevents lung
The research work of cancer has made great progress.From overall, treatment lung cancer has relied on merely the treatments such as operation excision, radioactive ray
Sense is insufficient, is difficult especially to prove effective to the treatment of medium and advanced lung cancer.
Chinese medicine thinks, lung cancer category " lung product ", " cough ", " hemoptysis ", " pectoralgia " scope.It primarily forms the reason is that the positive deficiency of vital energy
Damage, the heresy of imbalance of yin and yang, the six external factors which cause diseases are seized the opportunity and the criminal's of intrusion lung, lung qi retardance, a surname's drop mistake department, functional activity of QI being not smooth, and blood is obstructed, aqueous fortune
Row obstacle, Tianjin are gathered for phlegm, phlegm and qi stasis, qi depression to blood stasis, mental disorder ecchymosis, and heresy product in the heart, forms tangible long-pending block then.This disease disease position master
It is close with the Zang-Fu relationships such as spleen, kidney in lung.Always belong to asthenia in origin and asthenia in superficiality, deficiency of vial QI is this, and phlegm and blood stasis is mark, therefore righting
Qi-restoratives is its total principle of reatment, is aided with Eradicates phlegm, stagnation resolvation, its mark is removed in promoting the circulation of qi etc..Lung cancer disease is badly in need of effective medicine at present
Object.
Samson St Johswort is the herb of Hypericaceae hypericum Samson St Johswort Hypericum sampsonii Hance.Summer and autumn are adopted
It receives, cleans and dry or using fresh herb.Ingot steppe plant is perennial herb, 0.5~1 meter high.Fibrous root is very thin and short.Stem is cylindrical,
There are branch, entire body Glabrous.Single leaf interaction is to life, and the complete symphysis one of two leaf bases is like ship shape, and stem is through centre, blade
Oblong shape lanceolar.Cyme is taken out on autumn stem top, and bennet is very thin, petal 5, oval, yellow.Capsule is oval, and tool is yellowish-brown
Color body of gland.Seed is tiny.【Meridian distribution of property and flavor】It is pungent, bitter, it trembles with fear.The species are the poisonous plant that Chinese Plants spectrum data library is included,
Its toxicity is that herb is toxic.【Major function】It is clearing heat and detoxicating, clearing and activating the channels and collaterals, cooling blood and hemostasis.For infantile hyperpyrexia, dysentery, enteritis,
It spits blood, bleeding from five sense organs or subcutaneous tissue, irregular menstruation, leukorrhea；Traumatic hemorrhage, traumatic injury, mastitis, burn and scald, venomous snake bite are controlled in external application.Both at home and abroad
There is not yet treating the reports such as pertinent literature or the patent of lung cancer active component extraction purification technology in Samson St Johswort.
Invention content
The purpose of the present invention is to provide the extraction purification technologies that lung cancer active component is treated in a kind of Samson St Johswort.The present invention
It is achieved by the following technical solution：
Samson St Johswort used in the present invention is the complete of Hypericaceae hypericum Samson St Johswort Hypericum sampsonii Hance
Grass.
A kind of extraction purification technology for treating lung cancer active component, is achieved by the steps of:
（1）Samson St Johswort is taken, coarse powder is ground into, is solvent extraction 3 times with 70% ethyl alcohol, each soaking at room temperature is extracted 4 hours, 70% second
Alcohol dosage is 12 times of Samson St Johswort total weight, is filtered, and merging filtrate obtains extracting solution A；The dregs of a decoction after extraction are again molten with 30% ethyl alcohol
Agent is extracted 2 times, each heating and refluxing extraction 1.5 hours, and 30% ethanol consumption is 10 times of Samson St Johswort total weight, and filtration merges filter
Liquid obtains extracting solution B；The dregs of a decoction after extraction are solvent heating and refluxing extraction 1.5 hours with water again, and filtration, merging filtrate obtains extracting solution
E；
（2）By step（1）Obtained extracting solution A is concentrated under reduced pressure at 50 DEG C, the concentrate A that density is 1.20 is concentrated into, by step
（1）Obtained extracting solution B is concentrated under reduced pressure at 80 DEG C, is concentrated into the concentrate B that density is 1.20, concentrate A and concentrate B are closed
And obtain concentrate C；By step（1）Obtained extracting solution E is concentrated under reduced pressure at 80 DEG C, is concentrated into the concentrate C that density is 1.15；
（3）By step（2）95% ethyl alcohol is added in obtained concentrate C, makes ethyl alcohol weight accounting 60%, places 24 hours, filtration,
Filtrate is concentrated under reduced pressure at 50 DEG C, is concentrated into no ethyl alcohol, obtains concentrate D；
（4）By step（3）Obtained concentrate D is adsorbed by MCI gel chromatographic columns, is eluted with water, and elution amount is 5 chromatographies
Column volume, water elution discard, then with volume ratio 10:90、15:85、25:75 alcohol-water elutes successively, and total elution amount is
15 chromatography column volumes, eluent directly by the dry column of silica gel chromatographic column of chromatography column volume identical as MCI gel chromatographic columns,
The efflux of the 3-12 chromatography column volumes by the dry column of silica gel chromatographic column, recycling design are collected, low temperature drying obtains extract A；
（5）By step（2）Obtained concentrate C adds volume ratio 1:3 ethyl acetate-chloroforms extract 3 times, combining extraction liquid, place 8
Hour, filtration, filtrate recycling design, low temperature drying is to get extract B；
（6）By step（5）Obtained extract B is adsorbed by MCI gel chromatographic columns, is eluted with water, and elution amount is 5 chromatographies
Column volume, water elution discard, then with volume ratio 30:70 alcohol-water elution, elution amount are 5 chromatography column volumes, eluent
Directly by the dry column of silica gel chromatographic column of chromatography column volume identical as MCI gel chromatographic columns, collects and pass through the dry column of silica gel chromatographic column
The efflux of 3-4 chromatography column volumes, recycling design, low temperature drying obtain extract F；
（7）By step（4）Obtained extract A and step（6）Obtained extract F merges to get treatment lung cancer active component.
A kind of extraction purification technology for treating lung cancer active component, it is characterised in that the treatment lung cancer active component is same
Bis- Evil Sampsoniones A of Shi Hanyou, Samson St Johswort element E, xylocarpus granatum pyridine A.
A kind of extraction purification technology for treating lung cancer active component, it is characterised in that controlled with what the extraction purification technology obtained
It treats lung cancer active component Zhong bis- Evil Sampsoniones A, Samson St Johswort element E and the total content of xylocarpus granatum pyridine A accounts for the 65% of total effective parts
More than.
A kind of extraction purification technology for treating lung cancer active component, it is characterised in that controlled with what the extraction purification technology obtained
It treats lung cancer active component and can be used for preparing treatment lung-cancer medicament.
It is a kind of to treat lung cancer active component, it is characterised in that containing weight ratio be 12:6:1 bis- Evil Sampsoniones A, member
Precious grass element E, xylocarpus granatum pyridine A.
It is a kind of to treat lung cancer active component, it is characterised in that treatment lung cancer active component Zhong bis- Evil Sampsoniones A, Samson St Johswort
The total content of plain E and xylocarpus granatum pyridine A accounts for 65% of total effective parts or more.
The present invention establishes the extraction purification technology that lung cancer active component is treated in Samson St Johswort for the first time, using MCI gels-
The method that the dry column of silica gel is combined, especially silica gel dry column hydrous ethanol elution technique, realizing Samson St Johswort treatment lung cancer has
Prepared by the successful extraction purification for imitating position, it is 12 which contains weight ratio simultaneously:6:1 bis- Evil Sampsoniones A, member
Precious grass element E, xylocarpus granatum pyridine A.
Specific implementation mode
The extraction purification technology that lung cancer active component is treated in Samson St Johswort is done below by specific experiment example and embodiment
It further illustrates, but is not limited to the present invention.
Embodiment 1：The extraction purification of lung cancer active component is treated in Samson St Johswort
（1）Samson St Johswort is taken, coarse powder is ground into, is solvent extraction 3 times with 70% ethyl alcohol, each soaking at room temperature is extracted 4 hours, 70% second
Alcohol dosage is 12 times of Samson St Johswort total weight, is filtered, and merging filtrate obtains extracting solution A；The dregs of a decoction after extraction are again molten with 30% ethyl alcohol
Agent is extracted 2 times, each heating and refluxing extraction 1.5 hours, and 30% ethanol consumption is 10 times of Samson St Johswort total weight, and filtration merges filter
Liquid obtains extracting solution B；The dregs of a decoction after extraction are solvent heating and refluxing extraction 1.5 hours with water again, and filtration, merging filtrate obtains extracting solution
E；
（2）By step（1）Obtained extracting solution A is concentrated under reduced pressure at 50 DEG C, the concentrate A that density is 1.20 is concentrated into, by step
（1）Obtained extracting solution B is concentrated under reduced pressure at 80 DEG C, is concentrated into the concentrate B that density is 1.20, concentrate A and concentrate B are closed
And obtain concentrate C；By step（1）Obtained extracting solution E is concentrated under reduced pressure at 80 DEG C, is concentrated into the concentrate C that density is 1.15；
（3）By step（2）95% ethyl alcohol is added in obtained concentrate C, makes ethyl alcohol weight accounting 60%, places 24 hours, filtration,
Filtrate is concentrated under reduced pressure at 50 DEG C, is concentrated into no ethyl alcohol, obtains concentrate D；
（4）By step（3）Obtained concentrate D is adsorbed by MCI gel chromatographic columns, is eluted with water, and elution amount is 5 chromatographies
Column volume, water elution discard, then with volume ratio 10:90、15:85、25:75 alcohol-water elutes successively, and total elution amount is
15 chromatography column volumes, eluent directly by the dry column of silica gel chromatographic column of chromatography column volume identical as MCI gel chromatographic columns,
The efflux of the 3-12 chromatography column volumes by the dry column of silica gel chromatographic column, recycling design are collected, low temperature drying obtains extract A；
（5）By step（2）Obtained concentrate C adds volume ratio 1:3 ethyl acetate-chloroforms extract 3 times, combining extraction liquid, place 8
Hour, filtration, filtrate recycling design, low temperature drying is to get extract B；
（6）By step（5）Obtained extract B is adsorbed by MCI gel chromatographic columns, is eluted with water, and elution amount is 5 chromatographies
Column volume, water elution discard, then with volume ratio 30:70 alcohol-water elution, elution amount are 5 chromatography column volumes, eluent
Directly by the dry column of silica gel chromatographic column of chromatography column volume identical as MCI gel chromatographic columns, collects and pass through the dry column of silica gel chromatographic column
The efflux of 3-4 chromatography column volumes, recycling design, low temperature drying obtain extract F；
（7）By step（4）Obtained extract A and step（6）Obtained extract F merges to get treatment lung cancer active component.
Treat ingredient separation and the Structural Identification of lung cancer active component：
Treatment lung cancer active component is taken, methanol dissolving, 200-300 mesh silica gel mixed samples are dry, grind well, first pass through silicagel column color
Spectrum, in conjunction with polyamide column chromatography, gel column chromatography, isolates and purifies, 3 compounds is respectively obtained, by through physicochemical constant, matter
The methods of the spectroscopic datas such as spectrum and nuclear magnetic resonance chromatography, chemical reaction, confirm Wei bis- Evil Sampsoniones A respectively
(dioxasampsone A), Samson St Johswort element E (hyperisampsin E), xylocarpus granatum pyridine A（xylogranatopyridine
A）.It is detected through HPLC, treats the total content of lung cancer active component Zhong bis- Evil Sampsoniones A, Samson St Johswort element E and xylocarpus granatum pyridine A
65% or more, the Qie bis- Evil Sampsoniones A, Samson St Johswort element E, xylocarpus granatum pyridine A weight ratio for accounting for total effective parts are 12:6:1.
Embodiment 2：The extraction purification of lung cancer active component is treated in Samson St Johswort
（1）Samson St Johswort is taken, coarse powder is ground into, is solvent extraction 3 times with 70% ethyl alcohol, each soaking at room temperature is extracted 4 hours, 70% second
Alcohol dosage is 12 times of Samson St Johswort total weight, is filtered, and merging filtrate obtains extracting solution A；The dregs of a decoction after extraction are again molten with 30% ethyl alcohol
Agent is extracted 2 times, each heating and refluxing extraction 1.5 hours, and 30% ethanol consumption is 10 times of Samson St Johswort total weight, and filtration merges filter
Liquid obtains extracting solution B；The dregs of a decoction after extraction are solvent heating and refluxing extraction 1.5 hours with water again, and filtration, merging filtrate obtains extracting solution
E；
（2）By step（1）Obtained extracting solution A is concentrated under reduced pressure at 50 DEG C, the concentrate A that density is 1.20 is concentrated into, by step
（1）Obtained extracting solution B is concentrated under reduced pressure at 80 DEG C, is concentrated into the concentrate B that density is 1.20, concentrate A and concentrate B are closed
And obtain concentrate C；By step（1）Obtained extracting solution E is concentrated under reduced pressure at 80 DEG C, is concentrated into the concentrate C that density is 1.15；
（3）By step（2）95% ethyl alcohol is added in obtained concentrate C, makes ethyl alcohol weight accounting 60%, places 24 hours, filtration,
Filtrate is concentrated under reduced pressure at 50 DEG C, is concentrated into no ethyl alcohol, obtains concentrate D；
（4）By step（3）Obtained concentrate D is adsorbed by MCI gel chromatographic columns, is eluted with water, and elution amount is 5 chromatographies
Column volume, water elution discard, then with volume ratio 10:90、15:85、25:75 alcohol-water elutes successively, and total elution amount is
15 chromatography column volumes, eluent directly by the dry column of silica gel chromatographic column of chromatography column volume identical as MCI gel chromatographic columns,
The efflux of the 3-12 chromatography column volumes by the dry column of silica gel chromatographic column, recycling design are collected, low temperature drying obtains extract A；
（5）By step（2）Obtained concentrate C adds volume ratio 1:3 ethyl acetate-chloroforms extract 3 times, combining extraction liquid, place 8
Hour, filtration, filtrate recycling design, low temperature drying is to get extract B；
（6）By step（5）Obtained extract B is adsorbed by MCI gel chromatographic columns, is eluted with water, and elution amount is 5 chromatographies
Column volume, water elution discard, then with volume ratio 30:70 alcohol-water elution, elution amount are 5 chromatography column volumes, eluent
Directly by the dry column of silica gel chromatographic column of chromatography column volume identical as MCI gel chromatographic columns, collects and pass through the dry column of silica gel chromatographic column
The efflux of 3-4 chromatography column volumes, recycling design, low temperature drying obtain extract F；
（7）By step（4）Obtained extract A and step（6）Obtained extract F by weight proportion 3:1 merges to get treatment
Lung cancer active component.
Treat ingredient separation and the Structural Identification of lung cancer active component：
Treatment lung cancer active component is taken, methanol dissolving, 200-300 mesh silica gel mixed samples are dry, grind well, first pass through silicagel column color
Spectrum, in conjunction with polyamide column chromatography, gel column chromatography, isolates and purifies, 3 compounds is respectively obtained, by through physicochemical constant, matter
The methods of the spectroscopic datas such as spectrum and nuclear magnetic resonance chromatography, chemical reaction, confirm Wei bis- Evil Sampsoniones A respectively
(dioxasampsone A), Samson St Johswort element E (hyperisampsin E), xylocarpus granatum pyridine A（xylogranatopyridine
A）.It is detected through HPLC, treats the total content of lung cancer active component Zhong bis- Evil Sampsoniones A, Samson St Johswort element E and xylocarpus granatum pyridine A
75% or more, the Qie bis- Evil Sampsoniones A, Samson St Johswort element E, xylocarpus granatum pyridine A weight ratio for accounting for total effective parts are 18:9:1.
Embodiment 3：The extraction purification of lung cancer active component is treated in Samson St Johswort
（1）Samson St Johswort is taken, coarse powder is ground into, is solvent extraction 3 times with 70% ethyl alcohol, each soaking at room temperature is extracted 4 hours, 70% second
Alcohol dosage is 12 times of Samson St Johswort total weight, is filtered, and merging filtrate obtains extracting solution A；The dregs of a decoction after extraction are again molten with 30% ethyl alcohol
Agent is extracted 2 times, each heating and refluxing extraction 1.5 hours, and 30% ethanol consumption is 10 times of Samson St Johswort total weight, and filtration merges filter
Liquid obtains extracting solution B；The dregs of a decoction after extraction are solvent heating and refluxing extraction 1.5 hours with water again, and filtration, merging filtrate obtains extracting solution
E；
（2）By step（1）Obtained extracting solution A is concentrated under reduced pressure at 50 DEG C, the concentrate A that density is 1.20 is concentrated into, by step
（1）Obtained extracting solution B is concentrated under reduced pressure at 80 DEG C, is concentrated into the concentrate B that density is 1.20, concentrate A and concentrate B are closed
And obtain concentrate C；By step（1）Obtained extracting solution E is concentrated under reduced pressure at 80 DEG C, is concentrated into the concentrate C that density is 1.15；
（3）By step（2）95% ethyl alcohol is added in obtained concentrate C, makes ethyl alcohol weight accounting 60%, places 24 hours, filtration,
Filtrate is concentrated under reduced pressure at 50 DEG C, is concentrated into no ethyl alcohol, obtains concentrate D；
（4）By step（3）Obtained concentrate D is adsorbed by MCI gel chromatographic columns, is eluted with water, and elution amount is 5 chromatographies
Column volume, water elution discard, then with volume ratio 10:90、15:85、25:75 alcohol-water elutes successively, and total elution amount is
15 chromatography column volumes, eluent directly by the dry column of silica gel chromatographic column of chromatography column volume identical as MCI gel chromatographic columns,
The efflux of the 3-12 chromatography column volumes by the dry column of silica gel chromatographic column, recycling design are collected, low temperature drying obtains extract A；
（5）By step（2）Obtained concentrate C adds volume ratio 1:3 ethyl acetate-chloroforms extract 3 times, combining extraction liquid, place 8
Hour, filtration, filtrate recycling design, low temperature drying is to get extract B；
（6）By step（5）Obtained extract B is adsorbed by MCI gel chromatographic columns, is eluted with water, and elution amount is 5 chromatographies
Column volume, water elution discard, then with volume ratio 30:70 alcohol-water elution, elution amount are 5 chromatography column volumes, eluent
Directly by the dry column of silica gel chromatographic column of chromatography column volume identical as MCI gel chromatographic columns, collects and pass through the dry column of silica gel chromatographic column
The efflux of 3-4 chromatography column volumes, recycling design, low temperature drying obtain extract F；
（7）By step（4）Obtained extract A and step（6）Obtained extract F by weight proportion 1:3 merge to get treatment
Lung cancer active component.
Treat ingredient separation and the Structural Identification of lung cancer active component：
Treatment lung cancer active component is taken, methanol dissolving, 200-300 mesh silica gel mixed samples are dry, grind well, first pass through silicagel column color
Spectrum, in conjunction with polyamide column chromatography, gel column chromatography, isolates and purifies, 3 compounds is respectively obtained, by through physicochemical constant, matter
The methods of the spectroscopic datas such as spectrum and nuclear magnetic resonance chromatography, chemical reaction, confirm Wei bis- Evil Sampsoniones A respectively
(dioxasampsone A), Samson St Johswort element E (hyperisampsin E), xylocarpus granatum pyridine A（xylogranatopyridine
A）.It is detected through HPLC, treats the total content of lung cancer active component Zhong bis- Evil Sampsoniones A, Samson St Johswort element E and xylocarpus granatum pyridine A
55% or more, the Qie bis- Evil Sampsoniones A, Samson St Johswort element E, xylocarpus granatum pyridine A weight ratio for accounting for total effective parts are 10:5:2.
Experimental example 1：Treat the experimental study of lung cancer
1 material
1.1 medicine
Embodiment 1 treats lung cancer active component, embodiment 2 treats lung cancer active component, embodiment 3 treats lung cancer active component.
1.2 positive control medicine
Jin Xisu (hydrochloride for injection Hycamtin), Guizhou hanfang Pharmaceutical Co., Ltd, authentication code:Chinese medicines quasi-word
H20000428, specification:2mg/ bottles.
1.3 people's lung Small Cell Lung Cancer NCI-H446 cell strains
People's lung Small Cell Lung Cancer NCI-H446 cell strains are bought in Nanjing KaiJi Biology Science Development Co., Ltd,
Fostering requirement:Adhere-wall culture:
Culture medium:RPMI-1640+10% calf serums+1% are dual anti-
Digestive juice:0.25% trypsase
Frozen stock solution:Fetal calf serum:DMSO=9:1
Condition of culture:Carbon dioxide incubator: 5%CO2, 37 DEG C.
2 methods
2.1 medication
2.1.1 medicine ordinance sterilizes at the solution of concentration suitable concentration using autoclave sterilizer, and 4 DEG C of refrigerator storages are spare.
2.1.2 plain (hydrochloride for injection Hycamtin) human body of gold happiness is per consumption per day:1.2mg/m2。
The culture of 2.2 people's lung Small Cell Lung Cancer NCI-H446 cells
2.2.1 cellular identification
The cell strain received is identified, observes identification under inverted microscope by full-time pathology teacher, and combine Nanjing triumphant
The cell data that base biotechnology Development Co., Ltd provides, is accredited as NCI-H446 cell strains.
After cell strain receives, Tissue Culture Flask is excellent, and culture solution is without extravasation, and culture solution is without muddiness.It is micro- being inverted
Microscopic observation cell density, degree of converging are about 60%, the requirement for the degree of converging 80% that not up to cell passage requires, will be in culture bottle
Culture solution is sucked out, and 10 ml culture solutions is stayed to continue to cultivate, and round or shuttle is presented in cellular morphology.Cell confluency degree is super after having crossed 2d
After crossing 80%, passed on by cell passage requirement.Cell high malignancy, it is undifferentiated.Cancer cell size is small, core circle and oval
Shape, cancer cell endochylema is few, and caryoplasm ratio significantly increases, and core shape is irregular, and nuclear fission is common, and cancer cell boundary is unclear.
2.2.2 cell passes on
Continue to cultivate cell, when cell confluency degree reaches the requirement that cell passage is 80%, carries out cell passage.In NCI-
0.25% trypsase is added in H446 cell bottles, digests 3-6min, culture medium piping and druming is added, is fabricated to cell suspension by 1：2 pass
Generation.
It is passed on by cell culture condition, after 3 secondary cultures, cell one shares 8 bottles.Circle is presented in cellular morphology
Shape or shuttle, will wherein 4 bottles freeze.It is used for subsequent experimental by 2 bottles, in addition 2 bottles of continuation secondary cultures.
2.3 cell growth curves are drawn
2.3.1 cell sample-adding calculates cell concentration 2 times, obtains cell concentration average value.Then adding cell suspension equivalent
In each hole for entering culture plate, 4 holes are observed daily, and incubation time is calculated as 7d.
2.3.2 digestive juice is added every the culture solution for sucking 4 holes for 24 hours in observation index, and suspension cell counts cell
Number.Each hole counts 2 times, obtains cell concentration average value.
2.3.3 it is incubation time to draw cell growth curve abscissa, and ordinate is cell concentration, draws cell growth
Curve.Cell doubling time is about 26h.
2.4 cell growth inhibition assay
2.4.1 the cell of cell sample-adding logarithmic growth phase, is made cell suspension, then cell is added in cell suspension by cell
Tally is counted, and 5000/ml cell concentrations are formulated as, by its inoculating cell culture plate.96 porocyte culture plates are per hole
100 μ l are put into 37 DEG C, 5%CO2Incubator continues to cultivate.
2.4.2 it is cultivated by 24 h after adding method thereof cell sample-adding, 96 hole cells is taken out from carbon dioxide incubator
Culture plate is observing cellular morphology by inverted microscope, after determining that cell growth is good, empirically grouping with sampler per hole
Each 5 μ l of respective liquid are added.Every group of 4 multiple holes.
2.4.3 after detection method is cultivated using 24 h, 10 μ l of MTT solution is added in 96 porocyte culture plates and continue
Cultivate 4 h.Culture solution is sucked, is added and the same amount of DMSO of culture solution.Then 10min is vibrated, crystallization is made fully to dissolve.In enzyme
Absorbance value is measured in connection immune detector.
2.4.4 experiment packet tests the drug IC found out according to drug cell poison50Value, obtains the IC of drug50Where value
Administration concentration range.This experiment IC50Administration concentration range where being worth is between 1-100 μ g/ml.Therefore experiment packet is such as
Under:
Medicine group:Take the intermediate concentration (50 μ g/ml) for the treatment of lung cancer active component group；
Blank control group (only adds cell culture fluid), totally 4 big group, each group of each 4 multiple holes.
2.4.5 observation index measures absorbance value in microplate reader, and measurement wavelength is 490nm, measures OD numerical value.It goes forward side by side
Row morphological observation.
2.4.6 cell growth inhibition assay chart cell survival rate (%)=experimental group absorbance value/control group light is drawn
Absorption value × 100%, control group is only plus the cell culture fluid of equivalent is not added with any drug.Cell proliferation inhibition rate (%)=(1-
Cell survival rate) × 100%.Using the time as horizontal axis, using cell proliferation inhibition rate as the longitudinal axis, cell growth inhibition assay figure is drawn
Table.
2.5 experiment conditions control all experimentss and are completed in Shandong central laboratory of medical college, cell culture and correlation
Detection is completed in its cell culture chamber.
Comparison in difference is used two-by-two for 2.6 statistical procedures method variance analysesLSD- t is examined, and is compared between the two with mean ratio
Compared with.Statistics software application MicrosoftExcel2000 is for statistical analysis.
3 results
3.1 various concentration embodiments 1 treat inhibited proliferation of the lung cancer active component to NCI-H446 cells
The cancer cell number that increases for treating lung cancer active component concentration with embodiment 1 significantly reduces.NCI-H446 cells are low
When 20 μ g/ml of concentration, the most cancer cells of cell quantity are mutually overstock and show deformation state.NCI-H446 cells are higher
When 50 μ g/ml of concentration, cancer cell is in oval, fusiformis；Core is big, endochylema is few；Kernel unobvious, nuclear fission unobvious.NCI-
In 75 μ g/ml of higher concentration, cell quantity continues to reduce H446 cells, and cancer cell is unclear in structure.
The proliferation inhibition rate of 3.2 pairs of NCI-H446 cells
Embodiment 1 is treated lung cancer active component effect 48h and is significantly inhibited to the proliferation of NCI-H446 cells, with
The extension inhibiting effect of action time gradually weakens, and with the increase of dosage, inhibiting effect enhancing.Embodiment 2 is treated
Lung cancer active component and embodiment 3 treat lung cancer active component effect 48h and inhibit to make without apparent to the proliferation of NCI-H446 cells
With no difference of science of statistics.
Have in vitro to people's lung Small Cell Lung Cancer NCI-H446 cells the result shows that embodiment 1 treats lung cancer active component
There is inhibiting effect, there is potential medical value.
Claims (6)
1. a kind of extraction purification technology for treating lung cancer active component, it is characterised in that prepare as follows:
（1）Samson St Johswort is taken, coarse powder is ground into, is solvent extraction 3 times with 70% ethyl alcohol, each soaking at room temperature is extracted 4 hours, 70% second
Alcohol dosage is 12 times of Samson St Johswort total weight, is filtered, and merging filtrate obtains extracting solution A；The dregs of a decoction after extraction are again molten with 30% ethyl alcohol
Agent is extracted 2 times, each heating and refluxing extraction 1.5 hours, and 30% ethanol consumption is 10 times of Samson St Johswort total weight, and filtration merges filter
Liquid obtains extracting solution B；The dregs of a decoction after extraction are solvent heating and refluxing extraction 1.5 hours with water again, and filtration, merging filtrate obtains extracting solution
E；
（2）By step（1）Obtained extracting solution A is concentrated under reduced pressure at 50 DEG C, the concentrate A that density is 1.20 is concentrated into, by step
（1）Obtained extracting solution B is concentrated under reduced pressure at 80 DEG C, is concentrated into the concentrate B that density is 1.20, concentrate A and concentrate B are closed
And obtain concentrate C；By step（1）Obtained extracting solution E is concentrated under reduced pressure at 80 DEG C, is concentrated into the concentrate C that density is 1.15；
（3）By step（2）95% ethyl alcohol is added in obtained concentrate C, makes ethyl alcohol weight accounting 60%, places 24 hours, filtration,
Filtrate is concentrated under reduced pressure at 50 DEG C, is concentrated into no ethyl alcohol, obtains concentrate D；
（4）By step（3）Obtained concentrate D is adsorbed by MCI gel chromatographic columns, is eluted with water, and elution amount is 5 chromatographies
Column volume, water elution discard, then with volume ratio 10:90、15:85、25:75 alcohol-water elutes successively, and total elution amount is
15 chromatography column volumes, eluent directly by the dry column of silica gel chromatographic column of chromatography column volume identical as MCI gel chromatographic columns,
The efflux of the 3-12 chromatography column volumes by the dry column of silica gel chromatographic column, recycling design are collected, low temperature drying obtains extract A；
（5）By step（2）Obtained concentrate C adds volume ratio 1:3 ethyl acetate-chloroforms extract 3 times, combining extraction liquid, place 8
Hour, filtration, filtrate recycling design, low temperature drying is to get extract B；
（6）By step（5）Obtained extract B is adsorbed by MCI gel chromatographic columns, is eluted with water, and elution amount is 5 chromatographies
Column volume, water elution discard, then with volume ratio 30:70 alcohol-water elution, elution amount are 5 chromatography column volumes, eluent
Directly by the dry column of silica gel chromatographic column of chromatography column volume identical as MCI gel chromatographic columns, collects and pass through the dry column of silica gel chromatographic column
The efflux of 3-4 chromatography column volumes, recycling design, low temperature drying obtain extract F；
（7）By step（4）Obtained extract A and step（6）Obtained extract F merges to get treatment lung cancer active component.
2. a kind of extraction purification technology for treating lung cancer active component according to claim 1, it is characterised in that described controls
It treats lung cancer active component and contains Er Evil Sampsoniones A, Samson St Johswort element E, xylocarpus granatum pyridine A simultaneously.
3. a kind of extraction purification technology for treating lung cancer active component according to claim 1, it is characterised in that with the extraction
The total content for the treatment of lung cancer active component Zhong bis- Evil Sampsoniones A, Samson St Johswort element E and xylocarpus granatum pyridine A that purification technique obtains
Account for 65% or more of total effective parts.
4. a kind of extraction purification technology for treating lung cancer active component according to claim 1, it is characterised in that with the extraction
The treatment lung cancer active component that purification technique obtains can be used for preparing treatment lung-cancer medicament.
5. a kind for the treatment of lung cancer active component, it is characterised in that containing weight ratio be 12:6:1 bis- Evil Sampsoniones A, ingot
Careless element E, xylocarpus granatum pyridine A.
6. a kind of according to claim 5 treat lung cancer active component, it is characterised in that treatment lung cancer active component Zhong bis- Evil
Sampsonione A, Samson St Johswort element E and the total content of xylocarpus granatum pyridine A account for 65% of total effective parts or more.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109180699A (en) * | 2018-09-08 | 2019-01-11 | 济南昊雨青田医药技术有限公司 | A kind of Leaves of Corylus heterophylla Fish. ex Bess active constituent technology of preparing |
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| CN1861101A (en) * | 2005-05-13 | 2006-11-15 | 中国科学院上海生命科学研究院 | Application of extractive of Hypericum sampsonii Hance to control RXR alpha acceptor target |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109180699A (en) * | 2018-09-08 | 2019-01-11 | 济南昊雨青田医药技术有限公司 | A kind of Leaves of Corylus heterophylla Fish. ex Bess active constituent technology of preparing |
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Application publication date: 20180810 |