CN104546952B - A kind of selaginella doederlleini active component and its production and use - Google Patents
A kind of selaginella doederlleini active component and its production and use Download PDFInfo
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- 238000004519 manufacturing process Methods 0.000 title abstract description 3
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- 235000011949 flavones Nutrition 0.000 claims abstract description 53
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Landscapes
- Medicines Containing Plant Substances (AREA)
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Abstract
The present invention discloses a kind of selaginella doederlleini active component and its production and use.This method is concentrated to give selaginella doederlleini liquid extract by the dregs of a decoction after selaginella doederlleini degreasing, with ethanol solution extraction.Take selaginella doederlleini liquid extract to cross macroporous absorbent resin after water redissolves, eluted with opposed polarity solution gradient, collection obtains selaginella doederlleini opposed polarity eluent, and antitumor component is obtained by tumour cell in-vitro screening respectively after concentration.The active component is enriched the flavones ingredient of selaginella doederlleini more than 70%.Obtained selaginella doederlleini active component can be made into various formulations by the method in pharmacy.Selaginella doederlleini active component preparation method provided by the invention, and prove that it has obvious inhibitory action to tumor cell lines such as lung cancer, leukaemia, colon cancer, nasopharyngeal carcinoma in vitro.Active component prepared by this method can be applied in antineoplastic or pharmaceutical composition is prepared.
Description
Technical field
The present invention relates to a kind of Chinese medical extract, more particularly to a kind of selaginella doederlleini active component preparation method and applications.
Background technology
Selaginella doederlleini is Selaginella（Selaginella）Plant selaginella doederleinii (Selaginella doederleinii
Hieron drying herb).Its main product is in Fujian, Taiwan, Guangdong and other places.Whole year can harvest, clean, using fresh herb or dry into
Medicine.With clearing heat and detoxicating, dispelling wind and eliminating dampness, hemostasis, anticancer and other effects.It is the cancer therapy drug commonly used in traditional Chinese medical science Fighting cancer club.It is modern
Pharmacological research is it has also been found that selaginella doederlleini or its prescription can strengthen cancer patient organism metabolism and reticuloendothelial system function, and not simultaneously
See the report of toxic side effect.
Research shows that its main component is biflavone compound：Amentoflavone（amentoflavone）, rubber tree pair
Flavones（heveaflavone）, 7,4 ', 7 ", the 4 " -0- of '-four methyl-amentoflavone（7, 4′, 7", 4"'-
tetra-0-methyl-amentoflavone）, delicaflavone etc..The flavones ingredient is efficient, low in selaginella doederlleini
Malicious anticancer active constituent, it is the active component for being worth further investigation exploitation.At present, to selaginella doederlleini active compound for anti tumor and
Component there is no the report that new drug development is studied, and have that material base is indefinite, and the drawbacks such as quality is uncontrollable, shortage prepares high content
The method and patent report of general flavone active component.
The content of the invention
It is an object of the invention to provide a kind of selaginella doederlleini active component.
It is a further object of the present invention to provide a kind of preparation method of above-mentioned selaginella doederlleini active component.
Another object of the present invention is to provide a kind of medicine or pharmaceutical composition containing above selaginella doederlleini active component.
Present invention also offers the preparation containing the selaginella doederlleini active component and the purposes of the component.
The object of the present invention is achieved like this, a kind of preparation method of described selaginella doederlleini antitumor component, its
It is characterised by comprising the following steps：（1）Degreasing：After the selaginella doederlleini of naturally dry is crushed, after 8 ~ 15 times of amount petroleum ether immersions
Backflow, filtering, volatilizes organic solvent and obtains the dregs of a decoction；（2）Constituents extraction：By step（1）The dregs of a decoction after selaginella doederlleini extraction, it is molten with ethanol
Liquid condensing refluxing extraction, extraction time is at least 1 ~ 3 time or extraction time is 1-3 times, and each extraction time is 1 ~ 3 hour,
Merge ethanol extract and be condensed into selaginella doederlleini liquid extract；（3）Column chromatography：Selaginella doederlleini liquid extract upper polyamide column after being redissolved with water,
Using water-ethanol solution system gradient elution；（4）Component category authentication：Step（3）Water-ethanol solution system gradient elution
6 opposed polarity positions are obtained, there are 2 components to contain flavone compound by detecting discovery；（5）Assay：Measure step
Suddenly（3）Obtain the content of the flavone compound of component.
The step（2）In every time ethanol extraction when, every time plus ethanol water amount is that 6-10 times of the dregs of a decoction is measured, preferably 8-
10 times of amounts.
The step（2）In, the volumes of aqueous ethanol percentage concentration 60%-85% that adds every time, or concentration is 60%-
85wt%, preferred volume percentage concentration 60%-85%, or preferred alcohol concentration are 70%-85wt%.
The step（3）In, water-ethanol liquor capacity percentage concentration gradient be respectively 0-10%, 20-30%, 40-50%,
60-70%, 80-90% and 100%, preferably 10%, 30%, 50%, 70%, 90% and 100%.
The step（4）In obtained 6 polar fractions, respectively Fr.1, Fr.2, Fr.3, Fr.4, Fr.5 and Fr.6,
Screened respectively by external activity, the blending ingredients for obtaining component Fr.4, Fr.5 or component Fr.4 and Fr.5 are anti-swollen for selaginella doederlleini
Tumor activity component.
The step（4）In in obtained 6 components Fr.4 and Fr.5 component general flavone contents account for selaginella doederlleini general flavone and contain
The 70-95% of amount.
The activity extract is extracted using the dregs of a decoction after selaginella doederlleini degreasing as initiation material, and extract solution filtering is dense
Contracting, column chromatography, ethanol water gradient elution, concentrate respectively, obtain 6 polar fractions, by anti tumor activity in vitro checking and
The assay of flavonoids, obtain the selaginella doederlleini active component that general flavone content accounts for selaginella doederlleini general flavone content 70-95%.
The medicine of selaginella doederlleini antitumor component and pharmaceutically acceptable carrier made from above-mentioned preparation method or
With other active medicines and pharmaceutically acceptable carrier.
Any pharmaceutically acceptable preparation is made in above-mentioned medicine or pharmaceutical composition.
The application of medicine or pharmaceutical composition in chemosensitive test is prepared made from above-mentioned preparation method.
Specifically, the present invention may also provide following technical scheme：
Flavones ingredient is mainly contained in the selaginella doederlleini activity extract of the present invention.Wherein, by weight percentage, flavones
The content of constituents is more than 70%, it is preferred that the content of flavones ingredient is 70%-95%.
The preparation method of selaginella doederlleini activity of the present invention, comprises the following steps：（1）Degreasing：By on the stone of naturally dry
After cypress crushes, with refluxing extraction after 8 ~ 15 times of amount petroleum ether immersions, filtering, volatilize organic solvent and obtain the dregs of a decoction；（2）Constituents extraction：
The dregs of a decoction after selaginella doederlleini is extracted, 8-10 times is added to measure the extraction of ethanol solution condensing reflux, extraction time is 1 ~ 3 time, extraction every time
Time is to merge within 1 ~ 3 hour ethanol extract to be condensed into liquid extract；（3）Column chromatography：Selaginella doederlleini liquid extract is upper poly- after being redissolved with water
Acid amides post, using ethanol water system（Ethanol content is respectively：0-10%, 20-30%, 40-50%, 60-70%, 80-90% and
100%）Gradient elution；（4）Component category authentication：Obtain 6 opposed polarity components（Fr.1, Fr.2, Fr.3, Fr.4, Fr.5,
Fr.6）.（5）Assay：Above-mentioned 6 components are determined using HPLC-DAD, it is found that Fr.4 may contain more than 8 flavonoids
Composition, its flavones content reach 60-80%, account for the 70-85% of medicinal extract general flavone content；Fr.5 less 2 biflavones containing polarized
Composition, general flavone content 70-95%, account for the 10-16% of medicinal extract general flavone content.Fr.4 and Fr.5 components are enriched selaginella doederlleini medicine
70-95% flavone compounds in material.External activity screens, it is found that Fr.4 and Fr.5 components are selaginella doederlleini antitumor components.
Selaginella doederlleini component Fr.4, Fr.5 and Fr.4 and Fr.5 blending ingredients of the present invention can be used as active component,
Acceptable auxiliary material in pharmacy is added, preparation is made according to the preparation method for the preparation recorded in pharmacy.
Selaginella doederlleini active component of the present invention is also used as one of active component, receives in addition pharmacy auxiliary
Material, preparation is made according to the preparation method for the preparation recorded in pharmacy.
Described preparation include parenteral solution, drip solution, powder-injection, granule, tablet, powder, oral agents, sugar coated tablet,
Film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, mouth containing agent, granule, pill, paste, sublimed preparation,
Spray, pill, disintegrant, mouth collapse agent, micropill etc..
The selaginella doederlleini active component and its preparation of the present invention can be applied in antineoplastic is prepared.
The invention has the advantages that：Hydrolysis kinetics method is that one kind is simple for process, recovery rate is high, can scale
Metaplasia is produced.
Embodiment
The substantive content and beneficial effect of the present invention is further illustrated below in conjunction with embodiment, the embodiment is only used for
The bright present invention rather than limitation of the present invention.
The preparation of the selaginella doederlleini active component of embodiment 1
（1）Degreasing：After the selaginella doederlleini of naturally dry is crushed, with 8 times of amount petroleum ethers（That is the weight of petroleum ether is selaginella doederlleini
8 times of weight after crushing）Refluxing extraction after immersion, filtering, volatilizes organic solvent and obtains the dregs of a decoction；（2）Constituents extraction：By selaginella doederlleini
The dregs of a decoction after degreasing, add the ethanol solution that 8 times of amount concentration expressed in percentage by volumes are 70%（The ethanol that i.e. concentration expressed in percentage by volume is 70% is molten
The weight of liquid is 8 times of the weight of the dregs of a decoction after selaginella doederlleini degreasing）Condensing reflux extracts, and extraction time is 1 time, and extraction time is
3 hours, merge ethanol extract and be condensed into liquid extract；（3）Column chromatography：Selaginella doederlleini liquid extract upper polyamide column after being redissolved with water,
It is respectively 10%, 30%, 50%, 70%, 90% and 100% aqueous solution using ethanol concentration expressed in percentage by volume content（That is absolute ethyl alcohol）System
Gradient elution, collects the eluent of opposed polarity respectively, and rotary evaporation flings to solvent, obtains the selaginella doederlleini group of 6 polar fractions
Point；（4）Component category authentication：6 opposed polarity positions obtained above, find that Fr.4 and Fr.5 component polarity is washed by detection
De- liquid contains flavone compound, and wherein Fr.4 may account for containing more than 8 flavones ingredients, its flavones content up to 78%
The 83% of medicinal extract general flavone content；Fr.5 less 2 biflavone ingredients containing polarized, general flavone content 92%, it is total to account for medicinal extract
The 15% of flavones content；（5）Assay：Detected by HPLC-DAD, Fr.4 and Fr.5 component flavonoid contents reach
The 70% of medicinal material general flavone content.
The preparation of the selaginella doederlleini active component of embodiment 2
（1）Degreasing：After the selaginella doederlleini of naturally dry is crushed, with refluxing extraction after 15 times of amount petroleum ether immersions, filtering, wave
Dry organic solvent obtains the dregs of a decoction；（2）Constituents extraction：By the dregs of a decoction after selaginella doederlleini degreasing, 10 times of amount ethanol solution condensing refluxes are added
Extraction, extraction time are 3 times, and each extraction time is 1 hour, merge ethanol extract and are condensed into liquid extract；（3）Column chromatography：
Selaginella doederlleini liquid extract upper polyamide column, the ethanol volume for using concentration expressed in percentage by volume to be configured to for 85% ethanol after being redissolved with water
Percentage concentration content is respectively 0%, 20%, 40%, 60%, 80% and 100% water solution system gradient elution, collects opposed polarity respectively
Eluent, rotary evaporation flings to solvent, obtains the selaginella doederlleini component of 6 polar fractions；（4）Component category authentication：Obtain 6
Opposed polarity position, find that 2 eluents contain flavone compound by detection, wherein Fr.4 may contain more than 8
Flavones ingredient, its flavones content account for the 70% of medicinal extract general flavone content up to 60%；Fr.5 less 2 biflavones containing polarized
The less 2 biflavone ingredient contents of flavone compound in composition and a small amount of Fr.4 components, wherein polarity are 70%, account for leaching
The 10% of cream general flavone content.The component general flavone content is 80%, accounts for the 19% of medicinal extract general flavone；（5）Assay：By
HPLC-DAD is detected, Fr.4 and Fr.5 component flavonoid contents reach the 88% of medicinal material general flavone content.
The preparation of the selaginella doederlleini active component of embodiment 3
（1）Degreasing：After the selaginella doederlleini of naturally dry is crushed, with refluxing extraction after 8 times of amount petroleum ether immersions, filtering, wave
Dry organic solvent obtains the dregs of a decoction；（2）Constituents extraction：By the dregs of a decoction after selaginella doederlleini degreasing, it is 85% to add 8 times of amount concentration expressed in percentage by volumes
Ethanol solution condensing reflux extracts, and extraction time is 3 times, and extraction time is 3 hours, merges ethanol extract and is condensed into stream leaching
Cream；（3）Column chromatography：Selaginella doederlleini liquid extract upper polyamide column after being redissolved with water, uses ethanol concentration expressed in percentage by volume to distinguish for content
For 10%, 30%, 50%, 70%, 90% and 100% water solution system gradient elution, the eluent of opposed polarity is collected respectively, rotates and steams
Solvent is removed in performance, obtains the selaginella doederlleini component of 6 polar fractions；（4）Component category authentication：6 opposed polarity positions are obtained, are passed through
Cross detection and find that 2 eluents contain flavone compound, wherein Fr.4 may be containing more than 8 flavones ingredients, and it is yellow
Ketone content accounts for the 85% of medicinal extract general flavone content up to 80%；Fr.5 less 2 biflavone ingredients containing polarized, general flavone content
For 95%, the 16% of medicinal extract general flavone content is accounted for；（5）Assay：Detected by HPLC-DAD, Fr.4 and Fr.5 component flavonoids
Compounds content reaches the 95% of medicinal material general flavone content.
The preparation of the selaginella doederlleini active component of embodiment 4
（1）Degreasing：After the selaginella doederlleini of naturally dry is crushed, with refluxing extraction after 8 times of amount petroleum ether immersions, filtering, wave
Dry organic solvent obtains the dregs of a decoction；（2）Constituents extraction：By the dregs of a decoction after selaginella doederlleini degreasing, it is 70% to add 8 times of amount concentration expressed in percentage by volumes
Ethanol solution condensing reflux extracts, and extraction time is 3 times, and each extraction time is 1 hour, merges ethanol extract and is condensed into stream
Medicinal extract；（3）Column chromatography：Selaginella doederlleini liquid extract upper polyamide column after being redissolved with water, uses ethanol concentration expressed in percentage by volume as content point
Not Wei 10%, 30%, 50%, 70%, 90% and 100% water solution system gradient elution, respectively collect opposed polarity eluent, rotation
Solvent is flung in evaporation, obtains the selaginella doederlleini component of 6 polar fractions；（4）Component category authentication：6 opposed polarity positions are obtained,
Finding that 2 eluents contain flavone compound by detection, wherein Fr.4 may be containing more than 8 flavones ingredients, its
Flavones content accounts for the 83% of medicinal extract general flavone content up to 79%；Fr.5 less 2 biflavone ingredients containing polarized, general flavone contain
Measure as 95%, account for the 16% of medicinal extract general flavone content；（5）Assay：Detected by HPLC-DAD, Fr.4 and Fr.5 component flavones
Kind compound content reaches the 93% of medicinal material general flavone content.
The anti-tumor biological screening test of embodiment 5：
By the tumour cell of logarithmic growth（Non-small cell lung carcinoma cell A549, human leukaemia K562, human colon cancer cell
SW480, KB cell CNE-1, human nasopharyngeal carcinoma cells CNE-2）With 0.25% Trypsin Induced, then use and contain 10%
（Volumn concentration）The RPMI1640 culture medium suspension cells of calf serum, cell suspension is made, and it is respectively that each tumour is dilute
Release to 1.0 × 105Individual/ml, then be seeded in 96 well culture plates, per the μ l of hole 200, in 37 DEG C, 5%CO2Incubator in train
24h is supported, then sucks each hole nutrient solution.
Selaginella doederlleini active component made from embodiment 4 is made into final concentration of 200,100,50,25,12.5 μ g/ml, simultaneously
If experimental group and blank control group are tested.The culture plate of above each group is placed in 37 DEG C simultaneously, 5%CO2Incubator in train
Support 48h.48h adds 1 mg/ml MTT of 50 μ l Fresh per hole backward（Methyl thiazoly tetrazolium assay）Solution, and at 37 DEG C,
5%CO2Under conditions of continue be incubated 4h；Supernatant is carefully removed, 100 μ l DMSO is added, fully dissolves MTT reduzates；
OD value of each hole at wavelength 492nm is determined on ELIASA（OD value）, growth of tumour cell inhibiting rate is calculated by formula.Suppression
Rate processed（%）=[1-（ODExperiment-ODBackground）/（ODBlank-ODBackground）]×100%.6 components of selaginella doederlleini are calculated by orign7.5 softwares
To the IC of people's difference tumour cell A549, K562, SW480, CNE-1 and CNE-2 inhibitory action50Value, wherein Fr.4, Fr.5.
And the result of Fr.4 and Fr.5 blending ingredients is as shown in table 1.
Selaginella doederlleini component Fr.4, Fr.5. of table 1 and Fr.4 and Fr.5 blending ingredients are to different JEG-3 Carbazole alkaloids
The IC of effect50Value（μg/ml）
The inhibitory action of selaginella doederlleini component Fr.4, Fr.5. and Fr.4 and Fr.5 blending ingredients to five kinds of tumour cells
Test result indicates that（Table 1）, selaginella doederlleini active component Fr.4 and Fr.5 is to Non-small cell lung carcinoma cell A549, human leukemia
K562, human colon cancer cell SW480, KB cell CNE-1, human nasopharyngeal carcinoma cells CNE-2 have good inhibiting effect.
Therefore, selaginella doederlleini active component Fr.4, Fr.5. and Fr.4 and Fr.5 blending ingredients can be used for preparing lung cancer, leukaemia, knot
The medicine of intestinal cancer, nasopharyngeal carcinoma.
The preparation of the pill of embodiment 6
Selaginella doederlleini active component made from Example 1（Fr.4 and Fr.5 blending ingredients）0.5g and 10.5g polyethylene glycol-
6000 is well mixed, heating melting, is moved to after material in dripping pill drip irrigation, decoction is dropped in 6-8 DEG C of atoleine, oil removing, is made
Dripping pill 400.
The preparation of the freeze drying powder injection of embodiment 7
Dalbergia heartwood oil 1.5g is taken, is added in the hydroxypropylβ-cyclodextrin of 13ml saturations, stirring and dissolving, is filtered, filtrate low temperature
The inclusion compound powder of dry dalbergia heartwood oil and hydroxypropylβ-cyclodextrin.Except above-mentioned dalbergia heartwood oil closes the inclusion compound of hydroxypropylβ-cyclodextrin
Outside powder, then selaginella doederlleini activity extract 0.5g, mannitol 5.5g, mosatil 0.9g and distillation made from Example 2
Water 2ml, after said components mix, freeze-drying, 300 are dispensed, is produced.
The preparation of the freeze drying powder injection of embodiment 8
Selaginella doederlleini active component made from Example 3（Fr.4 and Fr.5 blending ingredients）It is 0.5g, glucose 4.5g, thio
Sodium sulphate 0.9g and distilled water 1ml, after said components are well mixed, freeze-drying, 500 dispensed, is produced.
Claims (5)
1. a kind of preparation method of selaginella doederlleini antitumor component, it is characterised in that comprise the following steps：（1）Degreasing：Will be certainly
After the selaginella doederlleini so dried crushes, with being flowed back after 8 ~ 15 times of amount petroleum ether immersions, filter, volatilize organic solvent and obtain the dregs of a decoction；（2）Into
Divide extraction：By step（1）The dregs of a decoction after selaginella doederlleini extraction, are extracted, extraction time is at least 1 ~ 3 with ethanol solution condensing reflux
Secondary, each extraction time is 1 ~ 3 hour, merges ethanol extract and is condensed into selaginella doederlleini liquid extract；（3）Column chromatography：Selaginella doederlleini is flowed
Medicinal extract upper polyamide column after being redissolved with water, using water-ethanol solution system gradient elution；（4）Component category authentication：Step（3）
Water-ethanol solution system gradient elution obtain 6 opposed polarity positions, through detection discovery there are 2 components to contain flavonoids
Compound；（5）Assay：Determination step（3）Obtain the content of the flavone compound of component；
The step（2）In every time ethanol extraction when, every time plus ethanol water amount is that 8-10 times of the dregs of a decoction is measured；
The step（2）In, the volumes of aqueous ethanol percentage concentration added every time is 70%-85%；
The step（3）In, water-ethanol liquor capacity percentage concentration gradient is respectively 0-10%, 20-30%, 40-50%, 60-
70%th, 80-90% and 100%, preferably 10%, 30%, 50%, 70%, 90% and 100%；
The step（4）In obtained 6 polar fractions, respectively Fr.1, Fr.2, Fr.3, Fr.4, Fr.5 and Fr.6, respectively
Screened by external activity, the blending ingredients for obtaining component Fr.4, Fr.5 or component Fr.4 and Fr.5 are the antitumor work of selaginella doederlleini
Property component；
The step（4）In in obtained 6 components Fr.4 and Fr.5 component general flavone contents account for selaginella doederlleini general flavone content
70-95%。
2. selaginella doederlleini antitumor component made from the preparation method described in claim 1, it is characterised in that the activity carries
Taking thing is extracted using the dregs of a decoction after selaginella doederlleini degreasing as initiation material, extract solution filtering and concentrating, column chromatography, ethanol water
Gradient elution, concentrate respectively, obtain 6 polar fractions, by anti tumor activity in vitro checking and the assay of flavonoids, obtain
General flavone content accounts for selaginella doederlleini general flavone content 70-95% selaginella doederlleini active component.
A kind of 3. selaginella doederlleini antitumor activity group made from preparation method containing described in the pharmaceutically claim 1 of effective dose
Divide and pharmaceutically acceptable carrier or the medicine or medicine that are formed with other active medicines and pharmaceutically acceptable carrier
Composition.
4. medicine according to claim 3 or pharmaceutical composition, it is characterised in that be made any pharmaceutically acceptable
Preparation.
5. containing pharmaceutically prepared by medicine made from the preparation method described in the claim 1 of effective dose or pharmaceutical composition
Application in chemosensitive test medicine.
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| CN103450136A (en) * | 2013-08-19 | 2013-12-18 | 南京标科生物科技有限公司 | Method for extracting hinokiflavone from Cacumen Biotae |
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| CN103450136A (en) * | 2013-08-19 | 2013-12-18 | 南京标科生物科技有限公司 | Method for extracting hinokiflavone from Cacumen Biotae |
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