CN109369589A - The synchronization extraction process of smoke tree flavonoid glycoside and its application - Google Patents
The synchronization extraction process of smoke tree flavonoid glycoside and its application Download PDFInfo
- Publication number
- CN109369589A CN109369589A CN201811191191.9A CN201811191191A CN109369589A CN 109369589 A CN109369589 A CN 109369589A CN 201811191191 A CN201811191191 A CN 201811191191A CN 109369589 A CN109369589 A CN 109369589A
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- Prior art keywords
- ethyl alcohol
- smoke tree
- fisetin
- myricetin
- purification
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
- A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/40—Separation, e.g. from natural material; Purification
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The present invention provides the methods for separating simultaneously and extracting fisetin and myricetin in smoke tree.Method of the invention has that process route is simple, step is few, can specificity and efficiently separation and Extraction fisetin and myricetin simultaneously.The present invention also provides the pharmaceutical composition, food additives composition and the cosmetic compositions that contain common smoketree extract prepared by the method for the present invention.
Description
Technical field
The invention belongs to the preparation of Chinese tradition drug purification and application fields.Specifically, the present invention relates to extract Huang simultaneously
The preparation method and applications of fisetin and myricetin in smoke tree.
Background technique
Smoke tree (Cotinus coggygria scop.) is Anacardiaceae smoke tree category (Cotinus L.) plant.Smoke tree with root,
Branch and leaf are used as medicine；Its is cool in nature, acrid flavour is bitter.With clearing heat and detoxicating, the effect of removing blood stasis and acesodyne.Although the plant is not used as medicinal material also
Into Chinese Pharmacopoeia, but in recent years, many pharmacodynamic experiments show that it has antibacterial action, antihepatitic activity and antifatigue effect etc..
Smoke tree contains the multiple efficacies ingredient such as flavones, polysaccharide, microelement, amino acid, and there is antifatigue, enhancing human body to exempt from
Epidemic disease power, the strong kidney of liver protection, anti-aging, the effect for reducing blood glucose, antitumor etc..Its effective component flavone compound such as paints Huang
Element can inhibit the proliferation of kinds of tumor cells and apoptosis-induced, such as inhibit prostate cancer PC3 and LNCaP cell, cancer of pancreas AsPC-
1 cell, lung cancer A549 cell, colon cancer cell line HT-29 proliferation, it is apoptosis-induced, inhibit Nasopharyngeal neoplasms and invasion and
The effect of chemotherapy, radio therapy sensitization.Principle active component is flavone compound in the stem and root of smoke tree, although flavone compound
It is lower in the content of smoke tree, but it is with very high pharmacological activity, wherein flavone compound contained by smoke tree mainly effectively at
It is divided into fisetin and myricetin, can be used for treating icterepatitis, the prevention and treatment diseases such as hypertension and influenza.In recent years, with state
Inside and outside scholar deepens continuously to extracting and developing and the pharmacological effect research of smoke tree effective component, and clinical application and research are also more next
It is more extensive, as fisetin is applied to the antineoplaston of lung cancer and prostate cancer.Studies have shown that the content of smoke tree general flavone by
Leaf-stem branch-root successively decline trend, since FOLIUM ET RAMULUS COTINI is containing Multiple components such as a large amount of tannin, volatile oil and pigments, total flavonoid at
Point extraction separation method is complex, it is therefore desirable to smoke tree stem, branch are subjected to profound extraction, processing, with improve its added value and
Resource utilization.
Contain a large amount of flavones ingredient in smoke tree, it is known that flavone compound is that one kind is present in the important of plant kingdom
Bioactive substance, according to research reports, it not only serves as the drug of prevention and treatment cardiovascular and cerebrovascular disease, and has apparent anti-grease matter
Peroxidating, anti-aging remove free radical, and reducing blood lipid reduces cholesterol, is hypoglycemic, and the physiology such as anti-cancer and cancer-preventing, immunological regulation are living
Property is a kind of drug for having broad prospect of application in terms of the nutrition of the mankind, health and disease preventing and treating and widely used
Natural inhibitor and preservative can be used for health food and cosmetics, therefore have good development and application values.
Chinese patent " new application and preparation of total flavone of effective component of Cotinus coggygria " (application number: CN200910021046.0) is public
A kind of method that smoke tree general flavone is extracted from smoke tree is opened, but the yield of smoke tree general flavone is very low, and cannot efficiently separate
Main active fisetin, the myricetin of general flavone show there is most flavones during extracting smoke tree general flavone
It is lost.The above prior art does not fully consider the chemical structure of each ingredient in smoke tree, using unreasonable extracting method,
It causes recovery rate relatively low or makes troubles to industrialized production.
Dispersive liquid-liquid microextraction technology (Dispersive liquid-liquid microextration, DLLME) is close
The new Liquid-liquid extraction technology (Assadi etc., 2006) of year exploitation.It utilizes the addition of dispersing agent, improves organic extraction
Dispersion of the agent in water phase is taken, phase-contact surface between organic extractant microballon and water sample is extended, so that analyte is fast
Speed is transferred in extractant, can reach extraction equilibrium in several seconds.DLLME technology is continued to develop and is improved, and occurs passing through
Extractant is rapidly injected extraction system, or by the modes such as stirring that are vortexed, so that quickly forming extraction in dispersing agent-water phase
Agent microballon, is suspended in solution.In DLLME, the selection of the type and cooperation of extractant and dispersing agent is extraction efficiency and richness
The important factor in order of collection degree.
This field needs the active component of a kind of pair of smoke tree and effective active composition to carry out specific aim extraction, and it is yellow to improve smoke tree
Ketone active constituent overall availability, and the method for the extract with excellent activity can be more effectively provided.
Summary of the invention
Aiming at the problems existing in the prior art, the purpose of the present invention is to provide two most important active yellows in smoke tree
The technical solution of the separating and extracting process of ketoside, i.e. fisetin and myricetin and application thereof.Method provided by the invention has work
Skill is easy to operate, production cost is low, recovery rate and purity is high, pollution-free, be easy to the advantages that industrialization production, thus obtained to mention
It takes object that there is fine anti-oxidant and anti-tumor activity, is suitble to be used as traditional Chinese chemical contrast, medical material, it is also possible to do health care
Product, food additives etc..
Specifically, the present invention provides a kind of method of fisetin and myricetin in separation and extraction smoke tree, including it is following
Step:
1) smoke tree stem branch is taken, is dried, crushed into powder after slice；
2) by dry smoke tree stem branch powder, with 10~30 times of smoke tree dried powder 50~80% ethyl alcohol of amount, pH is 8.0~
Twice, each 1~2h of extraction time obtains smoke tree stem branch alcohol extract to 10.0,60-80 DEG C of refluxing extractions, and rotary evaporation decompression is dense
After contracting, water bath method obtains alcohol extract medicinal extract；
3) the alcohol extract medicinal extract is dissolved in ethyl alcohol, after upper macroreticular resin, with 95% ethyl alcohol of column volume of 10~20BV
It is eluted, collects eluent, rotary evaporation is concentrated under reduced pressure, and water bath method is dry, obtains 95% ethyl alcohol column eluate, color
In light yellow；
4) 95% ethyl alcohol column eluate, 95% ethyl alcohol is dissolved, by polyamide column chromatography, with the chlorine of about 2:1
Imitative: eluent is collected in acetone elution；
5) chloroform for obtaining step 4): after acetone eluant is collected, about 50% aqueous methanol is added, vortex oscillation is mixed
It closes；About 90-98% ethyl alcohol is added, emulsion is mixed to form；Centrifugation is stood, and takes lower ethanol position, after drying is concentrated under reduced pressure,
Smoke tree purification is obtained, the smoke tree purification contains fisetin and myricetin.
In one aspect of the invention, the step 5) of the above method, pH is adjusted after about 50% aqueous methanol is added
It is about 8.0- about 10.0.
In one aspect of the invention, the step 5) of the above method, pH is adjusted after about 50% aqueous methanol is added
It is about 9.0.
In one aspect of the invention, the step 5) of the above method, the about 90-98% ethyl alcohol is about 95% second
Alcohol.
It is further comprising the steps of in one aspect of the invention, the above method:
By ethyl alcohol position in step 5) by polyamide column chromatography, with 60% ethanol elution, drying is concentrated under reduced pressure in eluent
Afterwards, fisetin is obtained.
It is further comprising the steps of in one aspect of the invention, the above method:
By ethyl alcohol position in step 5), after drying is concentrated under reduced pressure, it is dissolved in after a small amount of 95% ethyl alcohol through Sephadex LH-
20 column chromatographys obtain myricetin after drying is concentrated under reduced pressure in eluent with 95% ethanol elution.
In one aspect of the invention, a kind of Pharmaceutical composition is provided, it is prepared into comprising method as discussed above
The smoke tree purification arrived, and the pharmaceutically acceptable excipient being mixed with, such as diluent or carrier.Described medicinal group
Closing object can be used for treating tumour, such as glioma etc..
Pharmaceutical composition of the invention can use customary pharmaceutical excipients well known in the art, obtain through conventional method.For example,
The excipient that the composition for being intended to be administered orally may include is one or more colorants, sweetener, corrigent and/or preservative
Deng.
Pharmaceutical composition of the invention can be used be suitble to oral form (such as tablet, pastille, hard or soft capsule, water or
Oil suspension, emulsion, dispersible pulvis or granula, syrup or elixir), be suitble to the form of local use (such as creme, soft
Paste, gelling agent or water or oily solution or suspension), be suitable for inhalation into form (such as finely dispersed pulvis or the liquid of administration
Aerosol), be suitble to be blown into the form (such as finely dispersed pulvis) of administration or suitable parenteral form (such as with
In it is intravenous, subcutaneous, in peritonaeum or the sterile water of intramuscular administration or oily solution or for the suppository of rectally).
The present invention also provides a kind of food additives compositions, wherein the method containing with good grounds aforementioned present invention is prepared into
The smoke tree purification and food auxiliary material arrived, the smoke tree purification contain fisetin and myricetin.
The present invention also provides a kind of cosmetic compositions, wherein what the method containing with good grounds aforementioned present invention was prepared
Smoke tree purification and auxiliary material used for cosmetic, the smoke tree purification contain fisetin and myricetin.
Synchronous extraction provided by the invention separates fisetin in smoke tree, the method for myricetin has the following beneficial effects: system
Toxic solvent, non-environmental-pollution are not used in standby technique, have the characteristics that economic security, it is environmentally protective, be easy to industrialization production；
Using macroreticular resin decoloration removal of impurities processing after extraction, the interference that pigment purifies later separation is eliminated, using polyamide column color
Spectrum processing, avoids the interference of a large amount of polysaccharide and tannin in extract；Obtained fisetin and myricetin yield is higher, and purity is equal
Reach 90% or more；Include fisetin and myricetin in the purification being prepared, there is significant anti-oxidant and antitumor work
Property, there is good development and application prospect, can be used as traditional Chinese chemical contrast, medical material, health care product, food additives etc..
Percentage composition involved in present specification unless otherwise stated, refers both to weight percentage.
Detailed description of the invention
The HPLC/U analysis chart of the mixture obtained after D101 macroporous resin purification in Fig. 1 exemplary method technique of the present invention.
The HPLC/UV map of flavones standard reference material mixture contained by Fig. 2 smoke tree.It is yellow that it is followed successively by rutin, paint from left to right
Element, myricetin and Quercetin.
The HPLC/U analysis chart for the mixture that polyamide column obtains after purification in Fig. 3 exemplary method technique of the present invention.
By the HPLC/U analysis chart of the mixture obtained after abstraction purification in Fig. 4 exemplary method technique of the present invention.
The mixture obtained after abstraction purification is further processed in Fig. 5 exemplary method technique of the present invention to obtain paint Huang
The HPLC/U analysis chart of element.
The mixture obtained after abstraction purification is further processed to obtain red bayberry in Fig. 6 exemplary method technique of the present invention
The HPLC/U analysis chart of element.
The HPLC/U analysis chart for the mixture that polyamide column obtains after purification in Fig. 7 comparative examples method and process.
The HPLC/U analysis chart for the mixture that polyamide column obtains after purification in Fig. 8 comparative examples method and process.
By the HPLC/U analysis chart of the mixture obtained after abstraction purification in Fig. 9 comparative examples method and process.
Figure 10 Apoptosis by Flow Cytometry result figure.Common smoketree extract each component makees human glioma C6 cell
With.The area D1 (upper left corner) is non-viable non-apoptotic cell, and the area D2 (upper right corner) is non-viable non-apoptotic cell and apoptosis late cell, and the area D3 (lower right corner) is
Living cells, the area D4 (lower left corner) are apoptotic cell.
Figure 11 Apoptosis result figure.Common smoketree extract each component is to human glioma C6 cytosis.It is compareed with DMSO
Group, blank control group compare, P < 0.01 * P < 0.05, * *.
Figure 12 LDH activity testing result figure.Common smoketree extract each component is to human glioma C6 cytosis.With DMSO pairs
Compare according to group, blank control group, P < 0.01 * P < 0.05, * *.
Specific embodiment
Now in conjunction with the embodiment of the present invention and correlation test, the invention will be further described.
The separation of fisetin and myricetin and purifying process 1 in 1 smoke tree of embodiment
The processing of 1 > raw material: taking smoke tree stem branch, to dry after the slice of crosspiece face, is ground into powder, spare；
2 > refluxing extractions: by dry smoke tree stem branch powder 20g, with about the 70% of about 20 times of equivalent smoke tree dried powder amounts
Ethyl alcohol, adjusting pH is about 9.0, and twice, about 1.5h of each extraction time obtains smoke tree stem branch alcohol extract to 70 DEG C of refluxing extractions, and rotation is steamed
After hair method is concentrated under reduced pressure, water bath method obtains alcohol extract medicinal extract；
The decoloration removal of impurities processing of 3 > macroreticular resins: alcohol extract medicinal extract is dissolved in 50% ethyl alcohol, upper D101 macroreticular resin (China
Shanghai Resin Factory), it is eluted with 95% ethyl alcohol of the column volume of about 15BV, collects eluent, rotary evaporation is concentrated under reduced pressure,
Water bath method, it is dry, 95% ethyl alcohol macroporous resin column eluate is obtained, color is in light yellow, weighing.Dissolution carries out HPLC/U points
Analysis.As a result such as Fig. 1.
Fig. 2 is with rutin, fisetin, myricetin and (the rutin China pharmaceutical biological product calibrating of Quercetin standard reference material
Institute lot number 100080-200306；Fisetin Shanghai Yuan Ye Biotechnology Co., Ltd lot number 20121009；The general reputation in myricetin Shanghai
Scientific & trading Co., Ltd.'s lot number 140801；Quercetin Nat'l Pharmaceutical & Biological Products Control Institute lot number 100081-200406) do
HPLC/UV map (is followed successively by rutin, fisetin, myricetin and Quercetin) from left to right.
Compared with Fig. 2, Fig. 1 show in 95% ethyl alcohol macroporous resin column eluate containing rutin, fisetin, myricetin and
Other ingredients such as the flavone compounds such as Quercetin and tannin, volatile oil and pigment fail to reach purifying purpose.
4 > polyamide columns isolate and purify: about 80% ethyl alcohol of above-mentioned macroporous resin column eluate being dissolved, polyamide is passed through
Column chromatography, with the chloroform of 2:1: acetone elutes, and collects eluent；HPLC/U analysis.As a result such as Fig. 3.
Fig. 3 shows that polyamide column separation still fails to be kept completely separate and purify fisetin and myricetin.
5 > dispersive liquid-liquid microextractions (DLLME): 4 > chloroform of collection step: acetone eluant is transferred to centrifuge tube, is added
50% aqueous methanol of about 10 times of volumes, adjusting pH is about 9.0, vortex oscillation mixing；Add isometric about 95% ethyl alcohol, mixing
Form emulsion；Centrifugation is stood, and takes the extraction phase at 95% ethyl alcohol position of lower layer；After drying is concentrated under reduced pressure in extraction phase, it must crystallize
Common smoketree extract mixture 1.
It carries out HPLC/UV inspection to the extraction phase at 95% ethyl alcohol position to know, as a result as shown in Figure 4.
Fig. 4 is shown, in the chloroform for passing through polyamide column 2:1:, can by dispersive liquid-liquid microextraction after acetone elution
Efficiently separate and purify to obtain the extract containing fisetin and myricetin.
For further 5 > of verification step obtain ingredient, to the extraction phase further progress following steps at 95% ethyl alcohol position
To separate and purify fisetin and myricetin therein.
The extraction phase at 95% ethyl alcohol position is by polyamide column chromatography in 6 > step 5 >, with 60% ethanol elution, eluent
After drying is concentrated under reduced pressure, Light yellow crystals are obtained, are weighed, about 400mg.
HPLC/UV analysis is carried out to eluent, by passing through step compared with the HPLC/UV analysis chart of fisetin reference substance
The flavone component that rapid 5 > is obtained contains fisetin, equals or exceeds about 400mg.As a result it can refer to Fig. 5.
After crystalline mixture obtained in 7 > step 5 > is dissolved in a small amount of 95% ethyl alcohol, pass through Sephadex LH-20 column color
Spectrum obtains Light yellow crystals after drying is concentrated under reduced pressure in eluent with 95% ethanol elution, weighs, about 275mg.By with red bayberry
The HPLC/UV analysis chart of plain reference substance compares, and myricetin is contained in the flavone component obtained by step 5 >, is equaled or exceeded about
275mg.As a result it can refer to Fig. 6.
The separation of fisetin and myricetin and purifying process 2 in 2 smoke tree of embodiment
The processing of 1 > raw material: taking smoke tree stem branch, to dry after the slice of crosspiece face, is ground into powder, spare；
2 > refluxing extractions: by dry smoke tree stem branch powder 20g, with about the 70% of about 20 times of equivalent smoke tree dried powder amounts
Ethyl alcohol, adjusting pH is about 9.0, and twice, about 1.5h of each extraction time obtains smoke tree stem branch alcohol extract to 70 DEG C of refluxing extractions, and rotation is steamed
After hair method is concentrated under reduced pressure, water bath method obtains alcohol extract medicinal extract；
The decoloration removal of impurities processing of 3 > macroreticular resins: alcohol extract medicinal extract is dissolved in 50% ethyl alcohol, upper D101 macroreticular resin (China
Shanghai Resin Factory), it is eluted with 95% ethyl alcohol of the column volume of about 15BV, collects eluent, rotary evaporation is concentrated under reduced pressure,
Water bath method, it is dry, obtain 95% ethyl alcohol macroporous resin column eluate.
4 > polyamide columns isolate and purify: about 80% ethyl alcohol of above-mentioned macroporous resin column eluate being dissolved, polyamide is passed through
Column chromatography, with the chloroform of 2:1: acetone elutes, and collects eluent；HPLC/U analysis.
5 > dispersive liquid-liquid microextractions (DLLME): 4 > chloroform of collection step: acetone eluant is transferred to centrifuge tube, is added
50% aqueous methanol of about 10 times of volumes, adjusting pH is about 8.0, vortex oscillation mixing；Add isometric about 95% ethyl alcohol, mixing
Form emulsion；Centrifugation is stood, and takes the extraction phase at 95% ethyl alcohol position of lower layer；After drying is concentrated under reduced pressure in extraction phase, it must crystallize
Common smoketree extract mixture 2.
The separation of fisetin and myricetin and purifying process 3 in 3 smoke tree of embodiment
The processing of 1 > raw material: taking smoke tree stem branch, to dry after the slice of crosspiece face, is ground into powder, spare；
2 > refluxing extractions: by dry smoke tree stem branch powder 20g, with about the 70% of about 20 times of equivalent smoke tree dried powder amounts
Ethyl alcohol, adjusting pH is about 9.0, and twice, about 1.5h of each extraction time obtains smoke tree stem branch alcohol extract to 70 DEG C of refluxing extractions, and rotation is steamed
After hair method is concentrated under reduced pressure, water bath method obtains alcohol extract medicinal extract；
The decoloration removal of impurities processing of 3 > macroreticular resins: alcohol extract medicinal extract is dissolved in 50% ethyl alcohol, upper D101 macroreticular resin (China
Shanghai Resin Factory), it is eluted with 95% ethyl alcohol of the column volume of about 15BV, collects eluent, rotary evaporation is concentrated under reduced pressure,
Water bath method, it is dry, obtain 95% ethyl alcohol macroporous resin column eluate.
4 > polyamide columns isolate and purify: about 80% ethyl alcohol of above-mentioned macroporous resin column eluate being dissolved, polyamide is passed through
Column chromatography, with the chloroform of 2:1: acetone elutes, and collects eluent；HPLC/U analysis.
5 > dispersive liquid-liquid microextractions (DLLME): 4 > chloroform of collection step: acetone eluant is transferred to centrifuge tube, is added
50% aqueous methanol of about 10 times of volumes, adjusting pH is about 10.0, vortex oscillation mixing；Isometric about 95% ethyl alcohol is added, is mixed
Conjunction forms emulsion；Centrifugation is stood, and takes the extraction phase at 95% ethyl alcohol position of lower layer；After drying is concentrated under reduced pressure in extraction phase, it must tie
Brilliant common smoketree extract mixture 3.
Comparing embodiment 1
The processing of 1 > raw material: taking smoke tree stem branch, to dry after the slice of crosspiece face, is ground into powder, spare；
2 > refluxing extractions: by dry smoke tree stem branch powder 20g, with about the 70% of about 20 times of equivalent smoke tree dried powder amounts
Ethyl alcohol, adjusting pH is about 9.0, and twice, about 1.5h of each extraction time obtains smoke tree stem branch alcohol extract to 70 DEG C of refluxing extractions, and rotation is steamed
After hair method is concentrated under reduced pressure, water bath method obtains alcohol extract medicinal extract；
The decoloration removal of impurities processing of 3 > macroreticular resins: alcohol extract medicinal extract is dissolved in 50% ethyl alcohol, upper D101 macroreticular resin (China
Shanghai Resin Factory), it is eluted with 95% ethyl alcohol of the column volume of about 15BV, collects eluent, rotary evaporation is concentrated under reduced pressure,
Water bath method, it is dry, obtain 95% ethyl alcohol macroporous resin column eluate.
4 > polyamide columns isolate and purify: about 80% ethyl alcohol of above-mentioned macroporous resin column eluate being dissolved, polyamide is passed through
Column chromatography, with the chloroform of 1:1 or 1.5:1: acetone elutes, and collects eluent；HPLC/U analysis.
5 > dispersive liquid-liquid microextractions (DLLME): 4 > chloroform of collection step: acetone eluant is transferred to centrifuge tube, is added
50% aqueous methanol of about 10 times of volumes, adjusting pH is about 8.0, vortex oscillation mixing；Add isometric about 95% ethyl alcohol, mixing
Form emulsion；Centrifugation is stood, and is taken the extraction phase at 95% ethyl alcohol position of lower layer to carry out HPLC/UV inspection and is known.
Wherein with the chloroform of 1:1 in step 4 >: the result for the technique that acetone is eluted is as shown in Figure 7.
Wherein with the chloroform of 1.5:1 in step 4 >: the result for the technique that acetone is eluted is as shown in Figure 8.
The display of Fig. 7 and 8, in the chloroform for passing through polyamide column 1:1 or 1.5:1: then acetone elution carries out dispersion liquid
After micro-extraction, it cannot efficiently separate and purifying obtains fisetin and myricetin.
Comparing embodiment 2
The processing of 1 > raw material: taking smoke tree stem branch, to dry after the slice of crosspiece face, is ground into powder, spare；
2 > refluxing extractions: by dry smoke tree stem branch powder 20g, with about the 70% of about 20 times of equivalent smoke tree dried powder amounts
Ethyl alcohol, adjusting pH is about 9.0, and twice, about 1.5h of each extraction time obtains smoke tree stem branch alcohol extract to 70 DEG C of refluxing extractions, and rotation is steamed
After hair method is concentrated under reduced pressure, water bath method obtains alcohol extract medicinal extract；
The decoloration removal of impurities processing of 3 > macroreticular resins: alcohol extract medicinal extract is dissolved in 50% ethyl alcohol, upper D101 macroreticular resin (China
Shanghai Resin Factory), it is eluted with 95% ethyl alcohol of the column volume of about 15BV, collects eluent, rotary evaporation is concentrated under reduced pressure,
Water bath method, it is dry, obtain 95% ethyl alcohol macroporous resin column eluate.
4 > polyamide columns isolate and purify: about 80% ethyl alcohol of above-mentioned macroporous resin column eluate being dissolved, polyamide is passed through
Column chromatography, with the chloroform of 1:1 or 1.5:1: acetone elutes, and collects eluent；HPLC/U analysis.
5 > dispersive liquid-liquid microextractions (DLLME): 4 > chloroform of collection step: acetone eluant is transferred to centrifuge tube, is added
50% aqueous methanol of about 10 times of volumes, adjusting pH is about 8.0, vortex oscillation mixing；Add isometric about 60%, 70% or
85% ethyl alcohol, is mixed to form emulsion；Centrifugation is stood, and is taken the extraction phase at 95% ethyl alcohol position of lower layer to carry out HPLC/UV inspection and is known.
As a result, it has been found that using 60%, 70%, 85% ethyl alcohol in step 5 >, all fail fully effective separation and purifying
Obtain fisetin and myricetin.Wherein, the result of the ethyl alcohol in step 5 > using 85% is as shown in Figure 9.
The measurement of the removing free radical OH ability of embodiment 4
Reaction system model is established referring to the Fenton method reacted, passes through H2O2With Fe2+Mixing generation OH, but by
There is very high reactivity in OH, the time-to-live is short, if salicylic acid is added in the reaction system effectively to be captured
OH, and generate color products.According to fixed reaction time methods, in reaction system (the 8.8mmol/L H of same volume2O21mL,
9mmol/L FeSO41mL, 9mmol/L salicylic acid-ethanol solution 1mL) in determinand 1mL is added, aquae destillata makees blank control,
Control solvent makees reference, measures absorbance at 510nm, takes the average value of measured value three times.
Clearance rate (%) D=D control-D drug/D control × 100.
Common smoketree extract mixture 1, the common smoketree extract that embodiment 1, embodiment 2 and embodiment 3 are prepared mix
Object 2 and common smoketree extract mixture 3 are purged the active measurement of free radical OH according to the above method.It the results are shown in Table 1, linearly
It returns and test of significance of coefficient of correlation, inspection level ∝=0.01, variance analysis is shown in Table 2.
1 common smoketree extract mixture of table removes result and linear regression (n=3) to free radical OH
2 smoke tree chromocor compound of table removes result variance analysis to free radical OH
F(2,9)=4.26
Table 1 is the results show that the common smoketree extract mixture 1, the common smoketree extract that are prepared according to the method for the present invention are mixed
Closing object 2 and common smoketree extract mixture 3 all has scavenging effect to free radical OH, and linearly related, effect and be significant (P
< 0.01).The crystallization of common smoketree extract mixture has stronger capture to act on hydroxyl radical free radical, is testing section, with
Its clearance rate of the increase of common smoketree extract mixture concentration increases.Show that the crystallization of common smoketree extract mixture is removed free radical and made
With there are positive correlations between the amount of chromocor extract.
And the variance analysis of table 2 shows the crystallization for the common smoketree extract mixture that three kinds of extraction conditions obtain to free radical
There were significant differences for OH scavenging effect (P < 0.05).This illustrates in the common smoketree extract mixture obtained under various extracting conditions
The difference of flavones type and content has certain difference, has a significant impact to free radical OH scavenging effect.The results show that at this
Use pH for about 9.0 condition in the 4th > of invention smoke tree purifying technique step, the common smoketree extract finally obtained is to free radical OH
The activity of removing is relatively best.
The measurement of 5 anti-tumor activity of embodiment
1. experimental group and cell administration:
1 > method for cell count:
Digestive juice digests ware floor cells completely, blows and beats cell with serum RPMI1640 culture medium 5ml, is made unicellular outstanding
Liquid.It is micro to be added on blood cell counting plate, the sum of the cell number in the block plaid of quadrangle is counted under microscope, is substituted into following formula and is obtained
Cell density: cell number/ml=(the sum of 4 big lattice cells/4) × 10000
The grouping of 2 > cells:
The cell of logarithmic growth phase is with every hole 5 × 104A cell inoculation is in six orifice plates, when cell fusion degree is 80%
Myricetin group (50,100,200,400 μm of ol/L), implementation that 1 step 7 > of embodiment of various concentration is purified is added in grouping
The fisetin group (50,100,200,200 μm of ol/L) that 1 step 6 > of example is purified.Separately set blank control group and DMSO control group.
100ul RPMI1640 cell culture fluid is added in control group, and the DMSO cell culture fluid of equivalent is added in DMSO control.Also set quercitrin
The control group (50,100,200,400 μ g/ml) of plain standard items.Make every hole final volume 2ml, at 37 DEG C, 5%C02Incubator
Culture.After culture 24 hours, group of cells, row Flow cytometry cell cycle and apoptosis are collected.
2. Fluorescein activated cell sorter detects Rat C 6 Glioma Cells apoptosis
6 orifice plates suction is first discarded into culture solution, PBS washed once, the trypsin digestion cell 1min of rear addition 0.25%.It blows
Cell suspension suction is set in centrifuge tube after beating, 2000rpm is centrifuged 3min.Liquid is discarded supernatant, after 2mlPBS flushing is added
200rpm is centrifuged 3min, repeats primary.The combination liquid in cell apoptosis detection kit is added in supernatant after discarding centrifugation
400ul, reagent A nnexin-v-FITC dyeing liquor 5ul is added in every pipe after blowing and beating uniformly.After 4 DEG C are protected from light incubation 15min, PI is added
Dyeing liquor 10ul is protected from light in 4 DEG C is incubated for 5min.In being detected on flow cytometer.
The toxic effect of 3.LDH activity detection detection Rat C 6 Glioma Cells
Single cell suspension first is made by propagating method in cell, carries out cell count with blood counting chamber.Culture medium dilution
Make final concentration of the 1 × 10 of cell6/ml.Every hole takes 100ul to be placed in 96 orifice plates.96 orifice plates are set into 37 DEG C, 5%CO2Stand training
It supports, cell is adherent after 2h.Myricetin group (50,100,200,400 μ that 1 step 7 > of embodiment of various concentration is purified are added
Mol/L the fisetin group (50,100,200,200 μm of ol/L) that) 1 step 6 > of embodiment is purified.Separately set blank control group and
DMSO control group.Culture medium is sucked out to the activity for being placed on automatic biochemical detector and detecting LDH in culture solution afterwards for 24 hours.Data are used
Means standard deviationIt indicates, significant difference variance analysis between group.Experimental data is united through SPSS 17.0
Meter processing, P < 0.05 think with significant difference.
4. test result
Apoptosis by Flow Cytometry result
The result is shown in Figure 10 and 11.The result shows that myricetin and fisetin administration group that the method for the present invention is prepared and sky
White group and solvent blank group more have significant difference (P < 0.05).It is poor without conspicuousness between blank group and two groups of blank solvent group
Different (P > 0.05).
LDH activity testing result
The result is shown in Figure 12.The result shows that myricetin and fisetin 50umol/L, 100umol/L, 200umol/L administration group with
Blank group and solvent blank group more have significant difference (P < 0.05).Without conspicuousness between blank group and two groups of solvent control group
Difference (P > 0.05).
The result shows that certain density myricetin and fisetin can significantly induce C6 glioma apoptosis, to C 6 glioma in rats
Cell has significant cytotoxic effect.
6 acute toxicity testing of embodiment
Experimental method
Selection Kunming kind weight 20~22g cleaning grade mouse 24, is randomly divided into 4 groups, and every group 6, half male and half female.4
Experimental group is respectively the crystallization for the common smoketree extract mixture 1 that 1 step 5 > of embodiment is obtained, by 100%, 50% and 20%3
Concentration group and control group.Fasting 12 hours, 0.5ml/kg weight is pressed to each group mouse, gastric infusion several times in 24 hours.It gives
Experimental animal poisoning manifestations and death condition are observed after medicine, are observed continuously 14 days.None is dead, and survival mice dissection does not find different
Often.
7 long term toxicity test of embodiment
Rat 80 are selected, half male and half female, long term toxicity test is divided into 4 groups, the Huang that respectively 1 step 5 > of embodiment is obtained
The crystallization of smoke tree extract mixtures 1, by 100%, 50% and 20%3 concentration groups and control groups.Distilled water stomach-filling, daily administration
Once, successive administration 90 days pay attention to situations such as observing activity, the hair color, feed, weight of rat, after administration 90 days during administration
It is discontinued, carries out pathological examination, high, medium and low dosage group is administered compared with blank control group, unknown significance difference, after 30 days,
Above-mentioned inspection is repeated, does not also find toxic side effect.
It is demonstrated experimentally that the smoke tree purification small toxicity that technique of the invention is prepared, clinical application are safe and reliable.
Present inventor provides the new synchronous method for extracting fisetin, myricetin in separation smoke tree, and exceeds
Expect ground and finds that method of the invention has that process route is simple, step is few, it can efficiently and specifically separation and Extraction paint simultaneously
Flavine and myricetin both relatively most effective components in smoke tree, lose other objects when avoiding a certain ingredient of single extraction
The problem of matter, the comprehensive utilization of smoke tree flavonoids is greatly improved, and it is small to demonstrate mixture toxicity obtained, clinical application peace
Entirely, fisetin and myricetin therein have fine anti-oxidant and anti-tumor activity.
The above is the explanation carried out to the present invention, cannot be regarded as the limitation carried out to the present invention.Unless in addition referring to
Out, practice of the invention will use the routine techniques of organic chemistry, polymer chemistry, biotechnology etc., it is clear that except stating upper
Except being particularly described in bright and embodiment, the present invention can also be realized otherwise.Other aspects within the scope of the present invention
It will be apparent to those skilled in the art in the invention with improving.Introduction according to the present invention, many changes and variation are
It is feasible, therefore it is within the scope of the present invention.
DEG C if without particularly showing, the unit " degree " of herein presented temperature refers to degree Celsius, i.e.,.
Claims (10)
1. the method for fisetin and myricetin, includes the following steps: in a kind of separation and extraction smoke tree
1) smoke tree stem branch is taken, is dried, crushed into powder after slice；
2) by dry smoke tree stem branch powder, with 10~30 times of smoke tree dried powder 50~80% ethyl alcohol of amount, pH is 8.0~
Twice, each 1~2h of extraction time obtains smoke tree stem branch alcohol extract to 10.0,60-80 DEG C of refluxing extractions, and rotary evaporation decompression is dense
After contracting, water bath method obtains alcohol extract medicinal extract；
3) the alcohol extract medicinal extract is dissolved in ethyl alcohol, after upper macroreticular resin, is carried out with 95% ethyl alcohol of column volume of 10~20BV
Eluent is collected in elution, and rotary evaporation is concentrated under reduced pressure, and water bath method is dry, obtains 95% ethyl alcohol column eluate, color is in shallow
Yellow；
4) 95% ethyl alcohol column eluate, 95% ethyl alcohol is dissolved, by polyamide column chromatography, with the chloroform of about 2:1: third
Ketone elution, collects eluent；
5) after acetone eluant is collected, about 50% aqueous methanol, vortex oscillation mixing the chloroform for obtaining step 4): is added；Again
About 90-98% ethyl alcohol is added, is mixed to form emulsion；Centrifugation is stood, and takes lower ethanol position, after drying is concentrated under reduced pressure, is obtained yellow
Smoke tree purification, the smoke tree purification contain fisetin and myricetin.
2. according to the method described in claim 1, adjusting pH is about 8.0- after about 50% aqueous methanol is added wherein in step 5)
About 10.0.
3. according to the method described in claim 2, adjusting pH is about 9.0 after about 50% aqueous methanol is added wherein in step 5).
4. according to the method described in claim 1, the about 90-98% ethyl alcohol is about 95% ethyl alcohol wherein in step 5).
5. method according to any of claims 1-4, wherein further comprising the steps of:
By ethyl alcohol position in step 5) by polyamide column chromatography, obtained after drying is concentrated under reduced pressure in eluent with 60% ethanol elution
Fisetin.
6. method according to any of claims 1-4, wherein further comprising the steps of:
By ethyl alcohol position in step 5), after drying is concentrated under reduced pressure, it is dissolved in after a small amount of 95% ethyl alcohol through Sephadex LH-20 column
Chromatography obtains myricetin after drying is concentrated under reduced pressure in eluent with 95% ethanol elution.
7. a kind of pharmaceutical composition, wherein the smoke tree being prepared containing method according to claim 1 to 6
Purification and pharmaceutically acceptable excipient, the smoke tree purification contain fisetin and myricetin.
8. pharmaceutical composition according to claim 7 is used for treating cancer, such as glioma.
9. a kind of food additives composition, wherein being prepared containing method according to claim 1 to 6
Smoke tree purification and food auxiliary material, the smoke tree purification contain fisetin and myricetin.
10. a kind of cosmetic composition, wherein the Huang being prepared containing method according to claim 1 to 6
Smoke tree purification and cosmetic auxiliary material, the smoke tree purification contain fisetin and myricetin.
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