CN106333974A - Preparation and novel application of gynura cusimbua extract - Google Patents
Preparation and novel application of gynura cusimbua extract Download PDFInfo
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- CN106333974A CN106333974A CN201610725907.3A CN201610725907A CN106333974A CN 106333974 A CN106333974 A CN 106333974A CN 201610725907 A CN201610725907 A CN 201610725907A CN 106333974 A CN106333974 A CN 106333974A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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Abstract
The invention relates to preparation and novel application of a gynura cusimbua extract, and belongs to the field of natural plant chemistry. The prepared gynura cusimbua extract is applied to the activity of resisting a hepatitis B virus and inhibiting canine vascular smooth muscle cell proliferation inhibition. The gynura cusimbua extract is obtained by selecting medicinal and edible gynura cusimbua as raw materials through a modern extraction and separation technique, the extracted gynura cusimbua extract is applied to the activity of resisting the hepatitis B virus and the activity of inhibiting canine vascular smooth muscle cell proliferation inhibition activity and has an anti-HBV effect and a certain inhibition effect on canine vascular smooth muscle cell proliferation, and the research result has an important guiding effect on development and preparation of anti-arteriosclerosis drugs.
Description
Technical field
The invention belongs to natural plants chemical field is and in particular to a kind of extraction of muercai (vegetable) and its new application.
Background technology
Herba basellae rubrae, another name bean curd dish, kermes dish, because of the approximate Auricularia of its leaf, plump and glutinous cunning, therefore named Herba basellae rubrae.Wood
A kind of ancient vegetable of Er Caishi China, is distributed widely in China's most area.China's traditional Chinese medical science shows, Herba basellae rubrae has cunning
Intestinal, removing pathogenic heat from blood and toxic substance from the body, connect muscle symplectic bone, detumescence the effects such as, be usually used in treat breast intestinal accumulated heat gloomy, oliguria is puckery, dysentery, have blood in stool, speckle
The diseases such as rash, treatment skin ulcer, burn and scald, traumatic injury, rheumatic arthritis.Modern pharmacological research shows, Herba basellae rubrae have antiviral,
The pharmacologically active such as antioxidation and anti-inflammation.Herba basellae rubrae is nutritious, containing multiple chemical compositions, mainly includes saponin, waves
Hair oil, polysaccharide and various vitamin.We consult lot of documents and find, put down in anti-hepatitis virus and dog blood vessel with regard to Herba basellae rubrae
Sliding muscle cell multiplication inhibitory activity aspect there are no relevant report, and therefore, the present invention chooses Herba basellae rubrae and carries out above-mentioned activity research
There is wide DEVELOPMENT PROSPECT.
It is known that Chinese herbal medicine resource is national strategy resource, it is the basis of China's Chinese medicine sustainable development.Ten
After eight is big, China has successively worked out " Chinese medicine developmental strategy planning outline (2016-2030) " and " Chinese crude drug protection and development
Planning (2015-2020) ", the modernization of Chinese medicine is the grand strategy target of China, is also the emphasis neck of the current new drug development of China
Domain.The present invention chooses the plant Herba basellae rubrae of medicine-food two-purpose as object of study, using modern technologies, Herba basellae rubrae is extracted
Separate and obtain a kind of extract, and to this extract in terms of anti-hepatitis virus and inhibition against proliferation of dog vascular smooth muscle cells
Carry out systematic study, be that China develops treatment hepatitis B from Herba basellae rubrae and atherosclerosis drug has established important theoretical base
Plinth.
Content of the invention
In order to overcome the shortcomings of background technology presence, the present invention provides a kind of preparation of extraction of muercai (vegetable) and its new use
On the way, the Herba basellae rubrae from " medicine-food two-purpose " is raw material, obtains extraction of muercai (vegetable) using modern extracting and developing technology, and this
The extraction of muercai (vegetable) that invention is extracted has new use in terms of anti-hepatitis virus and inhibition against proliferation of dog vascular smooth muscle cells
On the way.
In order to achieve the above object, the following technical scheme of present invention proposition:
The preparation of described extraction of muercai (vegetable) includes step in detail below:
Step (1): take fresh Herba basellae rubrae, dry under 25-35 DEG C of low temperature environment, be crushed to 25-40 mesh, be immersed in
After 15-18h in the ethanol of 80-95%, it is placed in ultrasonic extraction instrument, mistake after ultrasonic 30-45min under 220-240w power
Filter, merging filtrate, in 45-50 DEG C of environment, concentrating under reduced pressure obtains extractum, standby;
Step (2): the extractum that step (1) is obtained is dispersed in distilled water, stands 24-28h, removes water insoluble
Thing, obtains filtrate, for future use;
Step (3): obtain adding petroleum ether continuous extraction 3 times, combining extraction liquid in filtrate in step (2), obtain oil
Ether extract layer and water layer;
Step (4): obtain adding ethyl acetate continuous extraction 3 times, combining extraction liquid in water layer filtrate in step (3), obtain
To ethyl acetate extract layer and water layer;
Step (5): step (4) is obtained acetic acid ethyl ester extract, drying under reduced pressure, obtains thick paste, made with 98% methanol
It fully dissolves, sephadex lh-20 post in wet method, sequentially adds 90%~100% methanol and carries out gradient elution, concentration evaporates
Divide standby;
Step (6): step (5) is obtained 200-300 mesh silica gel column chromatography on 90%~100% methanol-eluted fractions fraction, according to
Secondary volume ratio is that the petroleum ether-ethyl acetate of 10:1~4:1 carries out gradient elution, obtains different eluting fractions;
Step (7): toyopearl hw-40 gel column on petroleum ether-ethyl acetate eluting fraction will be obtained in step (6)
Chromatograph, carries out eluting with 95%meoh, eluting fraction concentrating under reduced pressure is dried, obtains final product extraction of muercai (vegetable).
Further, in step (1), material and the volume ratio of ethanol are material: ethanol=1:4-6.
Further, in step (2), extractum and the volume ratio of distilled water are extractum: distilled water=1:1.5.
Further, in step (3), filtrate and the volume ratio of petroleum ether are filtrate: petroleum ether=1:1.5.
Further, in step (4), filtrate and the volume ratio of ethyl acetate are filtrate: ethyl acetate=1:1.5.
Further, prepared extraction of muercai (vegetable) is in anti-hepatitis virus and inhibition against proliferation of dog vascular smooth muscle cells
The application of aspect.
Beneficial effects of the present invention:
The Herba basellae rubrae that the present invention chooses " medicine-food two-purpose " is raw material, obtains Herba basellae rubrae using modern extracting and developing technology and carries
Take thing, the extraction of muercai (vegetable) of extraction be applied to anti-hepatitis B activity and inhibition against proliferation of dog vascular smooth muscle cells,
Extraction of muercai (vegetable) has anti-hepatitis B virus and has certain inhibitory action to dog vascular smooth muscle cell proliferation, this
Bright achievement in research has important directive function for developing arteriosclerosis medicine.
Brief description
Fig. 1 is the Technology Roadmap of the present invention.
Specific embodiment
Below in conjunction with embodiments of the invention and accompanying drawing, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation description is it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments, is based on
Embodiment in the present invention, it is all other that this area comparison technical staff is obtained under the premise of not making creative work
Embodiment, broadly falls into the scope of protection of the invention.
Embodiment 1
Take fresh Herba basellae rubrae, low temperature (25 DEG C) dries, and is crushed to 30 mesh, be immersed in 85% ethanol (material: ethanol=
1:4, v/v) in after 15h, be placed in ultrasonic extraction instrument, filter after ultrasonic 30min under 220w power, merging filtrate, low temperature
(45 DEG C) concentrating under reduced pressure obtains extractum.The extractum obtaining is dispersed in distilled water (extractum: distilled water=1:1.5, v/v),
Standing 24h, removes water-insoluble, obtains filtrate.Petroleum ether (filtrate: petroleum ether=1:1.5, v/v) is added in obtaining filtrate
Continuous extraction 3 times, combining extraction liquid, obtain petroleum ether extraction layer and water layer.In obtaining water layer add ethyl acetate (filtrate:
Ethyl acetate=1:1.5, v/v) continuous extraction 3 times, combining extraction liquid, obtain ethyl acetate extract layer.By the acetic acid obtaining second
Ester extract, drying under reduced pressure, obtain thick paste.It is made fully to dissolve with 98% methanol again, sephadex lh-20 post in wet method,
Carry out eluting with 90% methanol, enriched fraction is standby.By silica gel (200-300 mesh) post color on obtain 90% methanol-eluted fractions fraction
Spectrum, the petroleum ether-ethyl acetate gradient elution being 9:1 with volume ratio, obtain eluting fraction.By on this eluting fraction
Toyopearl hw-40 gel column chromatography, carries out eluting with 95%meoh, eluting fraction concentrating under reduced pressure is dried, obtains final product Auricularia
Dish extract.
Embodiment 2
Take fresh Herba basellae rubrae, low temperature (30 DEG C) dries, and is crushed to 35 mesh, be immersed in 90% ethanol (material: ethanol=
1:5, v/v) in after 17h, be placed in ultrasonic extraction instrument, filter after ultrasonic 38min under 230w power, merging filtrate, low temperature
(48 DEG C) concentrating under reduced pressure obtains extractum.The extractum obtaining is dispersed in distilled water (extractum: distilled water=1:1.5, v/v),
Standing 26h, removes water-insoluble, obtains filtrate.Petroleum ether (filtrate: petroleum ether=1:1.5, v/v) is added in obtaining filtrate
Continuous extraction 3 times, combining extraction liquid, obtain petroleum ether extraction layer and water layer.In obtaining water layer add ethyl acetate (filtrate:
Ethyl acetate=1:1.5, v/v) continuous extraction 3 times, combining extraction liquid, obtain ethyl acetate extract layer.By the acetic acid obtaining second
Ester extract, drying under reduced pressure, obtain thick paste.It is made fully to dissolve with 98% methanol again, sephadex lh-20 post in wet method,
Carry out eluting with 95% methanol, enriched fraction is standby.By silica gel (200-300 mesh) post color on obtain 95% methanol-eluted fractions fraction
Spectrum, the petroleum ether-ethyl acetate gradient elution being 6:1 with volume ratio, obtain eluting fraction.By on this eluting fraction
Toyopearl hw-40 gel column chromatography, carries out eluting with 95%meoh, eluting fraction concentrating under reduced pressure is dried, obtains final product Auricularia
Dish extract.
Embodiment 3
Take fresh Herba basellae rubrae, low temperature (35 DEG C) dries, and is crushed to 40 mesh, be immersed in 95% ethanol (material: ethanol=
1:6, v/v) in after 18h, be placed in ultrasonic extraction instrument, filter after ultrasonic 45min under 240w power, merging filtrate, low temperature
(50 DEG C) concentrating under reduced pressure obtains extractum.The extractum obtaining is dispersed in distilled water (extractum: distilled water=1:1.5, v/v),
Standing 28h, removes water-insoluble, obtains filtrate.Petroleum ether (filtrate: petroleum ether=1:1.5, v/v) is added in obtaining filtrate
Continuous extraction 3 times, combining extraction liquid, obtain petroleum ether extraction layer and water layer.In obtaining water layer add ethyl acetate (filtrate:
Ethyl acetate=1:1.5, v/v) continuous extraction 3 times, combining extraction liquid, obtain ethyl acetate extract layer.By the acetic acid obtaining second
Ester extract, drying under reduced pressure, obtain thick paste.It is made fully to dissolve with 98% methanol again, sephadex lh-20 post in wet method,
Carry out eluting with 100% methanol, enriched fraction is standby.By silica gel (200-300 mesh) post on obtain 100% methanol-eluted fractions fraction
The petroleum ether-ethyl acetate gradient elution that chromatograph is 4:1 with volume ratio, obtains eluting fraction.By on this eluting fraction
Toyopearl hw-40 gel column chromatography, carries out eluting with 95%meoh, eluting fraction concentrating under reduced pressure is dried, obtains final product Auricularia
Dish extract.
Experimental analysiss:
1. the anti-hepatitis B activity of extraction of muercai (vegetable)
(1) instrument and material
Hepg 2 cell, Diagnostic Test Kit for Hepatitis B Surface Antigen(Elisa), hepatitis B virus e resists
Former diagnostic kit (euzymelinked immunosorbent assay (ELISA)), Lamivudine (3tc), microplate reader (spectra max m4), incubator (heraeus
bk800)
(2) experimental technique
(1) hepg 2 cell is placed in 96 porocyte culture plates and cultivates after one week, add the embodiment of 0.15 μm of ol/ml
The extraction of muercai (vegetable) of 2 extractions, in 37 DEG C, 25%co2Culture 24h, with the detection hbv antigen change of elisa method.
(2) 10 μ l supernatant and 5% confining liquid 40 μ l are added in 96 orifice plates and duplicate hole, every hole adds 50 μ l enzymes to combine
Thing, fully mixes, in 37 DEG C of water-bath 30min.Remove liquid, add 200 μ l pbst in every hole, stand 20s, remove liquid again
Body, repeats five times;It is eventually adding 50 μ l nitrite ions, fully mixes, in 37 DEG C of water-bath 15min, every hole adds terminate liquid 50 μ l.With
Microplate reader reads od450 (nrn) value under reference wavelength 650 (nrn).
(3) experiment conclusion
The change of hbsag and hbeag after being processed with elisa method detection compound, experimental result is shown in Table 1,
The inhibitory action to hbsag and hbeag for table 1 extraction of muercai (vegetable)
As can be seen from Table 1, extraction of muercai (vegetable) is above sun in 0.15 μm of ol/ml to the suppression ratio of hbsag and hbeag
Property comparison medicine Lamivudine (3tc), point out extraction of muercai (vegetable) to have the effect of anti-hepatitis B virus.
2. extraction of muercai (vegetable) is to inhibition against proliferation of dog vascular smooth muscle cells
(1) experiment material
Dog aorta vessel, dmem culture fluid contain 20% calf serum, 0.3% L-Glutamine, 100u/ml penicillin and
The streptomycin of 100 μ g/ml;Use nahco3Adjust the hank's liquid of ph to 7.2;Trypsin;pbs;96 orifice plates.
(2) experimental technique
(1) the thoracoabdominal aorta section of experimental dog is placed in the plate containing pbs liquid under aseptic condition and rinses 3 times, divest
The fibrof atty layer of adventitia, longitudinal incision blood vessel, strikes off inner membrance, is cut into the wide little bar of about 1mm, is immersed in the hank's containing serum
In liquid, and the small tissue blocks torn are planted in 25cm2Culture bottle wall, in 37 DEG C of calorstats culture 2h about.
(2) add the culture fluid dmem of 20% hyclone in culture bottle, be placed on incubator to cultivate 4 days, treat that cell spreads
During full culture bottle bottom 90% about, with trypsin and edta had digestive transfer culture.
(3), when had digestive transfer culture is to seven generations, makes thin suspension inoculation and pull in 96 holes, density 2 × 104Individual/ml, every hole 170 μ
l;It is placed in 37 DEG C, 5%co2Incubator is cultivated 24 hours.
(4) the extraction of muercai (vegetable) 10 μ l adding embodiment 2 to extract in every hole, cultivates 24 hours;Add insulin
20 μ l, continue culture 24 hours;Finally, add mtt 20 μ l in every hole, continue incubation 4h.Suction out culture fluid, add dmso
150 μ l/ holes, shaking table vibrates 15min;Under 490nm wavelength, measure od value with microplate reader.
(3) experiment conclusion
By extraction of muercai (vegetable) is carried out with inhibition against proliferation of dog vascular smooth muscle cells suppression screening, experimental result is shown in
Table 2:
Table 2 extraction of muercai (vegetable) is to inhibition against proliferation of dog vascular smooth muscle cells
Note: (1) model group is 0.85 ± 0.042;(2) * shows the effect with suppression smooth muscle cell proliferation.
As can be seen from Table 2, extraction of muercai (vegetable) has certain inhibitory action to dog vascular smooth muscle cell proliferation.Cause
This, the achievement in research of the present invention has important directive function for developing arteriosclerosis medicine.
The Herba basellae rubrae that the present invention chooses " medicine-food two-purpose " is raw material, obtains Herba basellae rubrae using modern extracting and developing technology and carries
Take thing, the extraction of muercai (vegetable) of extraction be applied to anti-hepatitis B activity and inhibition against proliferation of dog vascular smooth muscle cells,
Extraction of muercai (vegetable) has anti-hepatitis B virus and has certain inhibitory action to dog vascular smooth muscle cell proliferation, this
Bright achievement in research has important directive function for developing arteriosclerosis medicine.
Finally, above example and accompanying drawing be only in order to illustrate technical scheme and unrestricted, although by above-mentioned
Embodiment is described in detail to the present invention, it is to be understood by those skilled in the art that can in form and carefully
Various changes are made to it, without departing from claims of the present invention limited range on section.
Claims (6)
1. a kind of preparation of extraction of muercai (vegetable) it is characterised in that: the preparation of described extraction of muercai (vegetable) is included in detail below
Step:
Step (1): take fresh Herba basellae rubrae, dry under 25-35 DEG C of low temperature environment, be crushed to 25-40 mesh, be immersed in 80-
After 15-18h in 95% ethanol, it is placed in ultrasonic extraction instrument, filter after ultrasonic 30-45min under 220-240w power, close
And filtrate, in 45-50 DEG C of environment, concentrating under reduced pressure obtains extractum, standby;
Step (2): the extractum that step (1) is obtained is dispersed in distilled water, stands 24-28h, removes water-insoluble, obtains
To filtrate, for future use;
Step (3): obtain adding petroleum ether continuous extraction 3 times, combining extraction liquid in filtrate in step (2), obtain petroleum ether extraction
Take layer and water layer;
Step (4): obtain adding ethyl acetate continuous extraction 3 times, combining extraction liquid in water layer filtrate in step (3), obtain second
Acetoacetic ester extract layer and water layer;
Step (5): step (4) is obtained acetic acid ethyl ester extract, drying under reduced pressure, obtains thick paste, make it fill with 98% methanol
Divide dissolving, sephadex lh-20 post in wet method, sequentially add 90%~100% methanol and carry out gradient elution, enriched fraction is standby;
Step (6): step (5) is obtained 200-300 mesh silica gel column chromatography on 90%~100% methanol-eluted fractions fraction, uses body successively
The petroleum ether-ethyl acetate for 10:1~4:1 for the long-pending ratio carries out gradient elution, obtains different eluting fractions;
Step (7): toyopearl hw-40 gel column color on petroleum ether-ethyl acetate eluting fraction will be obtained in step (6)
Spectrum, carries out eluting with 95%meoh, eluting fraction concentrating under reduced pressure is dried, obtains final product extraction of muercai (vegetable).
2. a kind of extraction of muercai (vegetable) according to claim 1 preparation it is characterised in that: in step (1) material with
The volume ratio of ethanol is material: ethanol=1:4-6.
3. a kind of extraction of muercai (vegetable) according to claim 1 preparation it is characterised in that: in step (2) extractum with
The volume ratio of distilled water is extractum: distilled water=1:1.5.
4. a kind of extraction of muercai (vegetable) according to claim 1 preparation it is characterised in that: in step (3) filtrate with
The volume ratio of petroleum ether is filtrate: petroleum ether=1:1.5.
5. a kind of extraction of muercai (vegetable) according to claim 1 preparation it is characterised in that: in step (4) filtrate with
The volume ratio of ethyl acetate is filtrate: ethyl acetate=1:1.5.
6. the extraction of muercai (vegetable) according to prepared by claim 1-5 any one is thin in anti-hepatitis virus and dog vascular smooth muscle
The application of born of the same parents' proliferation inhibition activity aspect.
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