CN108670973A - A kind of Chloranthus glaber anti-influenza virus activity extract and preparation method thereof - Google Patents
A kind of Chloranthus glaber anti-influenza virus activity extract and preparation method thereof Download PDFInfo
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Abstract
The present invention provides a kind of Chloranthus glaber anti-influenza virus activity extracts,It contains the chlorogenic acid relative to 0.020~0.040 weight % of the extract total weight,The isofraxidin of 0.000~0.008 weight %,The Rosmarinic acid of Quercetin 3 the O β D glucuronides and 0.083~0.158 weight % of 0.025~0.060 weight %,Additionally provide a kind of preparation method of Chloranthus glaber anti-influenza virus activity extract,By by Chloranthus glaber deionized water refluxing extraction twice,Chloranthus glaber water extract dry extract is made after alcohol precipitation,Then the dry extract is configured to a certain concentration toward macroporous absorbent resin sample introduction,Then it is eluted with 20% ethanol solution,Collect eluent,Obtain the Chloranthus glaber anti-influenza virus activity extract,The advantage of the invention is that the application for being Herba Pileae Scriptae extract in terms of anti influenza provides help,Preparation method step is simple and easy to operate,It can quantify and reproducible,It is suitble to the later stage that the extract is applied to the large-scale commercial process of drug.
Description
Technical field
The present invention relates to a kind of extractive technique fields of Chinese medicine anti-influenza virus activity ingredient, more particularly to a kind of careless coral
Coral anti-influenza virus activity extract and preparation method thereof.
Background technology
Influenza virus abbreviation influenza virus is the representative species of orthomyxoviridae family (Orthomyxoviridae), packet
Include human influenza virus and animal influenza virus, contact between main through the air droplet, susceptible person and the infected or with
It is contaminated the contact of article and propagates.The frequency of various influenza viruses such as SARS, A type H7N9, H1N1, bird flu etc. in recent years
Numerous invasion, infectiousness is strong, it is rapid to propagate, and easily causes and is very popular, and influenza virus outburst each time all brings huge fear to the mankind,
Therefore it is significant efficiently to find suitable anti-influenza virus medicament using technological means.
Influenza virus is the enveloped virus with segment, sub-thread strand RNA genome, wherein on influenza A virus outer membrane
Inlay 3 kinds of memebrane proteins, respectively neuraminidase (NA), hemagglutinin (HA) and M2 albumen.Neuraminidase inhibitor at present
It is the hot spot for the treatment of of influenza drug research, neuraminidase is also known as one that sialidase is distributed across on influenza virus envelope and is in
Mushroom tetramer glycoprotein has antigenicity, can be catalyzed sialic acid hydrolysis, assists ripe influenza virus to be detached from host thin
Born of the same parents infect new cell, and important role is played in the life cycle of influenza virus, and therefore, neuraminidase becomes influenza
One action target spot of medicine, the Oseltamivir for neuraminidase design is one of foremost Tamiflu.
Western medicine action pathway is single, easily human body is made to generate drug resistance, and there are larger side effects.Chinese medicine is multiple for multicomponent
Miscellaneous system, the action character with multipath, too many levels, multiple target point, is increasingly subject in novel anti-influenza virus medicament development
Pay attention to.
Chloranthus glaber also known as Glabrous Sarcandra Herb, sarcandra, synthetism tree etc. are Chloranthaceae Sarcandra plant Chloranthus glaber
The drying herb of Sarcandra glabra (Thunb.) Nakai, main product each province on the south the Changjiang river such as Fujian, Jiangxi.Modern times doctor
It learns studies have shown that mainly containing the classes chemical composition such as flavones, cumarin, organic acid, terpene, volatile oil in Chloranthus glaber.Pharmacology
Show that Chloranthus glaber has antibacterial anti-inflammatory with toxicologic study, inhibits a variety of people and animals pathogens and influenza virus, antitumor, promotion
The various actives such as union, analgesia, safe and non-toxic have preferable therapeutic effect to a variety of people, animal diseases, are a kind of great
Research and develop the autonomic drug of utility value.Because Chloranthus glaber has clearing heat and cooling blood, promoting blood circulation ecchymose removing, dispelling wind and removing obstruction in the meridians, synthetism and detumescence
Analgesic and other effects is usually used in preparing the products such as rheumatism traditional Chinese medicine plaster, health-care cold tea, the promoting and tooth-building toothpaste that cleans the teeth, breeding feed.
Mentioning Chloranthus glaber in existing disclosed document has resisiting influenza virus effect, but is directed to the antiviral work of Chloranthus glaber at present
It researchs and analyses or the observation of the inhibition of infected by influenza mostly uses animal Inoculation Strain and during some are applied to
The method of herbal medicine fingerprint analysis.Ma Meixiang etc. observes the antivirus action of Chloranthus glaber, and refer to lung by antiviral study in vitro
It is index that number, virus, which cause mouse death rate etc., using Ribavirin as positive drug control, observes its therapeutic effect, finds grass
The water extract of coral has good internal resisiting influenza virus effect, and to the cellular immunity of influenza a virus infection mouse
System has good adjustment effect, can significantly lower the Lung Exponent of FM1 infecting mouses.Long Weiying etc. uses Caoshanhu extract pair
Chicken embryo carries out toxicity test, and with cold drug, Amantadine mixture, abitilguanide, column grass coral is crossed in drug, discovery 10% as a contrast
The coral medicinal extract liquid capital infected by influenza A/ section/1/68 (H3N2)15EID50Infective dose has deactivation.Chinese patent
CN200610163255.5 is using (CE-ED) quality analysis of capillary electrophoresis electrochemical method and differentiates herbal medicine and Chinese medicine, by Chinese medicine
Fingerprint map analyzing is combined with index components content measurement, to reach to herbal medicine Glabrous Sarcandra Herb and by three kinds made by it
Medicine：It the quality evaluation of the science of compound caoshanhu tablets, QINGRE XIAOYANNING JIAONANG and XUEKANG KOUFUYE and supervises.
Above method is mostly the qualitative or quantitative pharmacological evaluation for giving drug to living animal or in vitro tissue, there is influence
The shortcomings of factor is more, precision is low, costly, period is long and poor reproducibility, and be to Chloranthus glaber antivirus action or influenza
HIV suppression effect or using Chloranthus glaber as the quality evaluation of drug made of raw material, for anti-influenza virus activity in Chloranthus glaber and
Its separating and extracting process then has not been reported.
Duan Yinghui etc. is using macroreticular resin DiaionHP20 column chromatographys, reverse phase ODS column chromatographys, SephadexLH20 column colors
The means such as spectrum, reversed-phase HPLC are by a variety of column chromatography methods, isolated 10 compounds from the water extract of Chloranthus glaber, profit
The chemical constitution of the water extract is identified with method of spectroscopy.But their research purpose is to determine that the validation of Chloranthus glaber studies
Point, make element task to formulate Herba Pileae Scriptae extract quality standard, Chloranthus glaber resisiting influenza virus specific compound is not carried
It takes method to make targetedly to study.
Invention content
For this reason, it may be necessary to provide a kind of Chloranthus glaber anti-influenza virus activity and its separating and extracting process, come quickly, easy, essence
Anti-influenza virus activity in true ground separation and Extraction Chloranthus glaber, to fill up the sky for extracting anti-influenza virus activity from Chloranthus glaber
In vain, solution route is provided for popularization and application of the Chinese herbal and crude drugs preparations such as Chloranthus glaber in terms of resisiting influenza virus.
To achieve the above object, a kind of Chloranthus glaber anti-influenza virus activity extract, the anti influenza are inventor provided
Virus activity extract contains：
Relative to the chlorogenic acid of 0.020~0.040 weight % of the extract total weight, 0.000~0.008 weight %
Quercetin -3-O- β-D-Glucose aldehydic acid the glycosides and 0.083~0.158 weight % of isofraxidin, 0.025~0.060 weight %
Rosmarinic acid.
Further, the Quercetin -3-O- β-D-Glucose aldehydic acid glycosides contains relative to the extract total weight
Amount is 0.029~0.045 weight %.
It is different from the prior art, above-mentioned technical proposal has the following advantages that：Chloranthus glaber anti-influenza virus activity is provided to carry
The chlorogenic acid of object, the content of isofraxidin, Quercetin -3-O- β-D-Glucose aldehydic acid glycosides and Rosmarinic acid are taken, is carried for Chloranthus glaber
Application of the object in terms of anti influenza is taken to provide help.
Inventor additionally provides a kind of preparation method for extracting Chloranthus glaber anti-influenza virus activity extract from Chloranthus glaber,
It is characterized in that, the preparation method includes preparing Chloranthus glaber water extract and purifying elution,
The Chloranthus glaber water extract for preparing includes the following steps：Using Chloranthus glaber as raw material, powder is crushed into after dry
End obtains the first extracting solution with deionized water refluxing extraction, and ethyl alcohol is added into first extracting solution and carries out mixed precipitation,
Through being separated by solid-liquid separation, the second supernatant is obtained, the second supernatant is concentrated, dry extract is obtained；
The purifying elution includes the following steps：By the dry extract deionized water dissolving, by the dissolved matter toward macropore
Adsorption resin column sample introduction, the ethanol solution for being then 15%~25% with volume fraction are eluted, and eluent is obtained, and collect institute
Eluent is stated to get the Chloranthus glaber anti-influenza virus activity extract.
Further, the volumes of aqueous ethanol score of the purifying elution is 20%, and dosage is 3~5 times of institute
State adsorption resin column volume.
Further, the large pore resin absorption column be nonpolar macroporous adsorption resin, including HP20, HPD100,
It is one or more in HPD300 and D101 macroporous absorbent resins.
Further, it is described prepare water extract step be added the ethyl alcohol volume fraction be 50%~95%.
Further, the just extracting solution is added after ethyl alcohol mixed precipitation, stands 8~24 hours.
Further, 0.5~1g/mL of the mass concentration of solid content containing Chloranthus glaber in the dry extract.
Further, described when preparing Chloranthus glaber water extract with deionized water refluxing extraction, including with deionized water
Refluxing extraction and second of refluxing extraction, the process conditions with deionized water first time refluxing extraction are the powder
The mass ratio of end and deionized water is 1:40~1:20, reflux extracting time is 1~3 hour；It is described with deionized water second
The process conditions of secondary refluxing extraction are that the mass ratio of the powder and deionized water is 1:30~1:15, reflux extracting time
It is 0.5~2 hour.
A kind of Chloranthus glaber anti-influenza virus activity extract, contain relative to the extract total weight 0.020~
The Quercetin-of the chlorogenic acid of 0.040 weight %, the isofraxidin of 0.000~0.008 weight %, 0.025~0.060 weight %
The Rosmarinic acid of 3-O- β-D-Glucose aldehydic acid glycosides and 0.083~0.158 weight %, the extract is according to claim 3
It is prepared by method.
It is different from the prior art, above-mentioned technical proposal has the following advantages that：(1) influenza neuraminidase is used to inhibit
Bioactivity kit carries out Chloranthus glaber water extract the comparison that neuraminidase inhibits potency, compares traditional mouse or chicken
The animals Inoculation Strain such as embryo greatly shortens analysis to analyze the method that resisiting influenza virus acts in Chloranthus glaber water extract
Time；(2) influenza neuraminidase inhibits with strong points, the anti-influenza virus activity portion of separation of bioactivity kit
Position is more accurate than conventional method, and is avoided that other non-neural ammonia during Inoculation Strain test Chinese herbal medicine extract
The interference of sour enzyme inhibitor；(3) processing step when screening detaches most strong anti-influenza virus activity position from Herba Pileae Scriptae extract
It is simple and easy to operate, it can quantify and reproducible, be suitble to the later stage that the extract is applied to the large-scale industrial production mistake of drug
Journey.
Description of the drawings
Fig. 1 is that a kind of preparation method concrete operation step of Chloranthus glaber anti-influenza virus activity extract of the present embodiment is simple
Schematic diagram；
Fig. 2 is the present embodiment Content of Chlorogenic Acid, isofraxidin, Quercetin -3-O- β-D-Glucose aldehydic acid glycosides and Rosmarinic acid
Mix the HPLC chromatogram of reference substance；
Fig. 3 is 0% ethanol eluate HPLC chromatogram in the present embodiment；
Fig. 4 is 20% ethanol eluate HPLC chromatogram in the present embodiment；
Fig. 5 is 25% ethanol eluate HPLC chromatogram in the present embodiment；
Fig. 6 is 30% ethanol eluate HPLC chromatogram in the present embodiment；
Fig. 7 is 50% ethanol eluate HPLC chromatogram in the present embodiment；
Fig. 8 is 70% ethanol eluate HPLC chromatogram in the present embodiment；
1, chlorogenic acid；
2, isofraxidin；
3, Quercetin -3-O- β-D-Glucose aldehydic acid glycosides；
4, Rosmarinic acid.
Specific implementation mode
For the technology contents, method and step, the objects and the effects of technical solution are described in detail, below in conjunction with specific reality
It applies example and attached drawing is coordinated to be explained in detail.
The Chloranthus glaber crude drug source that the present embodiment uses is identified as Chloranthaceae before the rock of Sanming City, Fujian Province Sanyuan District
The drying herb of plant Chloranthus glaber Sarcandra glabra (Thunb.) Nakai.Macroporous absorbent resin (the Diaion of use
HP20, HPD100, HPD300 and D101) it is provided by Cangzhou Bon Adsorption Material Science and Technology Co., Ltd；Chlorogenic acid, isofraxidin,
Rosmarinic acid is purchased from the Shanghai bio tech ltd Yuan Ye；Quercetin -3-O- β-D-Glucose aldehydic acid glycosides is auspicious purchased from Chengdu
The bio tech ltd Fen Si；Methanol, acetonitrile be chromatographically pure, phosphoric acid, formic acid be analyze it is pure, methanol, acetonitrile, phosphoric acid, formic acid,
Petroleum ether, ethyl acetate and n-butanol are purchased from Sinopharm Chemical Reagent Co., Ltd.；Positive drug Peramivir injection
(3mg/mL) is purchased from Guangzhou Nan Xin pharmaceutical Co. Ltds；Deionized water is self-control ultra-pure water；Influenza neuraminidase inhibits
Bioactivity kit (neuraminidase, neuraminidase substrate, buffer solution and the terminate liquid that include purifying) is green purchased from Shanghai
Skies Bioisystech Co., Ltd (product batch number：170609, it is ground by the 302nd entire PLA of hospital Chinese medicine study of Beijing liberation army
System).
The instrument and equipment that the present embodiment uses is as follows：Agilent 1200series high performance liquid chromatographs, vacuum outgas
Machine, quaternary pump, binary pump, column oven, UV detector, HP1200 chem workstations are carried by Anjelen Sci. ＆ Tech. Inc of the U.S.
For；Numerical control ultrasonic cleaner KQ-500DE types are provided by Kunshan Ultrasonic Instruments Co., Ltd.；SHB-III circulating water types are multi-purpose
Vacuum pump is provided by Zhengzhou Greatwall Scientific Industrial ＆ Trading Co., Ltd.；HH-4 digital displays thermostat water bath is provided by Guo Hua Electrical Appliances Co., Ltd；
DKZ series electric heating constant temperature shaking water baths are provided by the permanent Science and Technology Ltd. in Shanghai one；RE-52 rotary evaporators are by Shanghai Asia Rong Sheng
Change instrument plant to provide；Low-temperature cooling fluid circulating pump is provided by Zhengzhou Greatwall Scientific Industrial ＆ Trading Co., Ltd.；AHAUS a ten thousandth electronics point
Analysis balance is provided by Ohaus Instrument (Changzhou) Co., Ltd.；(precision is 100,000 to METTLER TOLEDO electronic analytical balances
/ mono-)；Milli-Q ultrapure water systems are provided by Millipore companies of the U.S.；), the multi-functional extraction thickeners of TS-NS-100
Group is provided by Shanghai along instrument experimental facilities Co., Ltd, and ZF-2 types ultraviolet analysis instrument for three purposed is carried by Town in Shanghai pavilion Electronic Instruments Plant
For suppressing standby chromatography, Shimadzu LC-8A high pressure preparative chromatographs in sharp fringe；Multi-channel liquid transfer device (range 20-200 μ l, 2-10 μ
L), single channel pipettor (range 2-l0 μ l, 20-200 μ l, 100-1000 μ l) and each specification suction nozzle have fluorescence detection function
Microplate reader, 96 hole fluorescence ELISA Plates, constant-temperature incubation case, liquid feeding slot.
Embodiment one
The present embodiment provides a kind of separating and extracting process of Chloranthus glaber anti-influenza virus activity, including prepare Chloranthus glaber water and carry
Object and purifying is taken to elute；
The Chloranthus glaber water extract for preparing includes the following steps：Using Chloranthus glaber as raw material, it is placed in the drying of vacuum Chinese herbal medicine
It is 1 hour dry in machine, then the Chloranthus glaber after drying is crushed with pulverizer, mistake《Chinese Pharmacopoeia》(2015 editions) No. 3 standard medicines
Sieve, gained powder are the Chloranthus glaber powder that granularity is less than or equal to 50 mesh, and Chloranthus glaber powder 5g is taken to be placed in clean circle
In the flask of bottom, impregnated 30 minutes with 150mL deionized waters, then refluxing extraction 2 hours, collect first time extracting solution, spare；It is past
125mL deionized waters are added in round-bottomed flask to impregnate 30 minutes, second of extracting solution is collected in then refluxing extraction 2 hours, by the
The stirring of 95% ethyl alcohol is added into above-mentioned just extracting solution in extracting solution at the beginning of extracting solution and second of extracting solution are mixed to get
Mixing is allowed to precipitate, and stands 12 hours, using filtering, being concentrated under reduced pressure, is then evaporated in 65 DEG C of water-baths, obtains dry extract,
It is stored for future use in 4 DEG C of refrigerators after drying；
Has the effect of resisiting influenza virus to verify above-mentioned dry extract, with resisiting influenza virus inhibitor para in the present embodiment
Rice Wei injection (3mg/mL) is diluted to the positive reference liquid of 1mg/mL with deionized water；Precision weigh above-mentioned dry extract add from
Sub- water dissolution is made into the mother liquor of 1mg/ml, sonicated, and supernatant is taken after centrifugation, and medicinal material test solution is made.Using stream
Influenza Virus neuraminidase inhibits bioactivity kit, and (measuring principle of the kit is：Using the neuraminidase of purifying,
It is reacted with the fluorogenic substrate 4-methylumbelliferyl- α-D-N-acetylneuraminic acid of specificity, production production
Object 4-MU, under 355nm excitation wavelengths, 460nm absorbing wavelengths, fluorescent absorption strength assessment neuraminidase activity.) measure grass
Coral water extract anti-influenza virus activity, for verify Chloranthus glaber water extract resisiting influenza virus characteristic when done three batches
Chloranthus glaber is tested, and table 1 shows three batch Herba Pileae Scriptae extract yield and resisiting influenza virus potency, should be the result shows that Chloranthus glaber water
Extract has anti-influenza virus activity, and three batches of Chloranthus glaber medicinal material experimental result differences are little, and RSD values are less than 2%.
1 extract yield of table and resisiting influenza virus potency
Macroporous absorbent resin need to be pre-processed in purifying elution：20gDiaion HP20 macroreticular resins are taken, with 95%
Ethyl alcohol sealed soak 48 hours at room temperature, fully after swelling, wet method dress post flows cleaning on pillar with 95% ethyl alcohol, washes
It is washed till no alcohol taste without white opacity phenomenon, then with a large amount of pure water to eluate plus appropriate pure water, it is spare；
To weigh the above-mentioned mother liquors of 1.4g, it is 6 to adjust pH value for purifying elution, is 1BV to pretreatment with flow velocity per hour
Good Diaion HP20 macroporous absorbent resin sample introductions adsorb 0.5 hour, with 20% ethyl alcohol 4BV, are carried out with flow velocity 2BV per hour
Elution, collects the eluent, is concentrated under reduced pressure, and freeze-drying is at powder up to the Chloranthus glaber anti-influenza virus activity.
With extensively, having, quick, efficient, selectivity is good, clever in chemical composition of Chinese materia medica detaches for macroporous absorbent resin
Advantages, the effective elements of the medicines in capable of being effectively enriched with such as sensitivity is high, can be recycled make certain in Chinese medicine effective monomer component or compound
The index of one monomer component is improved.The present embodiment is being determined using 20% ethyl alcohol as eluant, eluent from Chloranthus glaber water extraction
Before Chloranthus glaber anti-influenza virus activity described in object separation and Extraction, a large amount of Comparative experiments and exploration have been carried out.It first passes through pair
0% ethyl alcohol of Sarcandra glabra aqueous extract, 20% ethyl alcohol, 25% ethyl alcohol, 30% ethyl alcohol, 50% ethyl alcohol, 70% ethyl alcohol carry out macropore suction
Attached purifying resin obtains different ethanol concentration gradient eluent.
Precision weighs isofraxidin, Rosmarinic acid, chlorogenic acid, Quercetin -3-O- β-D-Glucose aldehydic acid glycosides control respectively
Product 0.01g is placed in 10mL measuring bottles, and the constant volume graduation mark for adding methanol constant volume to measuring bottle is configured to reference substance storing solution.Then accurate
Pipette it is each this is placed according to product storing solution 1mL in 10mL measuring bottles, add methanol constant volume, every 1mL be made containing 101.5 μ g of chlorogenic acid, different
The mixing reference substance of 113.6 μ g of piperazine skin pyridine, Quercetin -3-O- β -81.30 μ g of D-Glucose aldehydic acid glycosides, 105.2 μ g of Rosmarinic acid
Solution.It is in chromatographic condition with HPLC with mixed reference substance solution by above-mentioned different ethanol concentration gradient eluent：DIKMA
Diamonsil octadecylsilane chemically bonded silicas (C18) chromatographic column (4.6mm × 250mm, 5 μm)；Mobile phase：Acetonitrile (A)-
0.2% phosphoric acid solution (B), and gradient elution (0~10 minute, 15%A；10~15 minutes, 15%A → 20%A；15~25 minutes,
20%A；25~40 minutes, 20%A → 40%A)；Flow velocity：1.0mL per minute；Column temperature：35℃；Detection wavelength：344nm, sample introduction
Amount：It is measured under 10 μ L, which elutes position active constituent content measuring result such as
Shown in table 2, the HPLC chromatogram of mixed reference substance solution is as shown in Fig. 2, different ethanol concentration elutes position eluent HPLC colors
For spectrogram respectively as shown in Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7 and Fig. 8, different ethanol concentration elutes position eluent resisiting influenza virus effect
The results are shown in Table 3 for valence.
2 Chloranthus glaber difference of table elutes position active constituent content measuring result (mg/g)
Then it, then by Chloranthus glaber water extract different ethanol concentration eluent is diluted to different quality concentration, use is above-mentioned
Influenza neuraminidase inhibits bioactivity kit to measure respectively, and inhibiting rate is calculated by formula (one)：
In formula, fluorescent value is the fluorescence reading in each hole in above-mentioned detection process.
3 different parts eluent resisiting influenza virus potency of table
It is compared by above-mentioned experimental result it is found that volume fraction is the eluent that 20% ethanol elution agent elutes that position is collected into
The content highest of middle Quercetin -3-O- β-D-Glucose aldehydic acid glycosides, the position are inhibited by the eluent of different multiples dilution gained
Rate is higher than other elution positions, and resisiting influenza virus potency is much higher than other elution positions.It thereby determines that from macroporous absorbent resin
Elute Chloranthus glaber water extract separation and Extraction purpose of the present invention product anti-influenza virus activity --- neuraminidase inhibitor
In the process, using 20% concentration of alcohol as eluant, eluent.
Embodiment two
The present embodiment provides a kind of separating and extracting process of Chloranthus glaber anti-influenza virus activity, including prepare Chloranthus glaber water and carry
Object and purifying is taken to elute；
What is different from the first embodiment is that the Chloranthus glaber water extract for preparing includes the following steps：Using Chloranthus glaber as raw material,
Drying 1.5 hours in vacuum Chinese herbal medicine drying machine are placed in, are then crushed the Chloranthus glaber after drying with pulverizer, mistake《Middle traditional Chinese medicines
Allusion quotation》(2015 editions) No. 3 standard medicine sieves, gained powder is the Chloranthus glaber powder that granularity is less than or equal to 50 mesh, takes the grass coral
Coral powder 6g is placed in clean round-bottomed flask, is impregnated 30 minutes with 180mL deionized waters, then refluxing extraction 2 hours, are collected
First time extracting solution, it is spare；150mL deionized waters immersion 30 minutes is added into round-bottomed flask, then refluxing extraction 2 hours,
Second of extracting solution is collected, extracting solution at the beginning of first time extracting solution and second of extracting solution are mixed to get just is carried toward above-mentioned
It takes 70% ethyl alcohol is added in liquid to be stirred and is allowed to precipitate, stand 12 hours, using filtering, being concentrated under reduced pressure, then at 65 DEG C
It is evaporated in water-bath, obtains dry extract, stored for future use in 4 DEG C of refrigerators after dry；
Purifying elution is carried out using Diaion HPD100 absorption resins in the present embodiment；
To weigh the above-mentioned dry extract powders of 2g, it is 1.0g/mL to be adjusted to mass concentration with deionized water for the purifying elution
Mother liquor, it is 6 to adjust pH value, is that 1BV adsorbs resin sample introduction, absorption to the Diaion HPD100 pre-processed with flow velocity per hour
It 0.5 hour, with 20% ethyl alcohol 4BV, is eluted with flow velocity 1.5BV per hour, collects the eluent, be concentrated under reduced pressure, freezing is dry
It is dry at powder up to the Chloranthus glaber anti-influenza virus activity.
Chloranthus glaber water extract different ethanol concentration elutes position active constituent content measuring result such as table 4 in the present embodiment
Shown, different ethanol concentration elutes position eluent resisiting influenza virus potency, and the results are shown in Table 5.
4 Chloranthus glaber difference of table elutes position active constituent content measuring result (mg/g)
5 different parts eluent resisiting influenza virus potency of table
It follows that different resins carries out purifying elution to water extract, it is still the elution portion of 20% concentration of alcohol
Position resisiting influenza virus active constituent content highest, the inhibiting rate highest of neuraminidase, resisiting influenza virus potency highest.
Chloranthus glaber anti-influenza virus activity made by technical solution using the present embodiment is verified through HPLC chromatogram method, together
Sample contains chlorogenic acid, isofraxidin, Quercetin -3-O- β-D-Glucose aldehydic acid glycosides and Rosmarinic acid.
It should be noted that although the various embodiments described above have been described herein, it is not intended to limit
The scope of patent protection of the present invention.Therefore, based on the present invention innovative idea, to embodiment described herein carry out change and repair
Change, or using equivalent structure or equivalent flow shift made by description of the invention and accompanying drawing content, it directly or indirectly will be with
Upper technical solution is used in other related technical areas, is included within the scope of patent protection of the present invention.
Claims (10)
1. a kind of Chloranthus glaber anti-influenza virus activity extract, which is characterized in that the anti-influenza virus activity extract contains：
Relative to the chlorogenic acid of 0.020~0.040 weight % of the extract total weight, the different piperazine of 0.000~0.008 weight %
Skin pyridine, 0.025~0.060 weight % Quercetin -3-O- β-D-Glucose aldehydic acid glycosides and 0.083~0.158 weight % fan
Repeatedly fragrant acid.
2. Chloranthus glaber anti-influenza virus activity extract according to claim 1, which is characterized in that the Quercetin-
3-O- β-D-Glucose aldehydic acid glycosides is 0.029~0.045 weight % relative to the content of the extract total weight.
3. a kind of preparation method for extracting Chloranthus glaber anti-influenza virus activity extract from Chloranthus glaber, which is characterized in that described
Preparation method includes preparing Chloranthus glaber water extract and purifying elution,
The Chloranthus glaber water extract for preparing includes the following steps：Using Chloranthus glaber as raw material, it is crushed into powder after dry, with
Deionized water refluxing extraction obtains the first extracting solution, and ethyl alcohol is added into first extracting solution and carries out mixed precipitation, through solid-liquid
Separation, obtains the second supernatant, the second supernatant is concentrated, dry extract is obtained；
The purifying elution includes the following steps：By the dry extract deionized water dissolving, by the dissolved matter toward macroporous absorption
Resin column sample introduction, the ethanol solution for being then 15%~25% with volume fraction are eluted, obtain eluent, washed described in collection
De- liquid is to get the Chloranthus glaber anti-influenza virus activity extract.
4. the preparation method of Chloranthus glaber anti-influenza virus activity extract according to claim 3, it is characterised in that：It is described
The volumes of aqueous ethanol score for purifying elution is 20%, the adsorption resin column volume that dosage is 3~5 times.
5. the preparation method of Chloranthus glaber anti-influenza virus activity extract according to claim 3, it is characterised in that：It is described
Large pore resin absorption column is in nonpolar macroporous adsorption resin, including HP20, HPD100, HPD300 and D101 macroporous absorbent resin
It is one or more.
6. the preparation method of Chloranthus glaber anti-influenza virus activity extract according to claim 3, it is characterised in that：It is described
It is 50%~95% to prepare water extract step and the volume fraction of the ethyl alcohol is added.
7. the preparation method of Chloranthus glaber anti-influenza virus activity extract according to claim 6, it is characterised in that：It is described
First extracting solution is added after ethyl alcohol mixed precipitation, stands 8~24 hours.
8. the preparation method of Chloranthus glaber anti-influenza virus activity extract according to claim 7, it is characterised in that：It is described
0.5~1g/mL of the mass concentration of solid content containing Chloranthus glaber in dry extract.
9. the preparation method of Chloranthus glaber anti-influenza virus activity extract according to claim 3, it is characterised in that：It is described
When preparing Chloranthus glaber water extract with deionized water refluxing extraction, including with deionized water first time refluxing extraction and second time
Stream extraction, the process conditions with deionized water first time refluxing extraction are the mass ratios of the powder and deionized water
It is 1:40~1:20, reflux extracting time is 1~3 hour；The process conditions with second of refluxing extraction of deionized water are,
The powder and the mass ratio of deionized water are 1:30~1:15, reflux extracting time is 0.5~2 hour.
10. a kind of Chloranthus glaber anti-influenza virus activity extract, contain relative to the extract total weight 0.020~
The Quercetin-of the chlorogenic acid of 0.040 weight %, the isofraxidin of 0.000~0.008 weight %, 0.025~0.060 weight %
The Rosmarinic acid of 3-O- β-D-Glucose aldehydic acid glycosides and 0.083~0.158 weight %, the extract is according to claim 3
It is prepared by method.
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