CN107033198A - A kind of separation method of pine needle chemical composition - Google Patents
A kind of separation method of pine needle chemical composition Download PDFInfo
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- CN107033198A CN107033198A CN201610079118.7A CN201610079118A CN107033198A CN 107033198 A CN107033198 A CN 107033198A CN 201610079118 A CN201610079118 A CN 201610079118A CN 107033198 A CN107033198 A CN 107033198A
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Abstract
The present invention relates to a kind of separation method of pine needle chemical composition, wherein, the chemical composition is betuloside and massonianoside C, it is characterised in that comprised the steps of：The preparation of need testing solution, is separated, purifying, again separation etc., and separation method of the present invention reduces the usage amount of organic solvent, and solvent is easily recycled, and method is simply amplified.
Description
Technical field
The present invention relates to medicinal chemistry art, and in particular to a kind of separation method of pine needle chemical composition.
Background technology
Pine needle (pine needle) is the topmost accessory substance of Pinaceae (Pinaceae) plant, is the medicinal representation position of Pinus.China pine
Section plant mainly has masson pine, Chinese pine, Huashan pine, pinus sylvestris var. mongolica, wet-land pine tree etc., wherein, masson pine is that south China is most main
The pine genus plant wanted.The research of last 100 yearses finds, extract from pine needles have analgesia, anti-inflammatory, calmness, antibechic, eliminating the phlegm, relieving asthma,
A series of pharmacological activity such as reducing blood lipid, anti-oxidant, anti-aging, anti-mutation, antitumor, because its aboundresources, effect are various
And cause further investigation of the scholars to pine needle chemical composition.The research main ingredient of the chemical composition of main ingredient in pine needle was concentrated in recent years
On the materials such as volatile oil, flavone compound, OPC, lignanoid, shikimic acid, vitamin, phenols；It is prepared by separation
Representative high-purity compound monomer turns into an important research contents in pine needle, and the especially part in pine needle is exclusive
Property chemical composition extraction separation for pine needle medicinal material quality research it is significant.Monomer chemistries composition in current pine needle
Separation method mainly from plant material extract separation, involved separation means based on silica gel and gel filtration chromatography, this
The monomeric compound purity that a little conventional methods are prepared is relatively low, and complex steps, cycle length, repeatability are poor, it is difficult to apply
In industrial big production.Therefore the separation method of compound monomer is badly in need of improving in pine needle, it is desirable to have one kind is quick, efficient, reappear
Property good separation means meet the demand prepared to high-purity monomer compound in pine needle.
The content of the invention
It is an object of the invention to provide a kind of simplicity, the method that separating monomer compound is fast and efficiently extracted from pine needle,
Its technical scheme is as follows：
One aspect of the present invention provides a kind of separation method of pine needle chemical composition, wherein, the chemical composition be betuloside and
Massonianoside C, specific method is comprised the steps of：
(1) preparation of need testing solution：Weigh Folium Pini extract appropriate, add organic solvent and extract, extract solution quiescent setting,
Supernatant is taken, is freeze-dried, desciccate is dissolved in ultra-pure water, filtering and collecting filter liquid is produced after concentration；
(2) separate：Need testing solution in step (1) is taken, using polarity inversion octadecylsilane chromatographic column as stationary phase, with
Methanol solution containing formic acid-aqueous solution containing formic acid is mobile phase, carries out gradient elution, and retention time is collected respectively and is
8.0-9.0min cut F1 and retention time is 20.5-22min cut F2；
(3) purify：Cut F1 and F2 in step (2) are taken, respectively using anti-phase octadecylsilane chromatographic column as stationary phase,
20:80 acetonitrile solution containing formic acid-aqueous solution containing formic acid be mobile phase, isocratic elution, respectively collect retention time be
3.8-4.6min cut F1-1 and retention time is 14.6-15.5min cut F2-1；
(4) separate：Cut F1-1 and cut F2-1 in step (3) is taken, respectively using hydrophilic filler chromatographic column as stationary phase,
90:10 acetonitrile solution containing formic acid-aqueous solution containing formic acid is mobile phase, carries out isocratic elution, collects in F1-1 and retains
The cut F2-2 that retention time is 16.7-17.3min in cut F1-2 and F2-1 that time is 12.3-12.7min；Wherein F1-2
For betuloside, F2-2 is massonianoside C.
Wherein, the methanol solution containing formic acid described in step (2) is preferably the methanol solution containing 0.1% formic acid；It is described to contain
The aqueous solution for having formic acid is preferably the aqueous solution containing 0.1% formic acid；The condition of gradient elution is preferably：0-35min, 25%-60%
Methanol solution containing 0.1% formic acid, 35-36min, 60%-95% contains the methanol solution of 0.1% formic acid, 36-55min,
95%-95% contains the methanol solution of 0.1% formic acid.
Acetonitrile solution containing formic acid described in the step (3) or (4) is preferably the acetonitrile solution containing 0.1% formic acid；Institute
It is preferably the aqueous solution containing 0.1% formic acid to state the aqueous solution containing formic acid.
The step (2), (3), (4) Detection wavelength are preferably 280nm；
Organic solvent described in the step (1) is preferably from one in methanol, ethanol, acetone, acetonitrile, ethyl acetate, water
Plant or a variety of mixtures, more preferably methanol solution or 70% ethanol solution；The filter type is preferably miillpore filter,
Wherein the aperture of micro porous filtration is 0.22 μm or 0.45 μm；The extraction step is preferably refluxing extraction or ultrasonic extraction.
The step (1) is most preferably to weigh Folium Pini extract, each in two times to add methanol, every time ultrasound 1 hour, and it is heavy to stand
Form sediment, take rotary evaporation in vacuo at supernatant, 60 DEG C, be freeze-dried after concentration, desciccate is dissolved in the water, cross 0.45 μm
Filter membrane, is produced；The mass volume ratio of wherein Folium Pini extract and methanol used in each ultrasonic extraction is 7:100；Or be most preferably to claim
Folium Pini extract 350g is taken, 70% ethanol is added, is heated to reflux 1 hour, staticly settles, takes vacuum at supernatant, 60 DEG C to revolve
Turn evaporation, be freeze-dried after concentration, desciccate is dissolved in 400mL ultra-pure waters, cross 0.45 μm of filter membrane, produce, its
The mass volume ratio of middle Folium Pini extract and 70% ethanol is 7:100.Polarity inversion octadecylsilane in the step (2)
Chromatographic column is preferably Silica Surface while having the chromatographic column of polar functional group and octadecyl functional group, qualified filled column
It can make or select commercially available into capo by oneself, such as selected from Atlantis dC18 series, Ultimate AQ-C18 series, XBridge C18
Series, the chromatographic column of XAqua C18 series can meet the implementation purpose of the present invention, wherein being imitated especially with XAqua C18 series
Preferably, the chromatographic column filler particle diameter is 5-60 μm, preferably 10 μm to fruit；
Anti-phase octadecylsilane chromatographic column is preferably the chromatographic column of Silica Surface bonding octadecyl functional group in step (3), can
Make or select commercially available into capo by oneself, such as selected from Xterra MS series, Inertsil ODS-SP series, Zorbax SB C18 series,
SunFire C18 series etc.；Most preferably SunFire C18 series, chromatographic column filler particle diameter is 5-60 μm, preferably 5 μm；
Hydrophilic filler chromatographic column is preferably the chromatographic column or silica gel chromatograph of Silica Surface bonding polar functional group in the step (4)
Post, can make or select commercially available into capo by oneself, such as selected from Atlantis HILIC Silica series, Venusil HILIC series, XAmide
Series, ClickXIon series etc., most preferably ClickXIon series, chromatographic column filler particle diameter is 5-60 μm, preferably 10 μm.
Folium Pini extract described in separation method of the present invention refers to using pine needle as raw material, using water or aqueous alcoholic solvent as extraction solution；
Applicants have found that water, 50% methanol or ethanol, 70% methanol or ethanol, 95% methanol or ethanol, methanol, ethanol can be with
The Extraction solvent of the present invention is done, it is especially optimal with methanol or 70% ethanol effect；It is preferred that taking fresh pine needle, add water to cook twice, first
Secondary plus 8 times of amount solvents, are decocted 1.5 hours, second plus 6 times of amount solvents, are decocted 1.5 hours；Filtrate decompression is condensed into relatively
Density is detected as 1.2 medicinal extract at 70 DEG C；Spray drying, is produced.
Because the content of both compositions of betuloside (betuloside) and massonianoside C in pine needle is relatively low, general
Extraction separation method hardly results in the monomer component of high-purity, and there is no feasible chemical synthesis process to be used to prepare both at present
Compound monomer.Though the system separation method studied using traditional plant can isolated a small amount of monomer component, method and step
Cumbersome, separation cycle is extremely long, and yield is extremely low, it is impossible to meet the demand that industry is produced greatly.The applicant gropes by long-term
Research, obtains the method that the present invention prepares betuloside and massonianoside C, and this method separative efficiency is high, and product is pure
Degree up to more than 95%, the yield of its comparable purity compound is 3-4 times or so of conventional method, and the preparation time cycle is only to pass
The 9% of system method；And with the increasing of preparation amount, the effect of its quick separating becomes apparent from.Meanwhile, separation method of the present invention is significantly
The usage amount of organic solvent is reduced, its isolating environment is relatively closed, and solvent is easily recycled, therefore more environment-friendly and safer；Also,
The present invention is simple and convenient, be easy to amplification, and full automation equipment separation can be achieved, time, the manpower of product preparation is greatlyd save
And financial cost, it is suitable for industrial big production application.The present invention pine needle chemical composition separation method can be applied to masson pine, it is wet
Betuloside in the pine needle raw material such as ground pine and massonianoside C extract separation, be prepare betuloside and
The desirable route of massonianoside C reference substances, compensate for both current compound control product market vacancys.
Embodiment
Pine needle raw material：
Pine needle medicinal material picks up from Sichuan Province China and saves Zigong City Fushun County, Guangyuan City Chaotian District, the Pinaceae Pinus horse hair of Bazhong City Nanjiang County
The pine needle of loose (Pinus massoniana Lamb.).
Reagent and material：
Methanol：HPLC, 4L/ bottles, Merck (Darmstadt, Germany)；
Acetonitrile：HPLC, 4L/ bottles, Merck (Darmstadt, Germany)；
Formic acid：HPLC, 500ml/ bottles, Sigma-Aldrich (St.Louis, MO, the U.S.)；
Deionized water is prepared by Milli-Q water purification system (Billerica, MA, the U.S.)；
Pure water comes from Wahaha company；
Macroreticular resin, supplier：Chengdu Ke Long chemical reagents factory, model：D101, (60-16 mesh) 0.3-1.25mm≤90%；
Aperture resin gel (MCI), supplier：The biological Co., Ltd of Chengdu science popularization, model：The anti-phase colors of SBC MCI GEL
Compose filler F types, 75-150 μm；
Sephadex, supplier：GE Healthcare Life Sciences, model：sephadex G25Medium, 50 μm；
C18 bonded silica gels (ODS), supplier：Sepax Technologies, Inc, model：GP-C18,50 μm；
Chromatographic column：Sunfire 50*150mm*10 μm (Waters, the U.S.)；XAqua C18 20*250mm*10 μm (China's spectrums
ACCHROM chromatographic columns, China)；Click XIon 4.6*250mm*10 μm (China's spectrum ACCHROM chromatographic columns, China)；
Click XIon 50*250mm*10 μm (China's spectrum ACCHROM chromatographic columns, China).
Instrument：
ZNTD-50 extracts concentration unit：Changsha Yuelu District Zhongnan (South Contral) Pharmacentical Machinery Fectory, China
B-191 spray dryers：BUCHI, Switzerland
Purification plant：Waters, the U.S..Pump, 2777 automatic sampler, 2757 are prepared including a 2525 binary high pressures
Fraction collector and 2489 dual wavelength detectors.
Analyze chromatogram：Waters, the U.S..Including Alliance e2695 liquid phase systems and 2998 PDADs.
Liquid chromatogram：Agilent, the U.S..Including a set of 1260 liquid phase separation and DAD detectors.
High resolution mass spectrum：Agilent, 6450 super-resolution degree level Four bars-time of-flight mass spectrometer (Q-Tof), the U.S..
Solid-state nuclear magnetic resonance instrument：Bruker Avance II 600MHz, Germany.
The preparation of the Folium Pini extract of embodiment one
Fresh pine needle, adds ZNTD-50 and extracts concentration unit, add water to cook twice, and 8 times of amount water are added for the first time, 1.5 are decocted small
When, second plus 6 times of amount water are decocted 1.5 hours；Filtrate decompression is condensed into the leaching that relative density is detected as 1.2 at 70 DEG C
Cream；Spray drying, is produced.
The traditional plant separation method of embodiment two prepares pine needle compound
(1) prepared by need testing solution
Folium Pini extract 1200g is taken, is extracted successively with ethyl acetate, water-saturated n-butanol, ethyl acetate extraction leaching is respectively obtained
Cream, water-saturated n-butanol extraction medicinal extract and the remaining medicinal extract of water layer, numbering is Pr.1~3.Time-consuming 5d.
(3) rough segmentation
Pr.2 (water-saturated n-butanol medicinal extract) is taken, is adsorbed through macroreticular resin D101,3 times of column volume water elutions, then through SBC MCI
Gel is separated, and with 3 times of isolated Pr.2A1 of ethanol elution of column volume 30%, is separated with 3 times of ethanol elutions of column volume 40%
To Pr.2A2.Time-consuming 3d.
Take Pr.2A2 (40% ethanol eluate) through Sephadex G25 posts, 3 times of column volume water elutions obtain 3 part Pr.
2A2A、Pr.2A2B、Pr.2A2C.Time-consuming 2d.
(4) essence point
①massonianoside C
Pr.2A2B obtains 1 part Pr.2A2B1 through SBC MCI posts, 3 times of ethanol elutions of column volume 20%.Time-consuming 1.5d.
2. betuloside
Pr.2A2C is through SBC MCI posts, and 20%-30% ethanol gradient elutions are isolated with 3 times of ethanol elutions of column volume 20%
Pr.2A2C1 is obtained, with 3 times of isolated Pr.2A2C2 of the ethanol elution of column volume 30%.Time-consuming 2d.
Take Pr.2A2C2 through C18 bonded silica gels (ODS) chromatographic isolation, with 20% ethanol elution of 3 times of column volumes, obtain
Pr.2A2C3.Time-consuming 0.5d.
(5) purify
①massonianoside C
Pr.2A2B1 is through silica gel column chromatography, successively with 3 times of column volume dichloromethane-ethanol (5: 1), petroleum ether-acetone (1: 3)
Elution, collects petroleum ether-acetone (1:3) eluent, obtains compound massonianoside C, purity be 95% (HPLC,
37mg).Time-consuming 1d.
2. betuloside
Pr.2A2C3 eluents are taken through silica gel column chromatography, successively with dichloromethane-ethanol (5: 1), the petroleum ether of 3 times of column volumes
- acetone (1: 3) is eluted, and collects petroleum ether-acetone (1:3) eluent, obtains compound betuloside, purity be 95% (HPLC,
93.67mg).Time-consuming 1d.
It is demonstrated experimentally that preparing massonianoside C from pine needle using system plant separation method and betuloside has operating process
Complexity, experimental procedure is more, the problems such as consumption of organic solvent is big, and is related to the poisonous examination such as dichloromethane, petroleum ether, acetone
Agent, security is relatively low.Also, this method, which is separately separated, obtains 2 compounds, takes altogether 19 days, manufacturing cycle is longer,
And product yield is extremely low, massonianoside C yields (relative to Folium Pini extract) are about 3/100000ths, and betuloside is received
Rate (relative to Folium Pini extract) is about 8/100000ths.
The preparation of the pine needle compound of embodiment three
(1) prepared by need testing solution
Folium Pini extract 35g is weighed, each in two times to add 500mL methanol, ultrasound 1 hour, staticly settles, take supernatant every time
Liquid, rotary evaporation in vacuo at 60 DEG C is freeze-dried after concentration, desciccate is dissolved in 40mL ultra-pure waters, crosses 0.45 μm
Filter membrane, is produced.Time-consuming 9h.
(2) one-dimensional preparation liquid phase separation
Instrument：Waters purification plants, chromatographic column：XAqua C18 (250mm × 20mm × 10 μm), each sample size is 8mL.
Gradient elution is carried out as mobile phase with the water (aqueous phase) containing 0.1% formic acid and the methanol (organic phase) containing 0.1% formic acid,
Elution requirement is 0-35min, 25%-60% organic phase, 35-36min, 60%-95% organic phases, 36-55min, 95%-95%
Organic phase, flow velocity is 20mL/min, and Detection wavelength is 280nm.Need testing solution is taken to be separated according to above-mentioned chromatographic condition,
Retention time 8.0-9.0min cut F1 and retention time 20.5-22min cut F2 are collected respectively.Time-consuming 7h.
(3) anti-phase octadecylsilane purifying
Instrument：Waters purification plants, chromatographic column：SunFire (150mm × 50mm × 5 μm), each sample size is 20mL.
Mobile phase：20:80 acetonitrile/water, two-phase adds 0.1% formic acid (v/v), and isocratic elution, flow velocity is 80mL/min, Detection wavelength
For 280nm.Cut F1 and F2 is taken to be isolated and purified by above-mentioned chromatographic condition, it is 3.8-4.6min that retention time is collected respectively
Cut F1-1 and retention time be 14.6-15.5min cut F2-1.Time-consuming 3.5h.
(3) two dimension prepares liquid phase separation
Instrument：Waters purification plants, chromatographic column：ClickXIon (250mm × 20mm × 10 μm), each sample size is 8mL.
Carried out with the water (aqueous phase) for containing 0.1% formic acid and acetonitrile (organic phase) solution for containing 0.1% formic acid as mobile phase isocratic
Elution, organic phase:Aqueous phase (90:10), flow velocity 80ml/min, Detection wavelength 280nm.Take cut F1-1 and F2-1 respectively by
Above-mentioned chromatographic condition is separated, and is received the cut of retention time 12.5-13.5min in F1-1, is obtained betuloside (F1-2)
11.15mg, purity 98% (HPLC), yield (relative to Folium Pini extract) 0.032% takes 13h.
The cut of 1 retention time 16.5-17.5min in F2-1 is collected, massonianoside C (F2-2) 3.8mg, purity is obtained
95% (HPLC), yield (relative to Folium Pini extract) 0.011% takes 17h.
The preparation of example IV pine needle compound
(1) preparation of need testing solution
Folium Pini extract 350g is weighed, 5L 70% ethanol is added, is heated to reflux 1 hour, staticly settles, take supernatant,
Rotary evaporation in vacuo at 60 DEG C, is freeze-dried after concentration, desciccate is dissolved in 400mL ultra-pure waters, crosses 0.45 μm of filter
Film, is produced.Time-consuming 10h.
(2) one-dimensional preparation liquid phase separation
Prepare liquid phase instrument：Waters purification plants, chromatographic column：XAqua C18 (250mm × 100mm × 10 μm), each sample size
For 40mL.Carried out with the water (aqueous phase) containing 0.1% formic acid and the methanol (organic phase) containing 0.1% formic acid as mobile phase
Gradient elution, elution requirement is：0-35min, 25%-60% organic phase, 35-36min, 60%-95% organic phase, 36-55min,
95%-95% organic phase, flow velocity is 300mL/min, and Detection wavelength is 280nm, takes need testing solution according to above-mentioned chromatostrip
Part is separated, and retention time 8.0-9.0min cut F1 and retention time 20.5-22min cut F2 are collected respectively.Consumption
When 12h.
(3) anti-phase octadecylsilane purifying
Instrument：Waters purification plants, chromatographic column：SunFire (150mm × 50mm × 5 μm), each sample size is 20mL.Stream
Dynamic phase：20:80 acetonitrile/water, two-phase adds 0.1% formic acid (v/v), and isocratic elution, flow velocity is 80mL/min, and Detection wavelength is
280nm.Cut F1 and F2 is taken to be isolated and purified by above-mentioned chromatographic condition, it is 3.8-4.6min's to collect retention time in F1
Retention time is 14.6-15.5min cut F2-1 in cut F1-1 and F2.Time-consuming 12h.
(4) two dimension prepares liquid phase separation
Instrument：Waters purification plants, chromatographic column：ClickXIon (250mm × 50mm × 10 μm), each sample size is 20mL.
Carried out with the water (aqueous phase) for containing 0.1% formic acid and acetonitrile (organic phase) solution for containing 0.1% formic acid as mobile phase isocratic
Elution, organic phase:Aqueous phase=10:90, flow velocity 80ml/min, Detection wavelength 280nm.Cut F1-1 and F2-1 are taken respectively by upper
Chromatographic condition separation is stated, the cut of retention time 12.5-13.5min in F1-1 is collected, obtains betuloside (F1-2) 100.8mg,
Compound purity 97% (HPLC), yield (relative to Folium Pini extract) 0.03%.Collect retention time 16.5-17.5min in F2-1
Cut, obtain massonianoside C (F2-2) 35.4mg, purity 96% (HPLC), yield (is extracted relative to pine needle
Thing) 0.01%.Time-consuming 12h.
The compound structure of embodiment five is verified
(1) liquid phase and mass spectrum
Isolated compound in Example three, four, is analyzed using the liquid chromatographs of Agilent 1260.Chromatographic column：
ClickXIon column (4.6 × 250mm, 10 μm), with the aqueous solution (aqueous phase) containing 0.1% formic acid and contain 0.1% formic acid
Acetonitrile solution (organic phase) be used as mobile phase carry out isocratic elution, organic phase:Aqueous phase (90:10), flow velocity 1.0ml/min.Liquid
Facies analysis thing is directly accessed Agilent 6540Q-TOF MS.Air temperature settings are 325 DEG C, and atomization gas pressure is set in
30psi, dry gas flow rate set is that 8L/min. capillary voltages are set in 3.5kV..
Purity and Liquid Chromatography data analysis software are Waters Empower software (version 3.0) and Masslynxsoftware
(version 4.1)(Milford,MA,USA)
MASS SPECTRAL DATA ANALYSIS software is Agilent Masshunter software (version 4.0) (Santa Clara, CA, USA)
(2) nuclear magnetic resonance
Analyzed using Bruker Avance II 600MHz NMRs.Hydrogen is composed：600MHz, sample solvent is MeOD.
Carbon is composed：150MHz, sample solvent is MeOD.
1H NMR and 13C NMR nuclear magnetic datas analysis software is Bruker Topspin software (version 3.0)
(3) qualification result
F1-2 (betuloside, betuloside) compound spectra, mass spectrum and nuclear magnetic data see the table below 1, and compound molecule formula is such as
Shown in formula I：
The compound F1-2 Structural Identification data of table 1
F2-2 (massonianoside C) compound spectra, mass spectrum and nuclear magnetic data see the table below 2, compound molecule formula such as formula II
It is shown：
The compound F2-2 Structural Identification data of table 2
Claims (10)
1. a kind of separation method of pine needle chemical composition, wherein, the chemical composition is betuloside and massonianoside C, its
It is characterised by comprising the steps of：
(1) preparation of need testing solution：Weigh Folium Pini extract appropriate, add organic solvent and extract, extract solution quiescent setting takes
Clear liquid, is freeze-dried after concentration, desciccate is dissolved in ultra-pure water, filtering and collecting filter liquid is produced；
(2) separate：Take need testing solution in step (1), using polarity inversion octadecylsilane chromatographic column as stationary phase, with containing
The methanol solution of formic acid-aqueous solution containing formic acid is mobile phase, carries out gradient elution, and it is 8.0-9.0min that retention time is collected respectively
Cut F1 and retention time be 20.5-22min cut F2；
(3) purify：Take cut F1 and F2 in step (2), respectively using anti-phase octadecylsilane chromatographic column as stationary phase, 20:80
The acetonitrile solution containing formic acid-aqueous solution containing formic acid be mobile phase, isocratic elution, respectively collect retention time be
3.8-4.6min cut F1-1 and retention time is 14.6-15.5min cut F2-1；
(4) separate：Take cut F1-1 and cut F2-1 in step (3), respectively using hydrophilic filler chromatographic column as stationary phase, 90:10
The acetonitrile solution containing formic acid-aqueous solution containing formic acid be mobile phase, carry out isocratic elution, collect F1-1 in retention time
The cut F2-2 for being 16.7-17.3min for retention time in 12.3-12.7min cut F1-2 and F2-1, wherein F1-2 are birch
Xyloside, F2-2 is massonianoside C.
2. separation method according to claim 1, it is characterised in that the methanol solution containing formic acid described in step (2) be containing
There is the methanol solution of 0.1% formic acid；The aqueous solution containing formic acid is the aqueous solution containing 0.1% formic acid；The gradient elution
Condition is：0-35min, 25%-60% contain the methanol solution of 0.1% formic acid, and 35-36min, 60%-95% contains 0.1% formic acid
Methanol solution, 36-55min, 95%-95% contains the methanol solution of 0.1% formic acid.
3. separation method according to claim 1, it is characterised in that the acetonitrile containing formic acid is molten described in step (3) or (4)
Liquid is the acetonitrile solution containing 0.1% formic acid；The aqueous solution containing formic acid is the aqueous solution containing 0.1% formic acid.
4. the separation method according to any one of claim 1-3, it is characterised in that the step (2) or (3) or (4)
Middle Detection wavelength is 280nm.
5. separation method according to claim 1, it is characterised in that organic solvent described in step (1) be selected from methanol, ethanol,
One or more mixtures in acetone, acetonitrile, ethyl acetate, water, preferably methanol solution or 70% ethanol solution；Institute
Filter type is stated for miillpore filter, wherein the aperture of micro porous filtration is 0.22 μm or 0.45 μm；The extraction step carries for backflow
Take or ultrasonic extraction.
6. separation method according to claim 5, it is characterised in that step (1) respectively adds in two times to weigh Folium Pini extract
Enter methanol, ultrasound 1 hour, staticly settles, take rotary evaporation in vacuo at supernatant, 60 DEG C every time, be freeze-dried after concentration,
Desciccate is dissolved in the water, 0.45 μm of filter membrane is crossed, produces；Wherein Folium Pini extract and methanol used in each ultrasonic extraction
Mass volume ratio is 7:100；Or Folium Pini extract 350g is taken, 70% ethanol is added, is heated to reflux 1 hour, staticly settles,
Rotary evaporation in vacuo at supernatant, 60 DEG C is taken, is freeze-dried, desciccate is dissolved in 400mL ultra-pure waters, mistake after concentration
0.45 μm of filter membrane, is produced, and wherein the mass volume ratio of Folium Pini extract and 70% ethanol is 7:100.
7. separation method according to claim 1, it is characterised in that polarity inversion octadecylsilane color in the step (2)
Spectrum post is the chromatographic column that Silica Surface has polar functional group and octadecyl functional group simultaneously；It is preferred that Atlantis dC18 are serial,
Ultimate AQ-C18 series, XBridge C18 series, XAqua C18 series chromatographic columns；Most preferably XAqua C18 systems
Row；The chromatographic column filler particle diameter is 5-60 μm, preferably 10 μm.
8. separation method according to claim 1, it is characterised in that anti-phase octadecylsilane chromatographic column in the step (3)
The chromatographic column of octadecyl functional group is bonded for Silica Surface；It is preferred that Xterra MS are serial, Inertsil ODS-SP series, Zorbax
SB C18 series or the serial chromatographic columns of SunFire C18；Most preferably SunFire C18 series；The chromatographic column filler particle diameter is
5-60 μm, preferably 5 μm.
9. preparation method according to claim 1, it is characterised in that hydrophilic filler chromatographic column is silica gel in the step (4)
The chromatographic column or silica gel chromatographic column of surface bond polar functional group；It is preferred that Atlantis HILIC Silica are serial, Venusil HILIC
Series, XAmide series or ClickXIon series, most preferably ClickXIon series chromatographic columns；Chromatographic column filler particle diameter
For 5-60 μm, preferably 10 μm.
10. preparation method according to claim 1, it is characterised in that the extract from pine needles be using pine needle as raw material, with water or
Aqueous alcoholic solvent is Extraction solvent, extracts obtained extract powder；It is preferred that taking fresh pine needle, add water to cook twice, 8 times are added for the first time
Solvent is measured, is decocted 1.5 hours, second plus 6 times of amount solvents are decocted 1.5 hours；Filtrate decompression is condensed into relative density and existed
1.2 medicinal extract is detected as at 70 DEG C；Spray drying, is produced.
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| CN110141576A (en) * | 2018-02-12 | 2019-08-20 | 成都康弘制药有限公司 | A kind of compound is used to prepare the application in treatment oxidative damage related disease drug |
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|---|---|---|---|---|
| CN103123344A (en) * | 2012-07-13 | 2013-05-29 | 成都康弘制药有限公司 | Method for measuring fingerprints of medicinal composition |
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| CN103123344A (en) * | 2012-07-13 | 2013-05-29 | 成都康弘制药有限公司 | Method for measuring fingerprints of medicinal composition |
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| JUN DANG ET AL.: "Antioxidative extracts and phenols isolated from Qinghai–Tibet Plateau medicinal plant Saxifraga tangutica Engl", 《INDUSTRIAL CROPS AND PRODUCTS》 * |
| YONG-LI LI ET AL.: "Chemical constituents of Abies fabri", 《PHYTOCHEMISTRY》 * |
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| CN110141576A (en) * | 2018-02-12 | 2019-08-20 | 成都康弘制药有限公司 | A kind of compound is used to prepare the application in treatment oxidative damage related disease drug |
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