CN107459467B - A kind of new amine D of Roripa montana, the new amine E of Roripa montana and the preparation method and application thereof - Google Patents
A kind of new amine D of Roripa montana, the new amine E of Roripa montana and the preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/22—Separation; Purification; Stabilisation; Use of additives
- C07C231/24—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
- C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
- C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
Abstract
The present invention relates to the new amine D of Roripa montana, the new amine E of Roripa montana and the preparation method and application thereof, it effectively solves the problems, such as to extract the new amine D of Roripa montana, the new amine E of Roripa montana from lepidii,semen and is used to prepare treatment cardiac muscle cell H9c2 damage medicine, lepidii,semen is fried, add water to cook extraction, extracting solution alcohol precipitation, supernatant is concentrated into no alcohol taste, upper Dianion HP-20 column, with water, ethanol elution, ethanol eluate silica gel mixed sample, methylene chloride, methyl alcohol mixed liquor elution, methylene chloride, methanol 10:1 fraction are component C3, and methylene chloride, methanol 5:1 fraction are component C4；Toyopearl HW-40 chromatographic column, methanol elution are crossed in the dissolution of component C3 methanol, and Sephadex LH-20 column chromatography, methanol elution are crossed in the fraction methanol dissolution of 150 ~ 400 mL, and retention time t is collected in the half preparation HPLC separation of fraction of 80 ~ 150 mLRThe fraction of=35.8 ~ 36.3min, it is dry, obtain the new amine E of Roripa montana；Component C4 is dissolved with water, crosses ODS column chromatography, water, methanol elution gradient, and retention time t is collected in 60% meoh eluate, half preparation HPLC separationRThe fraction of=66.5 ~ 66.8min, it is dry, obtain the new amine D of Roripa montana.
Description
Technical field
The present invention relates to medicine, the new amine D of especially a kind of Roripa montana, the new amine E of Roripa montana and the preparation method and application thereof.
Background technique
Lepidium seed is parts of generic medicinal plants, first recorded in Shennong's Herbal, is classified as careless subordinate's product, the flavour of a drug are pungent, bitter, and property is big
It is cold, return lung and bladder meridian, have effects that lead off relieving asthma, line water detumescence.2015 editions " Chinese Pharmacopoeia " " lepidium seeds " included have
Point in north and south, wherein the dry mature seed of crucifer Lepidium apetalum Lepidium apetalum Willd., which is practised, claims " north
Lepidium seed ", and the dry mature seed of Descurainia sophia Descurainia Sophia (L.) Webbex Prantl. is practised and claims " southern Roripa montana
Son ".
It is found from the test of research, benzamide compound is contained in lepidii,semen, i.e. the new amine D of Roripa montana, Roripa montana is new
Amine E, but how such compound extracts from lepidii,semen, and effective for preparation treatment cardiac muscle cell H9c2 damage
Drug, so far there are no is publicly reported.
Summary of the invention
For above situation, for the defect for overcoming the prior art, the purpose of the present invention be just to provide a kind of new amine D of Roripa montana,
New amine E of Roripa montana and the preparation method and application thereof can effectively solve to extract the new amine D of Roripa montana, the new amine E of Roripa montana and use from lepidii,semen
In preparation treat cardiac muscle cell H9c2 damage medicine the problem of.
The technical solution that the present invention solves is, a kind of new amine D of Roripa montana, the new amine E of Roripa montana and the preparation method and application thereof, wherein
The new amine D molecular formula of Roripa montana is C20H22N2O5, the new amine E molecular formula of Roripa montana is C18H20N2O4, molecular structural formula is shown in Fig. 1, and the Roripa montana is new
The preparation method of the new amine E of amine D, Roripa montana is:
Lepidii,semen is taken, is processed using method of frying, 240 DEG C of temperature, frying 5.5min takes the lepidii,semen of frying, every time
Adding the water boiling and extraction of 10 times of weight of lepidii,semen, each 1.5h, extracting solution, which is concentrated under reduced pressure into, is equivalent to 2g/mL containing crude drug three times
Medicinal extract, at room temperature, 5 times of volumes of medicinal extract medicinal extract, volumetric concentration be 80% ethyl alcohol alcohol precipitation, stand 1h, supernatant is fallen
Out, the ethyl alcohol alcohol precipitation that precipitating adds 5 times of volumes of medicinal extract, volumetric concentration is 80%, operates 4 times, repeatedly by gained supernatant
Liquid is concentrated into no alcohol taste, upper Dianion HP-20 column, successively with the water of 10 times of column volumes, volumetric concentration 20%, 40%, 60%
Ethanol gradient elution, flow velocity 6mL/min collect 60% ethanol eluate, obtain 60% alcohol elution A；At room temperature, 60%
The water of 5 times of volumes of alcohol elution A dissolves, and is centrifuged 15min with the revolving speed of 6000r/min, and 20 DEG C of temperature, gained supernatant
It is denoted as hydrotrope component B1, precipitating after centrifugation is denoted as water-insoluble B2；Component B2 is dissolved with methanol, silica gel mixed sample, component
B2 and silica gel dosage are 1:1, are eluted by eluent gradient of Er Lv Jia Wan ︰ methanol, flow velocity 10ml/min, Er Lv Jia Wan ︰ methanol
Volume ratio is followed successively by 100:0,20:1,10:1,5:1,1:1, and every 200ml inspection is known once, and the dosage of each gradient mobile phase is with fennel
Fragrant aldehyde-concentrated sulfuric acid thin layer inspection is known to judge, anisaldehyde-concentrated sulfuric acid thin layer inspection is known without color, then changes next ratio elution, and 3d is washed
It is de- to finish, merge methylene chloride: methanol=10:1 fraction, be designated as component C3, merges methylene chloride: methanol=5:1 fraction,
It is designated as component C4；
Component C3 70% methanol of volumetric concentration dissolves, by Toyopearl HW-40 chromatographic column, with 70% methanol
500mL elution, flow velocity 1.5ml/min are known with anisaldehyde-concentrated sulfuric acid thin layer inspection, and the fraction merged between 150~400mL is designated as
Component D2；Component D2 is dissolved with methanol, by Sephadex LH-20 column chromatography, is eluted with methanol 300mL, flow velocity 1ml/min,
Known with anisaldehyde-concentrated sulfuric acid thin layer inspection, the fraction for merging 80~150mL is designated as component E1；Half preparation HPLC separation of component E1,
Upper specifications and models are as follows: 250 × 10mm, 5 μm of partial size, the YMC-Pack ODS-AA chromatographic column of aperture 12nm, mobile phase is acetonitrile:
Water=11:89, flow velocity 3ml/min collect retention time tRThe fraction of=35.8~36.3min is concentrated and dried, obtains compound
The new amine E of Roripa montana；
Component C4 is dissolved with water, by ODS column chromatography, successively uses water, 10%, 20%, 30%, 60% methanol of volumetric concentration
Gradient elution, flow velocity 3ml/min are known with anisaldehyde-concentrated sulfuric acid thin layer inspection to judge to elute terminal, anisaldehyde-concentrated sulfuric acid thin layer
Inspection is known without color, then changes next ratio elution, and every 20ml inspection knowledge is primary, and 2d elution finishes, and merges 60% methanol elution fraction
It is designated as component D5；Component D5 half preparation HPLC separation, upper specifications and models are as follows: 250 × 20mm, 5 μm of partial size, aperture 12nm's
YMC-Pack ODS-AA chromatographic column, mobile phase is acetonitrile: water=25:75, flow velocity 5ml/min, collects retention time tR=32~
Fraction between 65min is designated as component E4；Component E4 passes through half preparation HPLC, upper specifications and models are as follows: 250 × 10mm, 5 μ of partial size
The YMC-Pack ODS-AA chromatographic column of m, aperture 12nm, mobile phase is acetonitrile: water=20:80, flow velocity 3ml/min, collects and retains
Time tRThe fraction of=66.5~66.8min is concentrated and dried, obtains the new amine D of compound Roripa montana.
The new amine D of the compound Roripa montana, the new amine E of Roripa montana can significantly improve cell viability, effective for preparation treatment cardiac muscle
The drug of cell H9c2 damage.
The new amine D of Roripa montana of the invention, the new amine E of Roripa montana are a kind of noval chemical compounds extracted from lepidii,semen, and raw material is rich
Richness, preparation method is scientific and reasonable, which has protective effect to cardiac muscle cell, can be effectively used for preparation treatment cardiac muscle cell
The drug of H9c2 damage has opened up the new way for the treatment of myocardial cell injury drug and medical value and business that lepidii,semen is new
Value, there is significant economic and social benefit.
Detailed description of the invention
Fig. 1 is the new amine D of the compounds of this invention Roripa montana, the new amine E molecular structural formula figure of Roripa montana.
Fig. 2 is the new amine D's of the compounds of this invention Roripa montana1H-NMR composes (in CD3OD) figure.
Fig. 3 is the new amine D's of the compounds of this invention Roripa montana13C-NMR composes (in CD3OD) figure.
The DEPT 135 that Fig. 4 is the new amine D of the compounds of this invention Roripa montana composes (in CD3OD) figure.
Fig. 5 is the new amine D's of the compounds of this invention Roripa montana1H-1H COSY spectrogram.
Fig. 6 is the hsqc spectrum figure of the new amine D of the compounds of this invention Roripa montana.
Fig. 7 is the HMBC spectrogram of the new amine D of the compounds of this invention Roripa montana.
Fig. 8 is the NOESY spectrogram of the new amine D of the compounds of this invention Roripa montana.
Fig. 9 is the HR-ESI-MS spectrogram of the new amine D of the compounds of this invention Roripa montana.
Figure 10 is the IR spectrogram of the new amine D of the compounds of this invention Roripa montana.
Figure 11 is the UV spectrogram of the new amine D of the compounds of this invention Roripa montana.
Figure 12 is the new amine E's of the compounds of this invention Roripa montana1H-NMR composes (in DMSO-d6) figure.
Figure 13 is the new amine E's of the compounds of this invention Roripa montana13C-NMR composes (in DMSO-d6) figure.
Figure 14 is the DEPT135 spectrogram of the new amine E of the compounds of this invention Roripa montana.
Figure 15 is the new amine E's of the compounds of this invention Roripa montana1H-1H COSY spectrogram.
Figure 16 is the hsqc spectrum figure of the new amine E of the compounds of this invention Roripa montana.
Figure 17 is the HMBC spectrogram of the new amine E of the compounds of this invention Roripa montana.
Figure 18 is the NOESY spectrogram of the new amine E of the compounds of this invention Roripa montana.
Figure 19 is the HR-ESI-MSMS spectrogram of the new amine E of the compounds of this invention Roripa montana.
Figure 20 is the IR spectrogram of the new amine E of the compounds of this invention Roripa montana.
Figure 21 is the UV spectrogram of the new amine E of the compounds of this invention Roripa montana.
Specific embodiment
It elaborates below in conjunction with concrete condition to a specific embodiment of the invention.
The present invention in specific implementation, a kind of new amine D of Roripa montana, the new amine E of Roripa montana and the preparation method and application thereof, wherein Roripa montana
New amine D molecular formula is C20H22N2O5, the new amine E molecular formula of Roripa montana is C18H20N2O4, molecular structural formula is:
The new amine D of the Roripa montana, the new amine E of Roripa montana preparation method be:
Lepidii,semen 10kg is taken, is processed using method of frying, 240 DEG C of temperature, frying 5.5min takes the lepidii,semen of frying,
Three times, each 1.5h, extracting solution is concentrated under reduced pressure into be equivalent to containing crude drug each plus 10 times of weight of lepidii,semen water boiling and extraction
The medicinal extract (5L) of 2g/mL, at room temperature, the ethyl alcohol 25L alcohol precipitation that medicinal extract volumetric concentration is 80% stands 1h, supernatant is fallen
Out, precipitating adds the ethyl alcohol 25L alcohol precipitation that volumetric concentration is 80%, operates 4 times repeatedly, gained supernatant is concentrated into nothing
Alcohol taste, upper Dianion HP-20 column are successively washed with the water of 10 times of column volumes, 20%, 40%, 60% ethanol gradient of volumetric concentration
De-, flow velocity 6mL/min collects 60% ethanol eluate, obtains 60% alcohol elution A；At room temperature, 60% ethanol elution portion
The water of 5 times of volumes of position A dissolves, and is centrifuged 15min with the revolving speed of 6000r/min, 20 DEG C of temperature, gained supernatant is denoted as the hydrotrope
Component B1, precipitating after centrifugation are denoted as water-insoluble B2；Component B2 is dissolved with methanol, silica gel mixed sample, and component B2 and silica gel are used
Amount is 1:1, is eluted by eluent gradient of Er Lv Jia Wan ︰ methanol, flow velocity 10ml/min, Er Lv Jia Wan ︰ methanol volume ratio is successively
For 100:0,20:1,10:1,5:1,1:1, every 200ml inspection is known once, and the dosage of each gradient mobile phase is with anisaldehyde-concentrated sulfuric acid
Thin layer inspection is known to judge, anisaldehyde-concentrated sulfuric acid thin layer inspection is known without color, then changes next ratio elution, and 3d elution finishes, merges
Methylene chloride: methanol=10:1 fraction is designated as component C3, and merge methylene chloride: methanol=5:1 fraction is designated as component C4；
Component C3 70% methanol of volumetric concentration dissolves, by Toyopearl HW-40 chromatographic column, with 70% methanol
500mL elution, flow velocity 1.5ml/min are known with anisaldehyde-concentrated sulfuric acid thin layer inspection, and the fraction merged between 150~400mL is designated as
Component D2；Component D2 is dissolved with methanol, by Sephadex LH-20 column chromatography, is eluted with methanol 300mL, flow velocity 1ml/min,
Known with anisaldehyde-concentrated sulfuric acid thin layer inspection, the fraction for merging 80~150mL is designated as component E1；Half preparation HPLC separation of component E1,
Upper specifications and models are as follows: 250 × 10mm, 5 μm of partial size, the YMC-Pack ODS-AA chromatographic column of aperture 12nm, mobile phase is acetonitrile:
Water=11:89, flow velocity 3ml/min collect retention time tRThe fraction of=35.8~36.3min is concentrated and dried, obtains compound
The new amine E of Roripa montana；
Component C4 is dissolved with water, by ODS column chromatography, successively uses water, 10%, 20%, 30%, 60% methanol of volumetric concentration
Gradient elution, flow velocity 3ml/min are known with anisaldehyde-concentrated sulfuric acid thin layer inspection to judge to elute terminal, anisaldehyde-concentrated sulfuric acid thin layer
Inspection is known without color, then changes next ratio elution, and every 20ml inspection knowledge is primary, and 2d elution finishes, and merges 60% methanol elution fraction
It is designated as component D5；Component D5 half preparation HPLC separation, upper specifications and models are as follows: 250 × 20mm, 5 μm of partial size, aperture 12nm's
YMC-Pack ODS-AA chromatographic column, mobile phase is acetonitrile: water=25:75, flow velocity 5ml/min, collects retention time tR=32~
Fraction between 65min is designated as component E4；Component E4 passes through half preparation HPLC, upper specifications and models are as follows: 250 × 10mm, 5 μ of partial size
The YMC-Pack ODS-AA chromatographic column of m, aperture 12nm, mobile phase is acetonitrile: water=20:80, flow velocity 3ml/min, collects and retains
Time tRThe fraction of=66.5~66.8min is concentrated and dried, obtains the new amine D of compound Roripa montana.
Through liquid chromatography for measuring, the new amine D molecular formula of compound Roripa montana is C20H22N2O5, the new amine E molecular formula of Roripa montana is
C18H20N2O4, molecular structural formula is:
Roripa montana new amine D, Light yellow crystals powder (CH3OH)。HR-ESI-MS gives
Quasi-molecular ion peak m/z:393.1443 [M+Na] out+(calcd.For C20H22N2O5Na 393.1426), determine its molecular formula
For C20H22N2O5；UV(MeOH)λmax:204(2.74),224(1.93),279(0.66)
nm；IR(KBr)νmaxcm-1: 3308,2930,2856,1641,1613,1516,1202,1027,720cm-1；
Roripa montana new amine E, Light yellow crystals powder (CH3OH).HR-ESI-MS provides quasi-molecular ion peak m/z:
351.1413[M+Na]+(calcd.For C18H20N2O4Na 351.1321), determine that its molecular formula is UV(MeOH)λmax:208(1.28),215(1.25),260
(0.42),295(0.25)nm；IR(KBr)νmaxcm-1: 3160,2931,2853,1675,1623,1184,1133,721cm-1；
The new amine D of the Roripa montana, the new amine E of Roripa montana have to myocardial cell protection effect, treat cardiac muscle cell effective for preparation
The drug of H9c2 damage, and extraordinary advantageous effects are achieved with test after tested, related testing data is as follows:
1 instrument and reagent
Nuclear magnetic resonance is used with III 500 Nuclear Magnetic Resonance of Bruker AVANCE (TMS internal standard) (Bruker), infrared spectroscopy
Nicolet is 10Microscope Spectrometer (Thermo Scientific, USA), high resolution mass spectrum are used
Bruker maxis HD mass spectrometer, ultraviolet spectra Shimadzu UV-2401PC apparatus, efficiently
The serial 2695 efficient liquid phase systems of phase chromatography-use Waters Alliance, are equipped with 2998 type diode array detector,
Empower3 Data Processing in Chromatography Workstation, LC50 type high pressure preparative liquid chromatograph, [match spectrum is sharp to think (north to UV200 type UV detector
Capital) Science and Technology Ltd.], YMC-Pack ODS-A chromatographic column (250 × 10mm.D.S-5mm, 12mm) (YMC Co., Ltd),
It is remaining to have N-1100 type Rotary Evaporators (Shanghai Ai Lang Instrument Ltd), A-1000S type water flow air exhauster (Shanghai Ai Lang instrument
Co., Ltd), N-1111 type freezes water circle device (Shanghai Ai Lang Instrument Ltd), FDU-2110 type freeze drier
(Shanghai Ai Lang Instrument Ltd), DFZ-60508 type vacuum oven (Shanghai Yiheng Scientific Instruments Co., Ltd), AB204-
N a ten thousandth analytical precision balances (METTLER TOLEDO), iMARK type microplate reader (U.S. BIO-RAD), carbon dioxide culture
Case (Shanghai STIK), superclean bench (Su Jing group), the super combined Superpure water machine (SARTORIUS) of 611 VF of Arium,
Inverted microscope (Nikon), PB-10 acidometer (German Sai Duolisi group).
Column chromatographic stuffing Diaion HP-20, MCI Gel CHP-20 (Mitsubishi chemical company), Toyopearl HW-
40 (Japanese TOSOH companies), Sephadex LH-20 (Parmacia Biotech company), column chromatograph used silica gel H (160-
200 mesh) it is that Haiyang Chemical Plant, Qingdao produces, culture dish, 96 well culture plates, cell cryopreservation tube (Corning company), chromatography used
The production of pure reagent position α Cygni friend's fine chemicals Co., Ltd, analytical reagents used are Beijing Chemical Plant and Tianjin third
Learn chemical reagent work's production.
DMEM high sugar culture solution (Gibco), fetal calf serum (Zhejiang Tian Hang Biotechnology Co., Ltd), trypsase
(Gibco), DMSO (Solarbio)；MTT (Biosharp), H2O2Solution (Tianjin Heng Xing chemical reagent Manufacturing Co., Ltd),
Water is ultrapure water, and PBS buffer solution etc. is autogamy.
Lepidii,semen picked up from Nanyang, henan in 2014, identified through Henan College Of Traditional Chinese Medicine professor Chen Suiqing and professor Dong Chengming
For the dry mature seed of crucifer Lepidium apetalum Lepidium apetalum Willd..
2 Structural Identifications
Roripa montana new amine D, Light yellow crystals powder (CH3OH)。HR-ESI-MS gives
Quasi-molecular ion peak m/z:393.1443 [M+Na] out+(calcd.For C20H22N2O5Na 393.1426), determine its molecular formula
ForUV(MeOH)λmax:204(2.74),224(1.93),279
(0.66)nm；IR(KBr)νmaxcm-1: 3308,2930,2856,1641,1613,1516,1202,1027,720cm-1.Its structure
Formula and nuclear magnetic data are as follows:
Roripa montana new amine E, Light yellow crystals powder (CH3OH).HR-ESI-MS provides quasi-molecular ion peak m/z:
351.1413[M+Na]+(calcd.For C18H20N2O4Na 351.1321), determine that its molecular formula is C18H20N2O4； UV(MeOH)λmax:208(1.28),215(1.25),260(0.42),295(0.25)
nm；IR(KBr)νmaxcm-1: 3160,2931,2853,1675,1623,1184,1133,721cm-1.Its structural formula and nuclear-magnetism number
According to as follows:
NMR data (the in CD of the new amine D of 1 compound Roripa montana of table3OD)
NMR data (the in DMSO-d of the new amine E of 2 compound Roripa montana of table6)
3 Activity determinations
3.1 experimental method
H9c2 cell is divided into 4 groups: normal, model (20 μ gmL of LPS-1), the north new amine D of Roripa [10 μM of+LPS (20 μ gmL-1)], the north new amine E of Roripa [10 μM of+LPS (20 μ gmL-1)] group.Cell density is 2 × 104A/mL is inoculated in 96 orifice plate cultures
12h, for 24 hours, mtt assay surveys absorbance for the rear culture medium culture with drug containing, calculates cell viability.Meanwhile ELISA method detection cell
The level of supernatant IL-6 and TNF-α are operated according to ELISA kit specification.
3.2 experimental result
Compared with normal group (Con), TNF-α and IL-6 in the significant decrease of model group (M) cell viability, cell supernatant
Horizontal conspicuousness increases；The northern new amine D of the Roripa and new amine E of northern Roripa then can be on conspicuousness elevation model group cell viability, reduction cell
TNF-α and IL-6 are horizontal in clear liquid, are shown in Table 3.
The 3 north new amine D of Roripa of table and the north new amine E of Roripa to cell viability, TNF-α, IL-6 influence (N=6)
The present invention separates from the water extract of lepidii,semen and identifies two new benzamide compounds, it may be assumed that north
The new amine D of the Roripa and new amine E of northern Roripa, experimental result show the two new amine compounds energy significant improvement lipopolysaccharides to H9c2 cell
Caused by cell viability reduces, TNF-α and IL-6 level increase, to have the function of myocardial preservation, abundant raw material, preparation side
Method is simple, has opened up the new way for the treatment of myocardial cell injury drug, has opened up the medical value and commercial value of lepidii,semen, be
The innovation on cardiac muscle cell's H9c2 damage medicine is treated, there is significant economic and social benefit.
Claims (4)
1. a kind of new amine D of the Roripa montana extracted in lepidii,semen, the new amine E of Roripa montana, the new amine D molecular formula of Roripa montana is C20H22N2O5, Roripa montana
New amine E molecular formula is C18H20N2O4, molecular structural formula is:
2. the preparation method of the new amine D of Roripa montana described in claim 1, the new amine E of Roripa montana, which is characterized in that take lepidii,semen, use
Method processing is fried, 240 DEG C of temperature, frying 5.5min takes the lepidii,semen of frying, adds the decocting of 10 times of weight of lepidii,semen every time
Boiling extraction three times, each 1.5h, extracting solution is concentrated under reduced pressure into the medicinal extract for being equivalent to the 2g/mL containing crude drug, at room temperature, medicinal extract leaching
5 times of volumes of cream, volumetric concentration be 80% ethyl alcohol alcohol precipitation, stand 1h, supernatant is poured out, precipitating add 5 times of volumes of medicinal extract,
The ethyl alcohol alcohol precipitation that volumetric concentration is 80% operates 4 times repeatedly, gained supernatant is concentrated into no alcohol taste, upper Dianion
HP-20 column, successively with the water of 10 times of column volumes, 20%, 40%, 60% ethanol gradient elution of volumetric concentration, flow velocity 6mL/min,
60% ethanol eluate is collected, 60% alcohol elution A is obtained；At room temperature, 60% alcohol elution A 5 times of volumes
Water dissolution is centrifuged 15min with the revolving speed of 6000r/min, and 20 DEG C of temperature, gained supernatant is denoted as hydrotrope component B1, is centrifuged it
Precipitating afterwards is denoted as water-insoluble B2；Component B2 is dissolved with methanol, silica gel mixed sample, and component B2 and silica gel dosage are 1:1, with dichloro
Jia Wan ︰ methanol be eluent gradient elution, flow velocity 10ml/min, Er Lv Jia Wan ︰ methanol volume ratio be followed successively by 100:0,20:1,
10:1,5:1,1:1, every 200ml inspection are known once, and the dosage of each gradient mobile phase is known with anisaldehyde-concentrated sulfuric acid thin layer inspection to sentence
Disconnected, anisaldehyde-concentrated sulfuric acid thin layer inspection is known without color, then changes next ratio elution, and 3d elution finishes, and merges methylene chloride: first
Alcohol=10:1 fraction is designated as component C3, and merge methylene chloride: methanol=5:1 fraction is designated as component C4；
Component C3 70% methanol of volumetric concentration dissolves, and by Toyopearl HW-40 chromatographic column, is washed with 70% methanol 500mL
De-, flow velocity 1.5ml/min is known with anisaldehyde-concentrated sulfuric acid thin layer inspection, and the fraction merged between 150~400mL is designated as component D2；
Component D2 is dissolved with methanol, by Sephadex LH-20 column chromatography, is eluted with methanol 300mL, flow velocity 1ml/min, with fennel
Aldehyde-concentrated sulfuric acid thin layer inspection is known, and the fraction for merging 80~150mL is designated as component E1；Component E1 half preparation HPLC separation, upper specification
Model are as follows: 250 × 10mm, 5 μm of partial size, the YMC-Pack ODS-AA chromatographic column of aperture 12nm, mobile phase is acetonitrile: water=11:
89, flow velocity 3ml/min collect retention time tRThe fraction of=35.8~36.3min is concentrated and dried, it is new to obtain compound Roripa montana
Amine E；
Component C4 is dissolved with water, by ODS column chromatography, successively uses water, 10%, 20%, 30%, 60% methanol gradient of volumetric concentration
Elution, flow velocity 3ml/min are known with anisaldehyde-concentrated sulfuric acid thin layer inspection to judge to elute terminal, and anisaldehyde-concentrated sulfuric acid thin layer inspection is known
Without color, then next ratio elution is changed, every 20ml inspection is known once, and 2d elution finishes, and merges 60% methanol elution fraction and is designated as
Component D5；Component D5 half preparation HPLC separation, upper specifications and models are as follows: 250 × 20mm, 5 μm of partial size, the YMC- of aperture 12nm
Pack ODS-AA chromatographic column, mobile phase is acetonitrile: water=25:75, flow velocity 5ml/min, collects retention time tR=32~
Fraction between 65min is designated as component E4；Component E4 passes through half preparation HPLC, upper specifications and models are as follows: 250 × 10mm, 5 μ of partial size
The YMC-Pack ODS-AA chromatographic column of m, aperture 12nm, mobile phase is acetonitrile: water=20:80, flow velocity 3ml/min, collects and retains
Time tRThe fraction of=66.5~66.8min is concentrated and dried, obtains the new amine D of compound Roripa montana.
3. the preparation method of the new amine D of Roripa montana according to claim 1, the new amine E of Roripa montana, which is characterized in that take lepidii,semen
10kg is processed using method of frying, and 240 DEG C of temperature, frying 5.5min takes the lepidii,semen of frying, adds 10 times of lepidii,semen every time
Three times, each 1.5h, extracting solution is concentrated under reduced pressure into the medicinal extract 5L for being equivalent to the 2g/mL containing crude drug to the water boiling and extraction of weight, in room
Under temperature, the ethyl alcohol 25L alcohol precipitation that medicinal extract volumetric concentration is 80% stands 1h, supernatant is poured out, and precipitating adds volumetric concentration
For 80% ethyl alcohol 25L alcohol precipitation, operates 4 times repeatedly, gained supernatant is concentrated into no alcohol taste, upper Dianion HP-20
Column, successively with the water of 10 times of column volumes, 20%, 40%, 60% ethanol gradient elution of volumetric concentration, flow velocity 6mL/min, collection
60% ethanol eluate obtains 60% alcohol elution A；At room temperature, 60% alcohol elution A, 5 times of volumes is water-soluble
Solution is centrifuged 15min with the revolving speed of 6000r/min, and 20 DEG C of temperature, gained supernatant is denoted as hydrotrope component B1, after centrifugation
Precipitating is denoted as water-insoluble B2；Component B2 is dissolved with methanol, silica gel mixed sample, and component B2 and silica gel dosage are 1:1, with dichloromethane
Wan ︰ methanol is eluent gradient elution, and flow velocity 10ml/min, Er Lv Jia Wan ︰ methanol volume ratio is followed successively by 100:0,20:1,10:
1,5:1,1:1, every 200ml inspection are known once, and the dosage of each gradient mobile phase is known with anisaldehyde-concentrated sulfuric acid thin layer inspection to judge,
Anisaldehyde-concentrated sulfuric acid thin layer inspection is known without color, then changes next ratio elution, and 3d elution finishes, merging methylene chloride: methanol=
The fraction of 10:1 is designated as component C3, and merge methylene chloride: methanol=5:1 fraction is designated as component C4；
Component C3 70% methanol of volumetric concentration dissolves, and by Toyopearl HW-40 chromatographic column, is washed with 70% methanol 500mL
De-, flow velocity 1.5ml/min is known with anisaldehyde-concentrated sulfuric acid thin layer inspection, and the fraction merged between 150~400mL is designated as component D2；
Component D2 is dissolved with methanol, by Sephadex LH-20 column chromatography, is eluted with methanol 300mL, flow velocity 1ml/min, with fennel
Aldehyde-concentrated sulfuric acid thin layer inspection is known, and the fraction for merging 80~150mL is designated as component E1；Component E1 half preparation HPLC separation, upper specification
Model are as follows: 250 × 10mm, 5 μm of partial size, the YMC-Pack ODS-AA chromatographic column of aperture 12nm, mobile phase is acetonitrile: water=11:
89, flow velocity 3ml/min collect retention time tRThe fraction of=35.8~36.3min is concentrated and dried, it is new to obtain compound Roripa montana
Amine E；
Component C4 is dissolved with water, by ODS column chromatography, successively uses water, 10%, 20%, 30%, 60% methanol gradient of volumetric concentration
Elution, flow velocity 3ml/min are known with anisaldehyde-concentrated sulfuric acid thin layer inspection to judge to elute terminal, and anisaldehyde-concentrated sulfuric acid thin layer inspection is known
Without color, then next ratio elution is changed, every 20ml inspection is known once, and 2d elution finishes, and merges 60% methanol elution fraction and is designated as
Component D5；Component D5 half preparation HPLC separation, upper specifications and models are as follows: 250 × 20mm, 5 μm of partial size, the YMC- of aperture 12nm
Pack ODS-AA chromatographic column, mobile phase is acetonitrile: water=25:75, flow velocity 5ml/min, collects retention time tR=32~
Fraction between 65min is designated as component E4；Component E4 passes through half preparation HPLC, upper specifications and models are as follows: 250 × 10mm, 5 μ of partial size
The YMC-Pack ODS-AA chromatographic column of m, aperture 12nm, mobile phase is acetonitrile: water=20:80, flow velocity 3ml/min, collects and retains
Time tRThe fraction of=66.5~66.8min is concentrated and dried, obtains the new amine D of compound Roripa montana.
4. the new amine D of Roripa montana described in claim 1, the new amine E of Roripa montana answering in preparation treatment cardiac muscle cell H9c2 damage medicine
With.
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