CN104761562B - Method for extracting and separating four or five alkaloids from the Tibetan medicine hypecoum leptocarpum - Google Patents
Method for extracting and separating four or five alkaloids from the Tibetan medicine hypecoum leptocarpum Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
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- C07C231/00—Preparation of carboxylic acid amides
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- C07C231/24—Separation; Purification
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- C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
- C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
- C07C235/32—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
- C07C235/34—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
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- C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
- C07D217/12—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring
- C07D217/18—Aralkyl radicals
- C07D217/20—Aralkyl radicals with oxygen atoms directly attached to the aromatic ring of said aralkyl radical, e.g. papaverine
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- C07D455/00—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
- C07D455/03—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing quinolizine ring systems directly condensed with at least one six-membered carbocyclic ring, e.g. protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
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Abstract
The present invention provides a method for extracting and separating four or five alkaloids from the Tibetan medicine Hypecoum leptocarpum. The method provided by the invention uses high speed countercurrent chromatography for extraction and separation four or alkaloids compounds from hypecoum leptocarpum, and has the advanategs of simple operation, obviously shortened separation time, and high purity of the obtained compounds compared with the traditional column chromatography method . At the same time, in the research process of the present invention, a novel compound-formula a-1 is separated and obtained, and the compound has certain antioxidant activity, and provides a new choice for clinical medication.
Description
Technical field
The present invention relates to extracting the method separating four kinds or five kinds alkaloids from Tibetan medicine Herba Hypecoi Leptocarpi.
Background technology
Tibetan medicine Radix Hypecol Erecti (Hypecoum leptocarpum Hook.f.et Thoms) is Papaveraceae Radix Hypecol Erecti platymiscium,
Another name throat grass, mountain Rhizoma Coptidis or wild Fructus Foeniculi etc., the entitled Ba Er of Tibetan medicine beats, Ba Erbada.It is grown on the grass of below height above sea level 4300m
On former, grassy marshland, sandy ground, bitter in the mouth is cool in nature, clearing away heat from blood, warm and scorchingly hot.《Chinese herbal medicine is commonly used in Tibet》It is claimed to have antipyretic-antalgic, disappear
Scorching merit of removing toxic substances effect, can control Common Cold, headache, extremities joint pain, cholecystitis, and solve alimentary toxicosis.《The sweet peaceful green grass or young crops medium-height grass in Shan
Medicine selects》Middle title：Herba Hypecoi Leptocarpi can control the diseases such as influenza, laryngopharynx swelling and pain, conjunctival congestion.Modern Analytical Chemistry and natural product chemistry research
Find：Multiple alkaloid components are contained, such as in Herba Hypecoi Leptocarpi：Protopine, cryptocavine, Sanguinarine etc..
Herba Hypecoi Leptocarpi alkaloid is to extract the alkaloid obtaining from Herba Hypecoi Leptocarpi herb.By consulting literatures, send out
Now also systematic study is not carried out to its active component.Research according to congener finds, the active component master of this platymiscium
Alkaloid to be.Up to the present only reported the report to Alkaloid separation in Herba Hypecoi Leptocarpi using traditional column chromatography
Road.
High speed adverse current chromatogram (High-speed counter-current chromatography, abbreviation HSCCC) separates
Technology is a kind of liquid luquid partition chromatography isolation technics of continuous high-efficient, and it, without the supporter of any solid-state or carrier, utilizes two
Phase solvent system sets up a kind of special unipolarity fluid dynamic equilibrium in the serpentine pipe of high-speed rotation, and wherein one mutually makees
For fixing phase, another as mobile phase, substantial amounts of fixing phase, the separation foundation of material can be retained during continuous eluting
Its difference in biphase middle partition coefficient and realize, be particularly suitable for the separation of natural product active ingredient.
Yet there are no someone using HSCCC technology, the alkaloid in Herba Hypecoi Leptocarpi to be isolated and purified.
Content of the invention
It is an object of the invention to provide a kind of using HSCCC technology extract from Tibetan medicine Herba Hypecoi Leptocarpi separately four kinds or
The method of five kinds of alkaloids.
Specifically, the invention provides extracting, from Tibetan medicine Herba Hypecoi Leptocarpi, the method separating four kinds of alkaloids, it includes
Following operating procedure：
(1) preparation of crude extract：
Take Herba Hypecoi Leptocarpi, be solvent extraction with 70%-95%v/v ethanol, extracting solution concentrates after removing ethanol, and gained soaks
The acid of cream salt adding, removes insoluble matter, and after solution defat, plus alkali adjusts pH to 9～10, then with chloroform extraction, collects chloroform layer, removes
After solvent, it is dried to obtain alkaloid crude extract；
(2) high-speed countercurrent chromatography is adopted to separate：
Take n-hexane-ethyl acetate-methanol-water=3: 5: 2: 5, it is placed in preparation two-phase solvent system in separatory funnel, take
Lower addition hydrochloric acid is as fixing phase during 5～10mM/L to its concentration, and upper addition triethylamine to its concentration is as stream during 10mM
Dynamic phase；Fixing phase is full of the chromatographic column of adverse current chromatogram, at 25-28 DEG C, main frame rotates forward, rotating speed is 800-900r/min, so
Afterwards mobile phase is entered with the flow pump of 1.5-1.8mL/min, reach poised state in post to two-phase solvent；Prepared by step (1)
Crude extract, is dissolved for solvent with fixing phase, prepares sample solution；By sample solution sample introduction, monitor under 280nm, receive respectively
Each target component, you can respectively obtain component a, (-)-N-methylanadine, oxohydrastinine,
hydroprotopine；
(3) component a, upper half preparative hplc are taken, you can separate and obtain leptopidine, wherein, half preparative hplc condition is such as
Under：
C18 post, methanol-water is mobile phase, and gradient condition is methanol：0-10min, 40-42%；10-30min, 42-
42%；Detection wavelength 280nm.
Further, in step (1), concentration of alcohol is 95%v/v, and Extracting temperature is 50-70 DEG C, preferably 60 DEG C.
Further, in step (1), the addition of hydrochloric acid is 4 times of extractum volume, and hydrochloric acid volume fraction is 2%.
Further, in step (1), ungrease treatment is to be realized using petroleum ether or chloroform extraction；Preferably use petroleum ether.
Further, in step (1), described alkali is ammonia.
Further, in step (2), crude extract consumption is 200mg.
Further, in step (2), receive the target component that appearance time is 49-57min, you can obtain component a；Connect
Receive the target component that appearance time is 70-75min, you can obtain (-)-N-methylanadine；Reception appearance time is 102-
The target component of 106min, you can obtain oxohydrastinine；Receive the target component that appearance time is 126-133min,
Can get hydroprotopine.
Present invention also offers extracting, from Tibetan medicine Herba Hypecoi Leptocarpi, the method separating five kinds of alkaloids, it includes grasping as follows
Make：
A, prepare according to the method described above component a, (-)-N-methylanadine, oxohydrastinine,
hydroprotopine；
B, take component a, upper half preparative hplc, separable successively obtain formula a-1 and leptopidine, wherein, partly prepare color
Spectral condition is as follows：C18 post, methanol-water is mobile phase, and gradient condition is methanol：0-10min, 40-42%；10-30min, 42-
42%；Detection wavelength 280nm.
Wherein, receive the target component that appearance time is 22.7-23.2min, you can obtain component a-1；When receiving appearance
Between for 24.8-25.4min target component, you can obtain leptopidine.
Present invention also offers the compound shown in formula a-1 or its pharmaceutically acceptable salt, hydrate, solvate or
Crystal formation：
Present invention also offers the compound shown in formula a-1 or its pharmaceutically acceptable salt, hydrate, solvate or
Purposes in preparing antioxidative medicine for the crystal formation.
Advantages of the present invention：Extracted from Herba Hypecoi Leptocarpi with high-speed countercurrent chromatography and separate four kinds or five kinds of alkaloidss
Compound, relatively conventional column chromatography is simple to operate, and disengaging time substantially shortens, and the compound purity obtaining is also relatively
High.
Meanwhile, separate in research process of the present invention and obtained new compound type a-1, this compound has certain resisting
Oxidation, is that clinical application provides new selection.
Brief description
Fig. 1 high speed adverse current chromatogram figure
The HPLC s of Fig. 2 adverse current component is study figure, and a, b, c, d are respectively the liquid phase figure of four adverse current components
Half preparative hplc figure of Fig. 3 adverse current component a
Fig. 4 HPLC figure s is the liquid phase figure preparing component for liquid phase figure a-1 and a-2 that study figure a is adverse current component
The C spectrum of Fig. 5 noval chemical compound a-1
The H spectrum of Fig. 6 noval chemical compound a-1
The DEPT figure of Fig. 7 noval chemical compound a-1
The HSQC figure of Fig. 8 noval chemical compound a-1
The HMBC figure of Fig. 9 noval chemical compound a-1
Figure 10 formula a-1 compound online DPPH antioxidant activity chromatogram
Specific embodiment
Embodiment 1 extraction separation method of the present invention
1st, the preparation of Herba Hypecoi Leptocarpi crude extract
2.0 kilograms of Herba Hypecoi Leptocarpies are added 20L 95% ethanol extraction 3h, is repeated 3 times.Merge 3 extracting solution and collect filter
After liquid, Rotary Evaporators concentrate and remove ethanol, obtain extractum.The extractum dissolving with hydrochloric acid of 2L 2%, filters, and removes not tolerant, obtains
Acid solution to alkaloid.Acid solution 2L petroleum ether extraction, in triplicate, removes oil-soluble impuritieses.Acid solution strong aqua ammonia adjusts PH
It is worth to 9-10.Then use chloroform extraction, obtain 12.6g alkaloid study, with high speed adverse current chromatogram later.
2nd, PH zone high-speed countercurrent chromatography combines half preparative chromatography and separates and prepares composition of alkaloids
Prepare n-hexane-ethyl acetate-methanol-water (3: 5: 2: 5, v/v/v/v) two-phase solvent system in separatory funnel,
Stand overnight after shake well.Lower addition hydrochloric acid make its concentration be 5mM as fixing phase, upper addition triethylamine makes its concentration be
10mM as mobile phase, using front difference ultrasonic degassing 30min.Take 200mg sample powder to be separated, concussion is dissolved completely in
In phase solution under 10mL, standby.
Main frame will mutually be pumped under solvent system and be full of separation solenoid with 20mL/min flow velocity, ON cycle water-bath simultaneously will
Temperature is set as 25 DEG C.Open host power supply to rotate forward with 850r/min, after stabilization of speed, with 1.5mL/min flow pump
Enter mobile phase, after mobile phase after tubing string exports outflow and baseline stability, sample solution is injected by sample introduction circle.Tubing string exit
Effluent continuous detecting at a wavelength of 280 nm, collects each chromatographic peak component manually according to adverse current chromatogram figure.Divide from 1.2g study
From obtaining 45mg hypecocarpine and leptopidine mixture thing (component a), 31mg (-)-N-methylanadine
(component b), 21mg oxohydrastinine (component c), 57mg hydroprotopine (four kinds of alkaloids of component d).
Wherein, receive the target component that appearance time is 49-57min, you can obtain component a；Reception appearance time is 70-
The target component of 75min, you can obtain (-)-N-methylanadine；Receive the target that appearance time is 102-106min to become
Point, you can obtain oxohydrastinine；Receive the target component that appearance time is 126-133min, you can obtain
hydroprotopine.
Component a can be efficiently separated through half preparative hplc, obtain 9mg hypecocarpine (component a-1) and
15mg leptopidine (component a-2).Wherein, receive the target component that appearance time is 22.7-23.2min, you can obtain
Component a-1；Receive the target component that appearance time is 24.8-25.4min, you can obtain leptopidine.
This in five alkaloid be all more than 91.7% through HPLC detection purity.Wherein hypecocarpine examines through SciFinder
Rope, and through one-dimensional, two-dimentional nuclear-magnetism and high-resolution and low-resolution Mass Spectrometric Identification, it is defined as new composition of alkaloids.
Half preparation condition：NU 3000SERIALS UV/VIS (Han Bang company) detector；NP 7000SERIALS (Chinese nation
Company) pump；Semi-preparative column：Dubhe C18 post (20*250mm) Han Bang Science and Technology Ltd.；Detection wavelength 280nm；Gradient strip
Part：Methanol：0-10min, 40-42%；10-30min, 42-42%.
HPLC analysis condition：HPLC/DAD system is Agilent 1200 system, is furnished with G1313A automatic sampler；
G1316A column oven；G1315B UV-vis detector.Chromatographic column be XBridge Shield RP18 analytical column (5 μm, 4.6 ×
250mm), sample size 15ul；Detection wavelength is：280；DAD scanning wavelength is 200-800nm.The condition of gradient elution：Methanol：
0-15min, 20-50%；20-25min, 50-80%；25-30min, 80-95%.
The nuclear magnetic data of five compounds is as follows
Compound a -1：
1H NMR (600MHz, CD3OD, δ ppm)：3.75 (s, 3H, N-Me), 3.97 (t, 2H, J=7.5Hz, H-3), 3.09
(t, 2H, J=7.5Hz, H-4), 6.91 (s, 1H, H-5), 7.17 (s, 1H, H-8), 3.80 (s, 3H, 4 '-OMe), 3.49 (s,
3H, 7-OMe), 3.87 (s, 3H, 6-OMe), 6.94 (d, 1H, J=7.5Hz, H-5 '), 6.68 (d, 1H, J=7.5Hz, H-6 ')
.13C NMR (600MHz, CD3OD, δ ppm)：178.30 (C-1), 44.30 (N-Me), 53.80 (C-3), 26.6 (C-4), 134.9
(C-4a), 111.4 (C-5), 156.5 (C-6), 149.0 (C-7), 114.5 (C-8), 120.6 (C-8a), 130.3 (C-1 '),
118.5 (C-2 '), 154.9 (C-3 '), 149.8 (C-4 '), 114.40 (C-5 '), 120.7 (C-6 '), 38.4C-7 '), 56.5
(4 '-OMe), 56.8 (6-OMe), 56.6 (7-OMe). through SciFinder retrieval, and through one-dimensional, two-dimentional nuclear-magnetism and high and low
Resolution mass spectrometric is identified, is defined as new composition of alkaloids, is named as hypecocarpine
Compound a -2
1H NMR (600MHz, CD3OD, δ ppm)：3.69 (s, 3H, N-Me), 3.97 (t, 2H, J=7.5Hz, H-3), 3.09
(t, 2H, J=7.5Hz, H-4), 6.85 (s, 1H, H-5), 6.00 (s, 2H, 6,7-OCH2-), 7.15 (s, 1H, H-8), 3.81
(s, 3H, 4 '-OMe), 6.91 (d, 1H, J=7.5Hz, H-5 '), 6.53 (d, 1H, J=7.5Hz, H-6 ').13C NMR
(600MHz, CD3OD, δ ppm)：178.30 (C-1), 44.42 (N-Me), 53.57 (C-3), 27.01 (C-4), 137.16 (C-
4a), 108.98 (C-5), 148.67 (C-6), 155.15 (C-7), 110.63 (C-8), 122.39 (C-8a), 129.30 (C-
1 '), 117.77 (C-2 '), 154.73 (C-3 '), 149.76 (C-4 '), 114.40 (C-5 '), 120.29 (C-6 '), 38.89 (C-
7 '), 104.22 (6,7-OCH2-), 56.39 (4 '-OMe). can determine this change according to its nuclear magnetic data with reference to pertinent literature
Compound is leptopidine.
Compound b
1NMR (600MHz, CD3OD, δ ppm) feruloyl moiety：3.91 (3H, s, C6-OCH3), 6.16 (1H, d, J
=15.6Hz, H-2), 6.82 (1H, d, J=8.0Hz, H-8), 7.03 (1H, dd, J=8.0,2.0Hz, H-9), 6.98 (1H, d,
J=2.0Hz, H-5), 7.43 (1H, d, J=15.6Hz, H-3)；tyramine moiety：2.80 (2H, t, J=6.8Hz, H-
2 '), 3.49 (2H, t, J=6.8Hz, H-1 '), 6.81 (2H, d, J=8.8Hz, H-5 ' and H-7 '), 7.05 (2H, d, J=
8.8Hz, H-4 ' and H-8 ')；13C-NMR (600MHz, CD3OD, δ ppm)：834.77 (C-13), 40.88 (C- α), 55.96
(OCH3), 109.53 (C-3), 114.66 (C-3 ", C-5 "), 115.57 (C-5 '), 118.10 (C-2 '), 122.18 (C-6 '),
126.75 (C-3 '), 129.92 (C-2 ", C-6 "), 129.59 (C-1 "), 141.05 (C-2), 147.26 (C-4 '), 147.86
(C-1 '), 155.13 (C-4 "), 167.23 (C-1). can determine this compound according to its nuclear magnetic data with reference to pertinent literature
For (-)-N-methylanadine
Compound c
13C NMR (600MHz, CD3OD, δ ppm)：) δ 164.42 (C-1), 150.17 (C-6), 146.76 (C-7),
133.35 (C-8a), 123.50 (C-4a), 108.12 (C-5), 106.75 (C-8), 101.34 (- OCH2O-), 48.14 (C-3),
35.06 (N-Me), 27.95 (C-4)
1H-NMR (600MHz, CD3OD, δ ppm)：3.53 (2H, t, J=7Hz, H-3), 2.89 (2H, t, J=7Hz, H-
4), 6.69 (1H, s, H-5), 7.31 (1H, s, H-8), 5.97 (2H, s ,-OCH2O-), 3.08 (3H, s, N-Me) are according to its nuclear-magnetism
Data and reference pertinent literature can determine that this compound is oxohydrastinine
Compound d
1H-NMR (600MHz, CD3OD, δ ppm)：7.14 (1H, s, H-1), 6.76 (1H, s, H-4), 3.03 (3H, s, N-
CH3), 3.67 (2H, m, H-8), 6.85 (1H, d, J=8Hz, H-11), 6.86 (1H, d, J=8Hz, H-12), 4.59 (2H, m,
H-13), 5.99 (2H, s, O-CH2-O), 6.02 (2H, s, O-CH2-O) .13C-NMR (600MHz, CD3OD, δ ppm))：
106.94 (C-1), 145.53 (C-2), 147.83 (C-3), 110.21 (C-4), 128.01 (C-4a), 25.0 (C-5), 56.25
(C-6), 43.67 (N-CH3), 55.16 (C-8), 123.43 (C-8a), 149.03 (C-9), 150.43 (C-10), 109.52 (C-
11), 123.92 (C-12), 125.73 (C-12a), 48.58 (C-13), 178.25 (C-14), 132.7 (C-14a), 103.17
(O-CH2-O), 103.40 (O-CH2-O). can determine that this compound is according to its nuclear magnetic data with reference to pertinent literature
hydroprotopine.
The present invention is measured to the antioxidant activity of new compound type a-1, and the online DPPH antioxidation of noval chemical compound is lived
Property chromatogram (Detection wavelength 280nm), concentration is 5.7mg compound dissolution in the methanol of 25mL.
(Figure 10) is understood by chromatogram, the negative peak after 12min is to prove that compound has antioxidant activity, after 3min
Negative peak be solvent peak.
Claims (9)
1. from Tibetan medicine Herba Hypecoi Leptocarpi extract separate four kinds of alkaloids method it is characterised in that：It includes operating as follows step
Suddenly：
(1) preparation of crude extract：
Take Herba Hypecoi Leptocarpi, be solvent extraction with 70%-95%v/v ethanol, extracting solution concentrates after removing ethanol, and gained extractum adds
Hydrochloric acid, removes insoluble matter, and after solution defat, plus alkali adjusts pH to 9～10, then with chloroform extraction, collects chloroform layer, removes solvent
Afterwards, it is dried to obtain alkaloid crude extract；
(2) high-speed countercurrent chromatography is adopted to separate：
Take n-hexane-ethyl acetate-methanol-water=3: 5: 2: 5v/v/v/v, it is placed in preparation two-phase solvent system in separatory funnel,
Taking off addition hydrochloric acid to its concentration is as fixing phase during 5～10mM/L, and upper addition triethylamine to its concentration is for making during 10mM/L
For mobile phase；Fixing phase is full of the chromatographic column of adverse current chromatogram, at 25-28 DEG C, main frame rotates forward, rotating speed is 800-900r/
Min, then enters mobile phase with the flow pump of 1.5-1.8mL/min, reaches poised state to two-phase solvent in post；Step (1)
The crude extract of preparation, is dissolved for solvent with fixing phase, prepares sample solution；By sample solution sample introduction, monitor under 280nm, connect
Receive the target component that appearance time is 49-57min, you can obtain component a；Receive the target that appearance time is 70-75min to become
Point, you can obtain (-)-N-methylanadine；Receive the target component that appearance time is 102-106min, you can obtain
oxohydrastinine；Receive the target component that appearance time is 126-133min, you can obtain hydroprotopine；
(3) take component a, upper half preparative hplc, receive the target component that appearance time is 24.8-25.4min, you can obtain
Leptopidine, wherein, half preparative hplc condition is as follows：
C18 post, Detection wavelength 280nm, methanol-water is mobile phase, and gradient condition is methanol：0-10min, 40-42%；10-
30min, 42-42%.
2. method according to claim 1 it is characterised in that：In step (1), concentration of alcohol is 95%v/v, Extracting temperature
For 50-70 DEG C.
3. method according to claim 2 it is characterised in that：In step (1), Extracting temperature is 60 DEG C.
4. method according to claim 1 it is characterised in that：In step (1), the addition of hydrochloric acid is the 4 of extractum volume
Times, hydrochloric acid volume fraction is 2%.
5. method according to claim 1 it is characterised in that：In step (1), ungrease treatment is using petroleum ether or chloroform
Extraction is realized.
6. according to the method described in claim 5 it is characterised in that：In step (1), ungrease treatment is real using petroleum ether extraction
Existing.
7. method according to claim 1 it is characterised in that：In step (1), described alkali is ammonia.
8. method according to claim 1 it is characterised in that：In step (2), crude extract consumption is 200mg.
9. from Tibetan medicine Herba Hypecoi Leptocarpi extract separate five kinds of alkaloids method it is characterised in that：It includes operating as follows：
A, according to claim 1 preparation component a, (-)-N-methylanadine, oxohydrastinine,
hydroprotopine；
B, take component a, upper half preparative hplc, receive the target component that appearance time is 22.7-23.2min, you can obtain component
a-1；Receive the target component that appearance time is 24.8-25.4min, you can obtain leptopidine, wherein, half preparative hplc
Condition is as follows：C18 post, Detection wavelength is 280nm, and methanol-water is mobile phase, and gradient condition is methanol：0-10min, 40-
42%；10-30min, 42-42%
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| CN105168214B (en) * | 2015-08-11 | 2018-03-13 | 中国科学院西北高原生物研究所 | The new application of benzylisoquinoline alkaloid |
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| CN113372354A (en) * | 2021-05-25 | 2021-09-10 | 中国科学院西北高原生物研究所 | Preparation method of violaxanthine |
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