CN104447656B - A kind of Leaf and twig of Common Jasminorange tonka bean camphor and extracting method thereof - Google Patents
A kind of Leaf and twig of Common Jasminorange tonka bean camphor and extracting method thereof Download PDFInfo
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Abstract
The invention belongs to medical art, relate to a kind of Leaf and twig of Common Jasminorange tonka bean camphor and extracting method thereof.The present invention is by Leaf and twig of Common Jasminorange through solvent extraction and solution extraction initial gross separation, then adopts high performance liquid phase to be separated, and utilizes half preparative chromatography to be separated further, and concentrated frozen is dry obtains finished product tonka bean camphor.Finally by the coumarin compound that Ultra Performance Liquid Chromatography-mass spectrometry detects.
Description
Technical field
The invention belongs to medical art, be specifically related to a kind of Leaf and twig of Common Jasminorange tonka bean camphor and extracting method thereof.
Background technology
Cause in the various diseases of serious harm to human life's situation, malignant tumour is wherein one of the most headachy arch-criminal, and the annual whole world has thousands of people to suffer from cancer.But the publicity and transfer of the malignant entity tumors such as the liver cancer of former, lung cancer and lymphoma is very general, cancer is once shift, and patient will certainly die undoubtedly.Many medicines that current chemotherapy uses are all that this chemotherapy on human body injury is larger by interference cell growth metabolism and breeding and inducing death of neoplastic cells to reach therapeutic purpose.Therefore, we focus on inhibition tumor cell metastasis above research just seem very necessary.
Various factor of influence participates in the process of metastases, suppresses and the transfer controlling tumour just must develop can the medicine of Tumor suppression transfer process, set about thus, be expected to obtain the novel drugs that prophylaxis of tumours safely and effectively shifts.For the various factors affecting metastases process, we can develop the medicine of the different mechanism of action to the transfer of the carrying out thus inhibition tumor cell that stop this process.The angiopoietic medicine TNP470 of such as Tumor suppression and thalidomide etc., but current medicine for anti transfer of tumor often has very large toxic side effect, also there is a lot of problem clinically.Herbal medicine generally has lower toxic side effect, and it can also improve the immunologic function of body simultaneously.Therefore, Chinese medicine has certain advantage at searching prophylaxis of tumours cell transfer party mask, therefrom finds new drug and has profound significance.Eye has been placed in the research to herbal medicine by many scientists, and this provides good opportunity for the development of China Chinese medicine.The plant amedica be made up of plant extract position gradually accept by various countries, China some distinctive medicinal plant and Chinese medicines thereof, be used to manufacture anticarcinogen obtain one seat in the international market.Along with the progress of technology, cognitive intensification, these systems will be more and more perfect, and Tumor suppression transfer will be no longer talk about stratagems on paper.
Leaf and twig of Common Jasminorange (
murrayaexotica(L.)) be Rutaceae Murraya plant.The same with most of natural phant, the chemical composition in Leaf and twig of Common Jasminorange is also very complicated, based on coumarins, flavonoid and alkaloids etc.Leaf and twig of Common Jasminorange in Murraya plant (
murrayaexotical.), Murraya paniculata (L.) Jack (
murrayapaniculate(L.) Jack) and winged petiole Leaf and twig of Common Jasminorange (
murrayaalata) root is all containing the tonka bean camphor of 8-amylene.Wherein a kind of as the coumarin kind compound in Leaf and twig of Common Jasminorange, murrayone is to the growth of tumour cell and all inhibited to the growth of animal subcutaneous transplantation knurl, and acute toxicity is lower.Kawaii etc. are studied analysis to comprising the come into leaves prophylaxis of tumours activity of 33 kinds of tonka bean camphors of coumurrayin of dehydration, find that they all have restraining effect in various degree to kinds of tumors (as people's lymph nodal metastasis cell TGBC11TKB etc.), but normal cell (as Human umbilical vein endothelial cells HUEVC etc.) is not had or only have very weak restraining effect; Analyze through structure effect, the isopentene group structure on discovery tonka bean camphor aromatic nucleus and its activity have close association.
Warfarin (coumarin kind compound) and heparin class anticoagulant, usually for the correction of cancer patients's blood coagulation symptom, can control metastases in some cases, extend the survival time of patient.Tonka bean camphor has blood coagulation resisting function, and the formation of the anti-hemostasis suppository of energy, it also has the effect comparatively affirmed in treatment and prevention of tumour in addition, and untoward reaction is little.In addition, the activeconstituents containing antifertility action in Leaf and twig of Common Jasminorange root, nearest research shows, contraceptive bian has the effect reducing related neoplasms incidence, namely has prophylactic effect to these cancers.
After having carried out a series of correlative study, we have found a kind of extracting and developing method effectively may with the Leaf and twig of Common Jasminorange tonka bean camphor single compound of anti metastasis, and completed the present invention accordingly.
Summary of the invention
The object of the present invention is to provide a kind of Leaf and twig of Common Jasminorange tonka bean camphor and preparation method thereof,
A kind of Leaf and twig of Common Jasminorange tonka bean camphor, its structural formula is:
。
The extracting method of described a kind of Leaf and twig of Common Jasminorange tonka bean camphor, preparation method comprises the following steps:
(1) fresh Leaf and twig of Common Jasminorange plant root clean, dry in the shade after by the separately process of each position, be ground into broken end, take 10-15g, respectively Plus acidic alcohol reflux 3 times, each refluxing extraction 5 hours, filter, merging filtrate;
(2) filtrate obtained in (1) is revolved steaming extremely without alcohol with Rotary Evaporators, (does is amount how many to add 250ml-300ml?) pure water dissolve after suction filtration, collect filtrate, and add ammoniacal liquor regulate pH=8-9, extract with different solvents; Each extract layer is spin-dried for and obtains the crude extract of common jasminorange root;
(3) adopt half preparative high-performance liquid chromatographic to be separated by crude extract in (2), obtain Leaf and twig of Common Jasminorange coumarin extract;
(4) adopt Ultra Performance Liquid Chromatography-mass spectrometry to be further analyzed comparison to extract in (3), determine that it comprises coumarin compound.
Acidic ethanol described in step (1) is pH3-5, and volume fraction of ethanol content is 70-90%.
Different solvents in step (2) uses chloroform, methylene dichloride, sherwood oil and ethyl acetate respectively.
Preparation liquid phase separation condition in step (4) is, chromatographic column: AgelaVenusilXBPC18; Flow velocity: 1-4ml/min; Column temperature: 35 ° of C; Determined wavelength: 350nm isocratic elution, moving phase is the volume ratio 1:3-1:6 of acetonitrile and pure water mixed solution, acetonitrile and pure water.
The application of described Leaf and twig of Common Jasminorange tonka bean camphor on metastases prophylactic agent.
The coumarin compound obtained by above extracting method also comprises Hydroxycoumarin, murrayone, trihydroxy-tonka bean camphor, murpanicin, 5,7-dimethoxy-8-(2-ketone group-3-methyl butyls) coumarin.
Adopt mtt assay that extract is carried out cytotoxicity test to human colon cancer cell (HT-29).After different concns (1-100 μ g/mL) acts on 72 hours, the IC50 value of extract and single compound, all higher than 100 μ g/mL, can find out that their cytotoxicity is little.Transwell Cell migration assay is adopted to carry out anti-migration capability study, choose adding consistency and be 50 μ g/mL, cultured continuously, after 24 hours, finds after measured, and Leaf and twig of Common Jasminorange coumarin extract has obvious restraining effect to human colon cancer cell (HT-29) Chemotaxis.The coumarins extract of common jasminorange root has obvious anti-cell migration ability and lower cytotoxicity, meets the basic characteristics of medicine for anti transfer of tumor.
The invention has the advantages that: this monomeric compound has obvious anti-cell migration ability and lower cytotoxicity, be different from existing most of cancer therapy drug (cell toxicity medicament), be therefore expected the metastases prophylactic agent developing applicable long-term taking.
Accompanying drawing explanation
The uv-visible absorption spectra of the different extraction agent extract of Fig. 1.
The fluorescence emission spectrum of Fig. 2 Leaf and twig of Common Jasminorange different sites extract.
Fig. 3 extracts the compositional analysis (HPLC figure) of rear Leaf and twig of Common Jasminorange different sites.
After Fig. 4 hatches 24 hours altogether, common jasminorange root, leaf extract are on the impact of HT29 cell proliferation.
After Fig. 5 hatches 24 hours altogether, Murraya jasminorage leaf, root extract are on the impact of HT29 cell migration ability.(*：p<0.05，**：p<0.01。)
The UPLC-MS-PDA spectrogram of Fig. 6 Murraya jasminorage leaf, root extract.
The mass spectrum of each component in Fig. 7 Leaf and twig of Common Jasminorange root extract.
In Fig. 8 Murraya jasminorage leaf extract each component mass spectrum.
Fig. 9 Leaf and twig of Common Jasminorange root extract half preparative chromatography figure.
The Purity assessment of Figure 10 Leaf and twig of Common Jasminorange root extract half preparative chromatography product.(, be followed successively by MP3, MP2, MP1 and root general extractive obtain HPLC analysis chart in figure from top to bottom).
Figure 11 A is 7-methoxyl group-8-(1,3-diene)-5 propenyloxy group tonka bean camphor structures; B be each atom numbering (left side) and
1h-NMR signals assignment.
Embodiment
The invention provides the composition composition research of described coumarins extract and the separation purification method of monomer new compound, this research and method comprise the following steps.Pre-separation operation is:
(1) fresh Leaf and twig of Common Jasminorange plant clean, dry in the shade after by the separately process of each position, be ground into broken end.Take common jasminorange root position powder 10-15g, add 80lv% acidic ethanol refluxing extraction 3 times respectively, each refluxing extraction 5 hours, filter, merging filtrate.
(2) filtrate obtained in (1) is revolved with Rotary Evaporators steam to without alcohol, suction filtration after the pure water adding 250ml-300ml dissolves, collection filtrate, and add ammoniacal liquor and regulate flat pH=8-9, extract with different solvents.Each extract layer is spin-dried for and obtains the crude extract of Leaf and twig of Common Jasminorange.
The acidic ethanol of described step (1) is pH=3.
Different solvents in described step (2) uses chloroform, methylene dichloride, sherwood oil and ethyl acetate respectively.
Described analysis and half preparative chromatography purification procedures are:
(3) root extract adopting Ultra Performance Liquid Chromatography-mass spectrometry (UPLC-MS) to analyze, the coumarin compound structure contained by supposition.
(4) adopt half preparative chromatography that the root crude extract in (2) is prepared separation.
Ultra Performance Liquid Chromatography mass spectrometry instrument operational condition in described step (3) is, (a) liquid chromatography parameter, moving phase: A-pure water (0.1% formic acid), B-acetonitrile (0.1% formic acid); Chromatographic column: ACQUITYUPLCBEHC18(2.1mm × 50mm, 1.7 μm); Flow velocity: 0.200mL/min; Elution program: isocratic elution A:B=4:1; Detector: PDA200-600nm; (b) mass spectrometry parameters, ion source: atmospheric pressure electrospray ion source (ESI); Scan pattern: positive ion mode full scan; Surface sweeping scope: 150-650
m/z; Spray voltage (sprayvoltage): 4.0kV; Sheath airshed (sheathgasflowrate): 35Arb; Assisted gas flow (auxgasflowrate): 5Arb; Ion transfer tube temperature (capillarytemperature): 350 DEG C; Caliber voltage (tubelens): 60.00V.
Liquid-phase condition of preparing in described step (4) is, chromatographic column: AgelaVenusilXBPC18(10mm × 250mm, 5 μm); Flow velocity: 1.25mL/min; Column temperature: 35 ° of C; Determined wavelength: 350nm isocratic elution and acetonitrile and pure water are according to the ratio of 1:4, and the sample DMSO solvent of sample size 100 μ L, 30mg/mL is prepared.
the extracting method of embodiment 1 Leaf and twig of Common Jasminorange effective constituent
Fresh Leaf and twig of Common Jasminorange plant wash clean, being placed on shady and cool ventilation place for some time makes moisture wherein enough few.By the separately process of each position after drying, be ground into broken end.Accurately take each 15g of each position powder of Leaf and twig of Common Jasminorange, be contained in respectively in four round-bottomed flasks, add 450mL80% acidic ethanol (pH=3) refluxing extraction respectively 3 times, each refluxing extraction 5 hours, filter, merging filtrate.Filtrate is revolved with Rotary Evaporators and steams to without alcohol, add appropriate pure water and dissolve rear suction filtration, collect filtrate.In the filtrate obtained, add ammoniacal liquor regulate pH=9, extract with the extraction agent that chloroform, methylene dichloride, sherwood oil and ethyl acetate four kinds is conventional respectively.Collected organic layer is for subsequent use.Each extract layer is spin-dried for the crude extract namely obtaining Leaf and twig of Common Jasminorange.Dissolve identical amount Folium Et Cacumen Murrayae extract with isopyknic DMSO, under fluorescence spectrophotometer, survey its photoluminescent property.
The uv-visible absorption spectra of Folium Et Cacumen Murrayae extract fluorescence spectrum and different extraction agent extract thereof illustrates that the extract fluorescence activity in tree root is the strongest, less for the hazardness of ethyl acetate to environment, therefore selects ethyl acetate as extraction agent.See Fig. 1, Fig. 2.
embodiment 2 efficient liquid phase chromatographic analysis
High-efficient liquid phase chromatogram condition is, instrument: Shimadzu LC-20A; Detector: Shimadzu SPD-20AV; Chromatographic column: GraceSmartRPC18 chromatographic column (150 × 2.1mm, 5 μ); Moving phase: B-pure water, A-acetonitrile; LC time-program(me): isocratic elution A:B=1:4; Flow velocity: 0.2mL/min; Column temperature: 35 ° of C; Determined wavelength: 350nm.All mobile phase solvents are all through the membrane filtration of 0.45 μm.All sample solutions all use 0.22 μm of membrane filtration.Adopt automatic sampling, each sample introduction 20 μ L, measures under these conditions.
As shown in Figure 3, the composition of common jasminorange root extract and leaf extract comparatively horn of plenty.Ethyl acetate mainly removes the larger water soluble component of polarity, retains fat-soluble component, is conducive to the extraction of tonka bean camphor.
embodiment 3 Leaf and twig of Common Jasminorange effective constituent is to the increment restraining effect of human colon cancer cell
Collect logarithmic phase HT-29 cell, adjustment concentration of cell suspension, every hole adds 100 μ L, and bed board makes cell to be measured adjust density 2000/ hole.5%CO
2hatch for 37 DEG C, make cell monolayer be paved with (96 hole flat underside) at the bottom of hole with this understanding, add Murraya jasminorage leaf extract wherein, root extract concentration is respectively 1 μ g/mL, 10 μ g/mL, 50 μ g/mL, 100 μ g/mL, every hole 200 μ L, if 3-5 multiple hole.5%CO
2, hatch after 0h, 6h, 24h, 48h, 72h hour for 37 DEG C and carry out MTT detection.Every hole adds 10 μ LMTT solution (5mg/mL, i.e. 0.5%MTT), continues to cultivate 4h.Stop cultivating, the careful nutrient solution going to inside, hole.Every hole adds 150 μ L dimethyl sulfoxide (DMSO), is positioned over low-speed oscillation 10min on shaking table, crystallisate is fully dissolved.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument OD492nm place.
The method of calculation of cell survival rate (I%) are as follows: I%=(A-A
0)/(A1-A
0) × 100%
In formula: A---tested sample group OD value;
A
1---solvent control group OD value;
A
0---blank group OD value
As table 1, shown in Fig. 4, both are lower to the toxicity of cell, and have concentration-dependent relation.Relatively the inhibiting rate of leaf extract and root extract on cell proliferation, can find out that the cytotoxicity of Leaf and twig of Common Jasminorange root extract is totally less than the extract of leaf.
The extract of table 172 hour different concns is to HT-29 growth inhibition ratio
Embodiment 4Transwell detects the ability of Leaf and twig of Common Jasminorange effective constituent on cell migration
After the soft rinsing of HT-29 cell serum free medium of growing fine twice, be suspended in serum-free matrix, cultivate 24 hours; Single cell suspension (5 × 10 is prepared with High/DMEM basic medium
5/ mL); Add the DMEM matrix of 600uL-800uL containing 20% serum and relative medicine concentration at lower room (namely bottom 24 orifice plates), upper room adds 100uL cell suspension, 5%CO
2continue to cultivate 24h; Take out Tanswell cell, discard nutrient solution in hole, wash 2 times with PBS, methyl alcohol fixes 30min; Abandon methyl alcohol, cell is suitably air-dry, and PBS washes 2 times, 0.1% violet staining 20min, dabs off the non-migrating cell in upper strata with wet cotton swab.Get under microscope and get 3 visual field countings at random, take the mean.
Mobility=dosing group migrating cell number/blank group migrating cell number × 100%
As table 2, shown in Fig. 5, Leaf and twig of Common Jasminorange leaf and root extract have obvious restraining effect to human colon cancer cell (HT-29) Chemotaxis; Relative to blank assay, both mobilities are respectively 56.04% and 15.14%, and root extract T suppression cell transporting action is stronger.
HT29 colon-cancer cell transport number after 24h cultivated by table 2
embodiment 5 Ultra Performance Liquid Chromatographies-mass spectrometry (UPLC-MS) is analyzed
Ultra Performance Liquid Chromatography mass spectrometry instrument operational condition is, (a) liquid chromatography parameter, instrument: ACQUITYUPLCsystem (Waters company); Moving phase: A-pure water (0.1% formic acid), B-acetonitrile (0.1% formic acid); Chromatographic column: ACQUITYUPLCBEHC18(2.1mm × 50mm, 1.7 μm); Flow velocity: 0.200mL/min; Elution program: isocratic elution A:B=4:1; Detector: PDA200-600nm; (b) instrument: XevoTQD triple level Four bar mass spectrum (Waters company); Mass spectrometry parameters, ion source: atmospheric pressure electrospray ion source (ESI); Scan pattern: positive ion mode full scan; Surface sweeping scope: 150-650
m/z; Spray voltage (sprayvoltage): 4.0kV; Sheath airshed (sheathgasflowrate): 35Arb; Assisted gas flow (auxgasflowrate): 5Arb; Ion transfer tube temperature (capillarytemperature): 350 DEG C; Caliber voltage (tubelens): 60.00V.
As shown in Fig. 3, Fig. 6, the UPLC-PDA of Leaf and twig of Common Jasminorange leaf and root extract schemes and substantially reappears with the color atlas that Shimadzu LC-20A instrument GraceSmartRPC18 chromatographic column is worked it out.The UPLC-PDA figure of each extract compares can find out with UPLC-TQ-MS, and mass spectrum total ion current detection signal is more than PDA signal, and the main peak on PDA figure, also there is corresponding signal to occur in corresponding time place total ion current figure.As shown in Fig. 7, Fig. 8, Leaf and twig of Common Jasminorange root and leaf extract are summed up summary in the mass signal of different time points.By reference to relevant bibliographical information and its mass signal, we infer and in compound to exist 5 kinds of structures.
embodiment 6 half preparative chromatography
Half preparative chromatography condition is, instrument: Shimadzu LC-20A; Detector: Shimadzu SPD-20AV; Chromatographic column: AgelaVenusilXBPC18(10mm × 250mm, 5 μm); Flow velocity: 1.25mL/min; Column temperature: 35 ° of C; Determined wavelength: 350nm; Isocratic elution and acetonitrile and pure water are according to the ratio of 1:4.Sample size is extended to 100 μ l quantitative loop, and DMSO compound concentration is the sample of 30mg/mL, scoops out, as Fig. 9 at the outflow end previously prepd container of detector.The component collected is replaced by analysis mode chromatographic column GraceSmartRPC18 chromatographic column (150 × 2.1mm, 5 μ), measure according to high-efficient liquid phase chromatogram condition, compare with the analysis color atlas of original Leaf and twig of Common Jasminorange root extract again, as Figure 10, to determine the analysis chromatographic peak that the composition respectively collected is corresponding and purity thereof.
embodiment 7 nuclear-magnetism determination monomeric compound structure
Utilize half preparative HPLC preparation can be separated from Leaf and twig of Common Jasminorange root extract and obtain some compounds.Component between one of them compound MP2(preparative chromatography retention time 27.66-29.61min), carry out nucleus magnetic resonance (DMSO, 500MHz) and characterized, its
1h-NMR spectrum as shown in figure 11.Chemical shift δ can be observed
hbetween 6.0-8.0, have 4 peaks to occur, be respectively δ 8.00 (d,
j=9.5Hz, 1H) and δ 6.29 (d,
j=9.4Hz, 1H), δ 7.61 (d,
j=8.7Hz, 1H) and δ 7.08 (d,
j=8.7Hz, 1H), be 2 in cis coupling alkene hydrogen signal, with reference to the substituted radical of common natural product in Leaf and twig of Common Jasminorange plant, it can be attributed to respectively the 3-H (δ 6.29) on tonka bean camphor parent nucleus, 4-H (δ 8.00), 5-H (δ 7.61), and 6-H (δ 7.08).δ
h3.89 (s, 3H) also according to common compounds structure in Leaf and twig of Common Jasminorange, can be speculated as 7-OCH
3proton signal, the strong inducing action of O and phenyl ring makes the chemical shift of methyl shift to low field.δ 1.81 (s, 3H) is the methyl proton signal without H in adjacent C; δ
h4.95 (s, 1H) and 4.86 (s, 1H) are unimodal, be the fignal center that the H spin coupling of 2 on terminal double link produces, and its adjacent carbon atom does not have hydrogen atom yet.Therefore can infer that compound has
fragment.δ 2.55 (s, 1H) may for being connected to-the CH of alkene.δ 5.12 (m, 2H) is-CH being connected to alkene, because of exist cis-trans isomerism and with being coupled of H in adjacent C and have complicated division.δ 4.59 (s, 1H) is coupled with δ 4.73 (s, 1H) and δ 4.64 (s, 1H) respectively, chemically displacement judges it may is two alkene, therefore can infer that compound has CH=CH-CH=fragment.The above analysis, supposition may be 7-methoxyl group-8-(1,3-diene)-5 propenyloxy group tonka bean camphors, structure as shown in A in Figure 11, each atom numbering (left side) and
1h-NMR signals assignment is as shown in B and table 3 in Figure 11.
The hydrogen nuclear magnetic resonance spectrum analysis of table 3 compound MP2
This structure therewith mass signal of compound and cracking situation is also mated.Signal as wherein m/z299.2 is then attributed to [M+H]
+, the signal of m/z259.1 then can be attributed to and continue to lose end CH
3-C=CH
2, the signal of m/z231.1 then can be attributed to m/z259.1 ion to be continued to lose CH
2oH.
The present invention's antitumor drug new to research and development provides lead compound, provides scientific basis for developing natural plant resource.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (6)
1. a Leaf and twig of Common Jasminorange tonka bean camphor, is characterized in that: described tonka bean camphor structure formula is:
.
2. an extracting method for a kind of Leaf and twig of Common Jasminorange tonka bean camphor as claimed in claim 1, is characterized in that: described preparation method comprises the following steps:
(1) fresh Leaf and twig of Common Jasminorange plant root clean, dry in the shade after by the separately process of each position, be ground into broken end, take 10-15g, respectively Plus acidic alcohol reflux 3 times, each refluxing extraction 5 hours, filter, merging filtrate;
(2) filtrate obtained in (1) is revolved steaming to without alcohol with Rotary Evaporators, add the rear suction filtration of pure water dissolving of 250ml-300ml, collect filtrate, and add ammoniacal liquor adjustment pH=8-9, extract with different solvents; Each extract layer is spin-dried for and obtains the crude extract of common jasminorange root;
(3) adopt half preparative high-performance liquid chromatographic to be separated by crude extract in (2), obtain Leaf and twig of Common Jasminorange coumarin extract;
(4) adopt Ultra Performance Liquid Chromatography-mass spectrometry to be further analyzed comparison to extract in (3), determine that it comprises coumarin compound.
3. the extracting method of a kind of Leaf and twig of Common Jasminorange coumarin substances according to claim 2, is characterized in that: the acidic ethanol described in step (1) is pH3-5, volume fraction of ethanol content 70-90%.
4. the extracting method of a kind of Leaf and twig of Common Jasminorange coumarin substances according to claim 2, is characterized in that: the different solvents in step (2) uses chloroform, methylene dichloride, sherwood oil and ethyl acetate respectively.
5. the extracting method of a kind of Leaf and twig of Common Jasminorange coumarin substances according to claim 2, is characterized in that: the preparation liquid phase separation condition in step (3) is, chromatographic column: AgelaVenusilXBPC18; Flow velocity: 1-4ml/min; Column temperature: 35 ° of C; Determined wavelength: 350nm isocratic elution, moving phase is the volume ratio 1:3-1:6 of acetonitrile and pure water mixed solution, acetonitrile and pure water.
6. the application of a kind of Leaf and twig of Common Jasminorange tonka bean camphor as claimed in claim 1 on the prophylactic agent of preparation metastases.
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