CN110294733A - One kind Oleracone I of key compound containing peroxide and its extraction separation method and application in purslane - Google Patents
One kind Oleracone I of key compound containing peroxide and its extraction separation method and application in purslane Download PDFInfo
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Abstract
The present invention relates to traditional Chinese medicine extractions, separation field, more particularly to extracting and developing and the noval chemical compound identified and its extraction separation method and application from purslane medicinal material, one kind Oleracone I of key compound containing peroxide and its extraction separation method and application specially in purslane.The extraction separation method of noval chemical compound successively uses water boiling and extraction, silica gel column chromatography, polyamide column chromatography, compression leg and Sephadex LH-20 separation preparation in ODS, successfully extracts and isolates a kind of new key compound containing peroxide.Its structure is accredited as a kind of new key compound containing peroxide by the method that mass spectrum, carbon spectrum, hydrogen spectrum and two-dimensional nuclear magnetic spectrum parse.There is noval chemical compound antitumor, anti-inflammatory effect, noval chemical compound and its salt or derivative can be used as the raw material of other compound synthesis primers and new drug development and pharmacology activity research, be used to prepare antitumor, anti-inflammatory drug.
Description
Technical field
The present invention relates to traditional Chinese medicine extraction, separation field, more particularly to extracting and developing and identifies from purslane medicinal material
Noval chemical compound and its extraction separation method, one kind Oleracone I of key compound containing peroxide and its extraction specially in purslane
Separation method and application.
Background technique
Purslane (Portulaca oleraceaL.) also known as horse three-coloured amaranth, long life dish, ant dish, it is Portulacaceae 1 year
Raw herbaceous plant.Purslane is widely distributed, resourceful, be 78 kinds of integration of drinking and medicinal herbs as defined in the Ministry of Public Health, China wild plant it
One.Purslane is recorded in 2015 editions Pharmacopoeias of the People's Republic of China, has clearing heat and detoxicating, cooling blood and hemostasis, stop dysentery and other effects, uses
In toxic-heat and blood stasis, carbuncle swells furunculosis, eczema, erysipelas, snakebite and bugbite, hematochezia, hemorrhoid blood, metrostaxis etc..
Purslane modern pharmacology research show its with anti-inflammatory, antibacterial, antiviral, lowering blood pressure and blood fat, it is anti-oxidant,
Anticancer, antitumor, relaxation skeletal muscle and smooth muscle adjust the effects of immune function.Purslane main chemical compositions include flavones
Class, cumarin, terpene, steroid, alkaloid, amino acid, lignanoids, volatile oil, polysaccharide, various pigments and minerals class etc.
Pharmacological action for its multiplicity provides material base.Wherein alkaloid is a kind of main chemical component in purslane, at present
Reported composition of alkaloids have norepinephrine, dopamine, a small amount of DOPA, adenosine, uracil, adenine, N, N- bis-
Cyclohexyl urea, allantoin, N- be trans--asafoetide acyl group tyrasamine；There are also Cyclic dipeptides alkaloid and alkaloids: oleracein A-I, K,
L、N-S。
Most of chemical component isolated from purslane at present is known, and structure novel is lower, therefore, right
The exploitation and separation of noval chemical compound urgently need in purslane.
Summary of the invention
In view of the above-mentioned problems, the present invention provides the Oleracone I of key compound containing peroxide and its extraction in a kind of purslane
Separation method, the noval chemical compound specially extracted from purslane, it has been investigated that novel compound of present invention have it is antitumor,
Anti-inflammatory effect, while a kind of extraction separation side easy, quick, environmentally friendly, with high purity for noval chemical compound of the present invention being provided
Method.
To achieve the above object, the present invention provides following technical scheme.
The Oleracone of key compound containing peroxide I, molecular formula C in purslane18H18O6, it is named as oleracone I, is changed
Learn structural formula are as follows:
。
Extraction separation method containing peroxide key compound Oleracone I in a kind of purslane, the specific steps are.
Step 1 takes the dry medicinal material of purslane, adopts and be extracted with water (water consumption be medicinal material 8~16 times) twice, Aqueous extracts filter
It crosses, merging filtrate directly heats concentration, cools to room temperature, it is spare to obtain medical fluid.
Step 2, upper silicagel column after being evaporated step 1 Chinese medicine liquid, are eluted with ethyl acetate, and ethyl acetate is recovered under reduced pressure to leaching
Cream obtains ethyl acetate extract.
Step 3 separates ethyl acetate extract in step 2 through polyamide column, using alcohol-water gradient elution, 50% second
Alcohol upper silicagel column after being evaporated, successively obtains several elution positions with acetate-methanol gradient elution, is examined through thin-layer chromatography
It surveys, colour developing merges the elution position of colour developing, spare by the elution position after merging through being concentrated to dryness.
Step 4, by the pretreated ODS column of gains in step 3, (Octadecylsilyl, octadecylsilane are bonded
Silica filler) chromatography obtains several elution positions, detected through thin-layer chromatography with methanol-water gradient elution, it develops the color,
The elution position of each colour developing is concentrated to dryness respectively, it is spare to obtain concentrate.
Step 5, by gains in step 4, pretreated sephadex column (Sephadex LH-20) chromatography divides again
From, it is eluted with methanol, obtains several elution positions, detected through thin-layer chromatography, developed the color, merge the elution position of colour developing, it will
Elution position after merging is spare through being concentrated to dryness.
Gained noval chemical compound in step 5 is passed through HPLC(efficient liquid phase by step 6) separation preparation, with -0.1% first of acetonitrile
Acid is that mobile phase carries out isocratic elution, finally obtains compound oleracone I.
The preprocessing process of the ODS and sephadex is that methanol impregnated 24 hours, and upper prop is washed till instillation with methanol
Without muddiness in water, then balanced each other with initial flow.
Beneficial effects of the present invention compared with prior art.
The separation of heretofore described purslane noval chemical compound containing peroxide bridge and pharmacology activity research be not by the existing paper phase
Periodical is reported；The present invention provides noval chemical compound containing peroxide bridge and a kind of mentioning for noval chemical compound of the present invention from purslane
Take separation method, successively using water boiling and extraction, silica gel column chromatography, polyamide column, compression leg in ODS, Sephadex LH-20 and
High performance liquid chromatograph is isolated and purified and is prepared, and is successfully extracted and is isolated noval chemical compound, this method operating procedure is only six
Step, operating method are easy and quickly, extract separation process and mainly adopt and are extracted with water and ethyl acetate elution, process environmental protection,
And be all larger than 90% through the isolated compound purity of this method is higher, furthermore research has shown that this compound have it is antitumor,
Anti-inflammatory effect, therefore noval chemical compound of the present invention and its salt and derivative can be used as other compound synthesis primers, Yi Jixin
The raw material of medicine exploitation and pharmacology activity research, also can be used for preparing antitumor, anti-inflammatory drug.
Detailed description of the invention
Fig. 1 is the ultraviolet spectrogram of noval chemical compound oleracone I of the present invention.
Fig. 2 is the infrared spectrogram of noval chemical compound oleracone I of the present invention.
Fig. 3 is the high resolution mass spectrum figure of noval chemical compound oleracone I of the present invention.
Fig. 4 is noval chemical compound oleracone I of the present invention1H-NMR spectrogram.
Fig. 5 is noval chemical compound oleracone I of the present invention13C-NMR spectrogram.
Fig. 6 is carbon-13 nmr spectra (DEPT) spectrogram of noval chemical compound oleracone I of the present invention.
Fig. 7 is the nuclear magnetic resonance of noval chemical compound oleracone I of the present invention1H-1H COSY spectrogram.
Fig. 8 is the nuclear magnetic resonance HMBC spectrogram of noval chemical compound oleracone I of the present invention.
Fig. 9 is the nuclear magnetic resonance HSQC spectrogram of noval chemical compound oleracone I of the present invention.
Figure 10 is the nuclear magnetic resonance NOESY spectrogram of noval chemical compound oleracone I of the present invention.
Specific embodiment
Embodiment.
The present invention provides noval chemical compound, molecular formula C18H18O6, it is named as oleracone I, chemical eliminant are as follows:
。
The noval chemical compound is named as oleracone I according to structure, and table 1 is the nuclear magnetic data of the noval chemical compound:1H-
NMR with13C-NMR is in deuterated DMSO.
1 noval chemical compound of table nuclear magnetic data (1H-NMR with13C-NMR is in deuterated DMSO).
The compounds of this invention Structural Identification refering to fig. 1-10.
Oleracone I: yellowish-brown powder is soluble in methanol.UV(MeOH)λmax: 284, 214 nm。IR (KBr)v max 2920, 2850, 2360, 1670, 1610, 1460, 1215, 1160, 831, 762 cm-1。HRESI(+)
TOFMS provides m/z:331.1141 [M+H]+Quasi-molecular ion peak, molecular weight 331.1137.In conjunction with1H-NMR,13C-
NMR and DEPT data, thus it is speculated that the possible molecular formula of the compound is C18H18O6, degree of unsaturation 10.13C-NMR spectrum and DEPT
Spectrum 18 carbon signals of display, respectively 2 OCH3(δ: 55.7；55.9), 3 CH2(δ: 33.2；72.1；72.5), 6 olefinic carbons
(δ: 92.9；93.3；115.0；118.6；127.5；132.4), 7 quaternary carbons (double key carbon of 5 company O,δ: 189.9；165.4；
163.9；162.3；155.7；2 double key carbons,δ: 122.1；103.5).
12 CH of H-NMR spectrum display3Signal is respectivelyδ3.78(3H, s)；δ3.80(3H, s)；3 methylene signals, respectively
For δ 2.89 (2H, dd, J=13.8,20.5)；δ 4.01 (1H, d, J=11.64), δ 4.07 (1H, d, J=11.5)；δ 4.10 (2H, d,
J=4.44)；6 methine signals are respectively (1H, d, J=2.28) δ 6.12；δ 6.20 (1H, d, J=2.28)；δ 6.69 (1H, m)；
δ 6.76 (1H, dd, J=0.78,7.92)；δ 7.02 (1H, m)；δ 7.11 (1H, dd, J=1.5,7.5).It can according to H-H COSY spectrum
Know, two methine δ 6.12 and δ 6.20 are coupled, and four methine δ 6.69, δ 6.76, δ, 7.02 and δ 7.11 are coupled, and say
It is bright that there are two the presence of phenyl ring.According to HMBC compose relevant peaks show that H δ 3.78 and the C-4 in methoxyl group is coupled, H δ 3.80 and
C-2 is coupled and C-4 (δ 162.3) and C-2 (δ 165.4) are located at low field area, prompts to be connected with O, illustrates two methoxyl groups point
Not on phenyl ring C-4 and C-2 be connected；Show that H and H-1 in two methoxyl groups, H-3 are coupled, say according to NOE spectrum
Bright and C-1, C-3 are associated；H-1 is coupled with C-3, and C-3(δ 163.9) it is located at low field area, it prompts to be connected with O；H-1ʹ
It is coupled with H-3 and C-4, H-1 and H-5 and C-3 are coupled, and C-5(δ 72.5) it is located at low field area, it prompts to be connected with O, say
Bright C-4 is connected with 6 O by C-5 methylene；H-5 is coupled with C-9, and the carbonyl C and C-9 of H-7 and C-8 are coupled, and C-7
(δ 72.1) is located at low field area, prompts to be connected with O, meanwhile, the carbonyl C of H-9 and C-8 are coupled, and are coupled with C-7, explanation
C-7 is connected with 6 O and is connected by the carbonyl C of C-8 with C-9；H-1, H-2 and H-3 are coupled with C-10, and H-9
It is coupled with C-10 and C-4, H-4 is coupled with C-9, illustrates that C-9 is connected with another phenyl ring；H-1, H-2, H-3
It is coupled with C-11 with H-4, and C-11(δ 155.7) it is located at low field area, it prompts to be connected with O；Show two according to NOE spectrum
Methine on a phenyl ring is coupled, and [M+H] provided by HRESI (+) TOFMS+Quasi-molecular ion peak evidence, 1
O and 2 O in position is connected to form peroxide bridge.According to information above, it may be determined that this noval chemical compound is above structure.
The present invention also provides the extraction separation method of above compound, the specific steps are.
Step 1: weighing dry 150 kg of medicinal material of purslane, using water refluxing extraction, water consumption (v/v) is the 10 of medicinal material
Times, twice, 2 h every time heats concentration at 90-100 DEG C, cools to room temperature, it is spare to obtain medical fluid refluxing extraction.
Step 2: separating after gained medical fluid in step 1 is evaporated through silica gel column chromatography, washed with ethyl acetate (115 L) is isocratic
De-, wherein silica gel is 100-200 mesh, and 40 DEG C or less are recovered under reduced pressure ethyl acetate to medicinal extract, obtain ethyl acetate extract.
Step 3: ethyl acetate extract in step 2 is separated through polyamide column, using alcohol-water (0/100,30/70,
50/50,70/30,100/0, v/v) gradient elution separates after being evaporated at 50% 90-100 DEG C of ethyl alcohol through silica gel column chromatography, wherein silicon
Glue is 200~300 mesh, successively uses acetate-methanol (5:1,2:1,1:2, v:v) gradient elution, 19 positions are obtained (i.e.
19 bottles, every bottle of 300 mL are obtained), it is detected, is developed the color through thin-layer chromatography, merged 1~14 elution position of colour developing, will close
40 DEG C of 1~14 position after and or less is concentrated to dryness, spare.
Step 4: by gains in step 3 again the separation of pretreated ODS medium pressure column chromatography (Octadecylsilyl, ten
Eight alkyl silane bonded silica gel fillers), wherein filler particle size be 20~40 μm, with methanol-water (50/50,60/40,70/30,
80/20,100/0, v/v) gradient elution (pressurization, make 1 mL/min of flow velocity, temperature is room temperature), it is (i.e. terraced to obtain 16 positions
Degree elutes to obtain 16 bottles, every bottle of 100 mL), it is detected, is developed the color through thin-layer chromatography, 7~9 positions of colour developing are retained, 50 DEG C
It is concentrated to dryness below, it is spare.
Step 5: by pretreated sephadex column chromatography for separation (the Sephadex LH- again of gains in step 4
20) it, is eluted with methanol, obtains 26 elution positions (26 bottles, every bottle of 50 mL are obtained), detected through thin-layer chromatography, shown
Color retains the position 5-11 of colour developing, and 50 DEG C or less are concentrated to dryness, spare, obtains noval chemical compound.The ODS and Portugal are poly-
The preprocessing process of sugared gel is that methanol impregnated 24 h, and upper prop is washed till with methanol and is instilled in water without muddiness, then with initial flow
It balances each other.
Step 6: gained noval chemical compound in step 5 being separated through HPLC and is prepared, with acetonitrile: 0.1% formic acid (40:60, v/v)
As mobile phase, Detection wavelength is 210 nm, 280 nm, and noval chemical compound of the present invention is prepared in separation, and normalization method measures purity
It is 90~99%.
The antitumor action of the compounds of this invention.
1 main material.
1.1 drugs and reagent: testing noval chemical compound used and prepared by the above method, and purity is 90~99%, and precision weighs,
Solution needed for being diluted to following each dosage groups with DMSO.DMEM high glucose medium, fetal calf serum (Hyclone company, the U.S.)；It is green
Mycin, streptomysin (Hangzhou Chinese holly company).
1.2 cell strains: Human colon adenocarcinoma cell line Caco-2, human breast cancer cell line Bcap-37, human gastric cancer cells BGC-823, people
Lung adenocarcinoma cell SPC-A1, human liver cancer cell BEL-7402, human cervical carcinoma cell Hela-229, ovarian cancer cell Ho-8910, people
Class oral cavity epidermoid carcinoma cell KB(Chinese Academy of Sciences Shanghai cell bank).
1.3 groupings: it is divided into control group, experimental group and zeroing group (culture solution of the solvent containing DMSO).
2 experimental methods.
The fetal calf serum of l0% is added in 2.1 cell culture, DMEM high glucose medium, and (100 U/mL are green for l% antibiotic
Mycin and 100 μ g/mL streptomysins), it is placed in 37 DEG C, 5%CO2It is cultivated in incubator.
2.2 MTI methods detect cell Proliferation, and logarithmic growth phase cell inoculation is in 96 well culture plates, cell density 1
× 104/mL, every 100 μ L of hole, 37 DEG C of temperature, 5% CO2Under the conditions of after overnight incubation, the sheet of various concentration is added in experimental group
Invention noval chemical compound, every group sets 3 multiple holes, and dosing is placed on 37 DEG C, 5%CO248 h are cultivated in incubator.By drug containing culture solution
Suck, it is 5 mg/mL that serum-free medium that volume ratio is 4:1 and MTT(end mass concentration, which is added) totally 100 mL, continues to be incubated for
4 h, after carefully sucking supernatant, DMSO150 μ L is added in every hole, is put on oscillator and is shaken so that crystallization is completely dissolved (5
Min), microplate reader detects absorbance (A) value in each hole under 570 nm wavelength.Then, it is raw to calculate each concentration compound on intracellular
Long inhibiting rate, inhibiting rate formula: inhibitory rate of cell growth=(1-AMedicine feeding hole/ AControl wells) × 100% reapplies the processing of SPSS software
Inhibiting rate is made curve to drug concentration, calculates IC by data50Value.
3 experimental results.
The experimental results showed that present invention noval chemical compound containing peroxide bridge is to Human colon adenocarcinoma cell line Caco-2, human breast cancer cell
MCF-7, human gastric cancer cells BGC-823, human lung adenocarcinoma cell SPC-A1, human liver cancer cell BEL-7402, human cervical carcinoma cell
Hela-229, ovarian cancer cell Ho-8910, the proliferation of Human Oral Cavity epidermoid carcinoma cell KB are inhibited, and with drug
Concentration increases, and inhibiting rate is also significantly raised, that is, is in concentration dependant.Noval chemical compound of the present invention is to above-mentioned eight kinds of tumour cell IC50Value
It is shown in Table 5.
Inhibiting effect of the noval chemical compound of the present invention of table 2 to tumour cell.
The anti-inflammatory effect of noval chemical compound of the present invention.
1, main material.
1.1, drug and reagent: testing noval chemical compound used and prepared by the above method, and purity is 90~99%, and precision weighs,
Solution needed for being diluted to following each dosage groups with DMSO.DMEM high glucose medium, fetal calf serum (Hyclone company, the U.S.)；It is green
Mycin, streptomysin (Hangzhou Chinese holly company)；LPS(Sigma Co., USA)；IL-6,TNF-α,PGE2ELISA kit
(Cayman company, the U.S.)；Cell pyrolysis liquid, Griess reagent (green skies Bioisystech Co., Ltd).
1.2 cell strains: RAW264.7 macrophage (U.S.'s ATCC cell bank).
1.3 groupings: being divided into control group, LPS group and experimental group, and each one group.
2 experimental methods.
The fetal calf serum of l0%, l% antibiotic (100U/mL mould is added in 2.1 cell culture, DMEM high glucose medium
Element and 100 μ g/mL streptomysins), it is placed in 37.5%, CO2It is cultivated in incubator.
2.2 MTT colorimetric method for determining cell viabilities, above-mentioned three groups respectively logarithmic growth phase RAW264.7 macrophage connect
For kind in 96 well culture plates, cell density is 1 × 104A/mL, every 100 μ L of hole, 37 DEG C of temperature, 5%CO2Under the conditions of overnight incubation
Afterwards, the noval chemical compound Oleracone I(1-50 μM of the present invention of various concentration is added in experimental group), it is incubated for after 1h to LPS group and reality
The LPS that group is separately added into final concentration of 1 μ g/mL is tested, zeroing group (culture solution of the solvent containing DMSO) is separately set, every group sets 3 multiple holes,
Investigate the influence being added after drug to cell.After above-mentioned group of cells culture for 24 hours, 5 mg/mL MTT are added in each hole cell
20 μ L, 37 DEG C of temperature, 5%CO2Under the conditions of continue after being incubated for 4h, terminate culture, inhale and abandon liquid in hole, 100 μ L diformazans are added in every hole
Base sulfoxide (DMSO) vibrates 10min, makes to crystallize sufficiently dissolution into the cell, each hole light absorption value of measurement at microplate reader 570nm wavelength.
2.3 measure the content of NO using Ge Lisi (Griess) method, and investigation noval chemical compound of the present invention induces LPS small
The inhibiting effect of the NO yield of mouse macrophage RAW264.7.Containing 10% tire ox after mouse macrophage RAW264.7 passage
It is cultivated in the sugared cell culture medium DMEM of the height of serum, the noval chemical compound Oleracone I of the present invention of various concentration is added in experimental group
(1-20 μM), at 37 DEG C, 5%CO2Under the conditions of be incubated for 1h after use the final concentration of 1 μ g/mL of LPS() induction inflammatory reaction, receive afterwards for 24 hours
Collect supernatant, every group of processing repeats 3 holes.Griess method measures the content of NO in cell supernatant, according to the various concentration present invention
Influence of the noval chemical compound to the LPS RAW264.7 cell release NO induced, to reflect NO level.
2.4 ELISA methods measure inflammatory factor IL-6, TNF-α and inflammatory mediator PGE2: by logarithmic growth phase RAW264.7
Macrophage is inoculated in 24 well culture plates, and cell density is 1 × 105A/mL, every hole 1mL, 37 DEG C of temperature, 5% CO2Under the conditions of
Noval chemical compound Oleracone I(1-20 μM of the present invention is added in overnight incubation, experimental group), after cultivating 1h, LPS is added in every hole
(final concentration of 1 μ g/mL) is incubated for for 24 hours altogether, and every group of processing repeats 3 holes.ELISA method measures the processing of purslane source noval chemical compound
IL-6, TNF-α and the PGE of RAW264.7 macrophages secrete afterwards2Content.
3 experimental results.
The experimental results showed that proliferation nothing of the present invention noval chemical compound containing peroxide bridge to the LPS macrophage RAW264.7 induced
It influences, it is safe and non-toxic；And excess inflammatory cytokine IL- produced by the macrophage RAW264.7 of LPS induction can be effectively suppressed
6, TNF-α and inflammatory mediator NO, PGE2, and be in concentration dependant.
Comparative survival rate of cells experimental result is as shown in table 3.
Influence of 3 present invention of table to RAW264.7 macrophage relative survival rate.
Note:*P < 0.05 compared with the control group (high concentration group has significant difference), is measured using Ge Lisi (Griess) method
The content experimental result of NO is shown in Table 4.
Influence (mean ± standard deviation, n=3) of 4 present invention of table to the RAW264.7 cell release NO of LPS induction.
Note:*P < 0.05 compared with the control group,#P < 0.05 is compared with LPS group.
ELISA method measures inflammatory factor IL-6, TNF-α and inflammatory mediator PGE2The results are shown in Table 5.
IL-6, TNF-α and PGE of 5 present invention of table to the RAW264.7 cell secretion of LPS induction2The influence of content is (
Number ± standard deviation, n=3).
Note:*P < 0.05 compared with the control group,#P < 0.05 is compared with LPS group.
In conclusion the present invention provides special compound and its extraction separation method, water refluxing extraction, silica gel are successively used
Column chromatography, polyamide column chromatography, compression leg, the purifying of sephadex column chromatography for separation in ODS, successfully isolated a kind of newization
Object is closed, this method is easy, quickly, environmental protection, and it is higher through the isolated compound purity of this method, due to gained chemical combination materialization
Unique structure is learned, is extracted from conventional Chinese medicine purslane, with antitumor and anti-inflammatory effect, therefore the present invention becomes privileged
It closes object and its salt and derivative can be used as natural products exploitation new Chinese medicine, have broad prospects.
Claims (10)
1. the Oleracone of key compound containing peroxide I, molecular formula C in purslane18H18O6, chemical structural formula are as follows:
。
2. the extraction separation method of compound Oleracone I as described in claim 1, which is characterized in that specific steps are as follows:
Step 1 takes the dry medicinal material of purslane to be cooled to room temperature using water boiling and extraction, it is spare to be obtained medical fluid；
Step 2, upper silicagel column after being evaporated step 1 Chinese medicine liquid, are eluted with ethyl acetate, ethyl acetate are recovered under reduced pressure to medicinal extract,
Obtain ethyl acetate extract；
Step 3 separates ethyl acetate extract in step 2 through polyamide column, using alcohol-water gradient elution, 50% ethyl alcohol portion
Divide upper silicagel column after being evaporated, successively obtains several elution positions with acetate-methanol gradient elution, examined through thin-layer chromatography
It surveys, colour developing merges the elution position of colour developing, spare by the elution position after merging through being concentrated to dryness；
Step 4, by gains in step 3 pretreated ODS column (Octadecylsilyl, octadecylsilane chemically bonded silica
Filler) chromatography obtains several elution positions, detected through thin-layer chromatography with methanol-water gradient elution, develops the color, and it will be each
The elution position of colour developing is concentrated to dryness respectively, and it is spare to obtain concentrate；
Step 5, by pretreated sephadex column (Sephadex LH-20) chromatography again of gains in step 4, use
Methanol elution, obtains several elution positions, is detected through thin-layer chromatography, develops the color, merges the elution position of colour developing, after merging
Elution position through being concentrated to dryness, it is spare；
Step 6 carries out HPLC (efficient liquid phase) separation preparation to gained concentrate in step 5, with the body of acetonitrile and 0.1% formic acid
A kind of key compound containing peroxide is prepared than being 45:55 as mobile phase in product.
3. extraction separation method as claimed in claim 2, which is characterized in that water boiling and extraction 2 times in the step 1 are decocted every time
It boils 2 hours, water is 8~16 times of medicinal material.
4. extraction separation method as claimed in claim 2, which is characterized in that the ODS and sephadex it is pretreated
Journey is that methanol impregnated 24 hours, upper prop, is washed till to instill in water with methanol and be balanced each other without muddiness, then with initial flow.
5. extraction separation method as claimed in claim 2, which is characterized in that mobile phase elution program used in the step 2
For isocratic elution.
6. extraction separation method as claimed in claim 2, which is characterized in that use the volume ratio of water and ethyl alcohol in the step 3
For 100:0,70:30,50:50,30:70 and 0:100 gradient elution.
7. extraction separation method as claimed in claim 2, which is characterized in that use ethyl acetate and acetic acid second in the step 3
The volume ratio of ester and methanol is 5:1,2:1 and 1:2 gradient elution.
8. extraction separation method as claimed in claim 2, which is characterized in that use the volume ratio of first alcohol and water in the step 4
For 50:50,60:40,70:30 and 80:20 gradient elution.
9. extraction separation method as claimed in claim 2, which is characterized in that be with methanol elution program in the step 5
Degree elution.
10. compound oleracone I as described in claim 1 is used to prepare antitumor, anti-inflammatory drug.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910266306.4A CN110294733B (en) | 2019-04-03 | 2019-04-03 | Peroxide bond-containing compound Oleracone I in purslane, and extraction separation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910266306.4A CN110294733B (en) | 2019-04-03 | 2019-04-03 | Peroxide bond-containing compound Oleracone I in purslane, and extraction separation method and application thereof |
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| Publication Number | Publication Date |
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| CN110294733A true CN110294733A (en) | 2019-10-01 |
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