CN103508912A - Method for separation and preparation of spermidines from safflower by employing high-speed counter-current chromatography - Google Patents
Method for separation and preparation of spermidines from safflower by employing high-speed counter-current chromatography Download PDFInfo
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Abstract
The invention relates to a separation method of natural medicines, and specifically relates to a method for separation and preparation of spermidines from safflower by employing high-speed counter-current chromatography. The method comprises the following steps: (1) mixing chloroform, methanol and aqueous solution to form a mixed solvent, layering; (2) dissolving safflower spermidine crude extract in the lower layer of the mixed solvent obtained in the step (1); (3) layering the mixed solvent in the step (1) for obtain a solid phase and a mobile phase, injecting the solution in the step (2) into chromatographic column via an admission valve, continuously pumping the mobile phase after injection is finished; and (4) collecting isolates and concentrating to obtain first spermidine, a second spermidine, third spermidine and fourth spermidine. By employing the high-speed counter-current chromatography and the reasonable solvent system, high-purity spermidine compounds are extracted from safflower; on solid-phase carrier is needed, and no irreversible adsorption exists, spermidines are prepared efficiently rapidly with low cost and without sample loss and without pollution. The method is simple in technology, convenient to operate, safe, good in repeatability, and applicable to industrialized production.
Description
Technical field
The present invention relates to the separation method of natural drug, be specifically related to a kind of by high-speed countercurrent chromatography 4 methods along anti-isometry spermidine of separated preparation from safflower.
Background technology
The dried floral of safflower composite family platymiscium safflower, can " invigorate blood circulation, moisturize, pain relieving, loose swollen stimulating the menstrual flow ".In the traditional Chinese medical science, think pungent, the micro-hardship of safflower taste, warm in nature, the thoughts of returning home and Liver Channel, there is the effect of promoting blood circulation to restore menstrual flow, blood stasis removing analgesic, hypotensive, reducing blood-fat, for through closing, the disease such as dysmenorrhoea, wound, coronary heart diseases and angina pectoris and hypertension.Safflower becomes the important Chinese medicine of the diseases such as prevention and treatment coronary heart disease, myocardial infarction and cerebral thrombosis in modern clinic.Modern pharmacology experiment shows: the pharmacological actions such as safflower can improve the supply of heart and brain blood oxygen, alleviates ischemia injury, anticoagulation, resists myocardial ischemia, anticoagulant, anti-oxidant, antitumor and anti-inflammatory.
There is numerous isomerss in the spermidine in safflower, traditional chromatographic separation means can not realize its efficient, sharp separation.In tradition research, conventionally use multistep silica gel column chromatography and coupling to prepare the method separation such as liquid phase and prepare spermidine in safflower, the length that not only expends time in, can not detect online, also can cause dead absorption, loses greatlyr, and yield is lower.
High speed adverse current chromatogram (high-speed countercurrent chromatography, HSCCC) be a kind of liquid luquid partition chromatography technology of continuous high-efficient, owing to not needing solid support, the separation of material realizes according to the difference of its partition ratio in two-phase, thereby avoided because of sample loss that irreversible adsorption causes, inactivation, sex change etc., the theoretical recovery of sample is 100%, is particularly suitable for the separation of Natural active component.Utilize high speed adverse current chromatogram separated suitability for industrialized production of preparing spermidine compounds from safflower, have not yet to see domestic and international related documents and patent report.
Summary of the invention
The invention discloses a kind of with high speed adverse current chromatogram from safflower 1,2,3 and 4 four spermidines of separating compound (
n 1,
n 5,
n 10-(E)-tri-
p-coumaroylspermidine (EEE),
n 1(E)-
n 5-(Z)-
n 10-(E)-tri-
p-coumaroylspermidine (EZE),
n 1(Z)-
n 5-(Z)-
n 10-(E)-tri-
p-coumaroylspermidine (ZZE) and
n 1,
n 5,
n 10-(Z)-tri-
pthe method of-coumaroylspermidine (ZZZ), four spermidines
Structural formula as Figure 1-Figure 4.
In the separated safflower of a kind of high speed adverse current chromatogram of the present invention, the method for four spermidines is achieved by following technical proposals:
In the separated safflower of a kind of high speed adverse current chromatogram, the method for four spermidines comprises the following steps:
1. chloroform, methyl alcohol and aqueous solution are become to mixed solvent, layering;
2. safflower spermidine crude extract is dissolved in step ① lower floor;
3. get 1. mixed solvent layering of step, lower to stationary phase, upper as moving phase, stationary phase and moving phase are successively pumped in high speed adverse current chromatogram, by step 2. solution through sampling valve, enter chromatographic column, after sample introduction, continue to pump into moving phase;
4. according to detector spectrogram, collect respectively isolate, concentrated, obtain 1,2,3 and 4 four along anti-isometry spermidine monomer.
Advantage of the present invention:
The present invention utilizes high speed adverse current chromatogram and has adopted rational solvent system from safflower, to obtain highly purified spermidine compound, this technology does not need to use solid phase carrier, without irreversible adsorption, sample nondestructive loses, pollution-free, efficient, quick, low-cost separation is prepared spermidine, and the spermidine purity of acquisition is more than 95%.
Technique of the present invention is simple, easy to operate, safe, reproducible, can suitability for industrialized production.
Accompanying drawing explanation
In the separated safflower of a kind of high speed adverse current chromatogram of the present invention, the method for four spermidines has following accompanying drawing:
Fig. 1 is method spermidine 1 chemical structural formula of four spermidines in the separated safflower of a kind of high speed adverse current chromatogram of the present invention;
Fig. 2 is method spermine 2 chemical structural formulas of four spermidines in the separated safflower of a kind of high speed adverse current chromatogram of the present invention;
Fig. 3 is method spermine 3 chemical structural formulas of four spermidines in the separated safflower of a kind of high speed adverse current chromatogram of the present invention;
Fig. 4 is method spermine 4 chemical structural formulas of four spermidines in the separated safflower of a kind of high speed adverse current chromatogram of the present invention;
Fig. 5 is the separated spermidine spectrogram of method high speed adverse current chromatogram of four spermidines in the separated safflower of a kind of high speed adverse current chromatogram of the present invention;
Fig. 6 is the method safflower spermidine crude extract Liquid Detection figure of four spermidines in the separated safflower of a kind of high speed adverse current chromatogram of the present invention;
Fig. 7 is the method spermidine 1 monomer Liquid Detection figure of four spermidines in the separated safflower of a kind of high speed adverse current chromatogram of the present invention;
Fig. 8 is the method spermidine 2 monomer Liquid Detection figure of four spermidines in the separated safflower of a kind of high speed adverse current chromatogram of the present invention;
Fig. 9 is the method spermidine 3 monomer Liquid Detection figure of four spermidines in the separated safflower of a kind of high speed adverse current chromatogram of the present invention;
Figure 10 is the method spermidine 4 monomer Liquid Detection figure of four spermidines in the separated safflower of a kind of high speed adverse current chromatogram of the present invention.
Embodiment
Below in conjunction with drawings and Examples, the method and technology scheme of four spermidines in the separated safflower of a kind of high speed adverse current chromatogram of the present invention is further described.
As shown in Fig. 1-Figure 10, a kind of high speed adverse current chromatogram separated method of preparing spermidine from safflower of using, is characterized in that described method comprises the following steps:
1. chloroform, methyl alcohol and aqueous solution are become to mixed solvent, layering;
2. safflower spermidine crude extract is dissolved in step ① lower floor;
3. get 1. mixed solvent layering of step, lower to stationary phase, upper as moving phase, stationary phase and moving phase are successively pumped in high speed adverse current chromatogram, by step 2. solution through sampling valve, enter chromatographic column, after sample introduction, continue to pump into moving phase;
4. according to detector spectrogram, collect respectively isolate, concentrated, obtain 1,2,3 and 4 four along anti-isometry spermidine monomer.
The chloroform of described step in 1., the volume ratio of first alcohol and water are 3～5: 4: 3～5.
The described step concentration that 2. middle safflower crude extract is dissolved in after mixed solvent is 6～15 mg/mL.
Described safflower spermidine crude extract is prepared by following method: by liquid ratio 10～20:1, add 60～80% alcohol in 45～65 ℃ of refluxing extraction 2～4 times safflower dried floral, each 1.5～2.5 hours, united extraction liquid is also evaporated to dry, by the medicinal extract deionized water dissolving of evaporate to dryness, then by isopyknic sherwood oil and ethyl acetate, extract respectively 3～6 times; By macroporous adsorbent resin on water, first use 25～35% ethanol elution, then use 45～55% ethanol elutions; 45～55% ethanol eluates are evaporated to dry, obtain.
Described high-speed counter-current chromatograph flow rates is 0.5～2 mL/min, and speed range is 600～900 r/min.
Described detection wavelength is 210～360nm.
1, safflower spermidine crude extract preparation
Getting safflower 500 g adds 70% alcohol in 60 ℃ of refluxing extraction 3 times by liquid ratio 15:1, each 2 h, united extraction liquid is also evaporated to dryly, by 250 mL deionized water dissolvings for the medicinal extract of evaporate to dryness, then by isopyknic sherwood oil and ethyl acetate, extracts respectively 5 times.Get 500 g AB-8 macroporous resins, pack the chromatography column of diameter 20 cm into, by water loading, 30% and 50% ethanol is wash-out successively.50% ethanol eluate is evaporated to dry, is stored in refrigerator and treats that further HSCCC is separated.
2, spermidine is prepared in high-speed counter-current separation
In separating funnel, prepare chloroform-methanol-water (1:1:1, v/v) two-phase solvent system, hold over night after shake well.Lower is stationary phase mutually, and upper is moving phase mutually, difference ultrasonic degas 30 min before using.Get 100 mg sample powder to be separated, concussion is dissolved under 10 mL in phase solution completely, standby.
With 15 mL/min flow velocitys, will under solvent systems, pump into mutually main frame and be full of separated solenoid coil, ON cycle water-bath be 25 ℃ by Temperature Setting.Open host power supply and be rotated in the forward with 850 r/min, after stabilization of speed, with 2.0 mL/min flow pump, enter moving phase, until moving phase, from tubing string outlet outflow and baseline stability, sample solution is injected by sample introduction circle.Tubing string exit effluent liquid is continuous detecting under 280 nm wavelength, according to color atlas, manually collect as shown in Figure 5 each chromatographic peak component, from 100 mg sample to be separated, disposable separation prepares 1.3 mg compound Isosorbide-5-Nitrae .4 mg compounds 2,7.2 mg compound 3,11.5 mg compounds 4.HPLC detects purity and is respectively 95.5%, 98.1%, 97.5% and 96.2%, as shown in Fig. 7-Figure 10.
HPLC analysis condition: chromatographic column: Eslipse XDB C
18(4.6 mm*250 mm, 5 μ m), moving phase: methanol-water (30:70～70:30,0～40 min), flow velocity 1.0 mL/min, 25 ℃ of column temperatures, detect wavelength 280 nm.
3, Structural Identification
Peak 1:ESI-MS:
m/z584 [M+H]
+;
1h-NMR (CD
3oD, 400 MHz)
δ=1.44/1.57 (2H, m, H-8), 1.48/1.64 (2H, m, H-7), 1.83/1.91 (2H, m, H-3), 3.03/3.21 (2H, m, H-9), 3.10/3.27 (2H, m, H-2), 3.30/3.33 (2H, m, H-6), 3.41/3.45 (2H, m, H-4), 6.36/6.41 (d
j=15.5 Hz, H-8
'), 6.50/6.53 (d,
j=15.5 Hz, H-8
' ' '), 6.65/6.72 (m,
j=8.5 Hz, H-3
' ', 5
' '), 6.79 (m,
j=8.5 Hz, H-3
', 5
'), 6.80 (m,
j=8.5 Hz, H-3
' ' ', 5
' ' '), 6.85/6.93 (d,
j=15.5 Hz, H-8
' '), 7.28/7.35 (d,
j=8.5 Hz, H-2
', 6
'), 7.38 (d,
j=8.5 Hz, H-2
' ' ', 6
' ' '), 7.44 (d,
j=15.5 Hz, H-7
'), 7.44/7.50 (d,
j=8.5 Hz, H-2
' ', 6
' '), 7.50/7.52 (d,
j=15.5 Hz, H-7
' ' '), 7.52/7.57 (d,
j=15.5 Hz, H-7
' ');
13c-NMR (CD
3oD, 400 MHz)
δ=25.5/26.8 (C-7), 27.2/27.7 (C-8), 28.3/29.6 (C-3), 37.7/38.1 (C-2), 39.4/39.7 (C-9), 44.8/46.5 (C-4), 47.1/48.5 (C-6), 113.4 (C-8
' '), 116.1 (C-3
', 3
' ', 3
' ' ', 5
', 5
' ', 5
' ' '), 117.9/118.1 (C-8
'), 118.2 (C-8
' ' '), 127.5 (C-1
', 1
' ' '), 128.0 (C-1
' '), 129.5 (C-2
', 6
', 2
' ' ', 6
' ' '), 130.2 (C-2
' ', 6
' '), 141.9/142.0 (C-7
', 7
' ' '), 143.9 (C-7
' '), 159.7/160.0 (C-4
', 4
' ', 4
' ' '), 169.2/169.3 (C-9
', C-9
' ' '), 169.4 (C-9
' '). be defined as compound (
n 1,
n 5,
n 10-(E)-tri-
p-coumaroylspermidine (EEE).As shown in Figure 7.
Peak 2:ESI-MS:
m/z584 [M+H]
+;
1h-NMR (CD
3oD, 400 MHz)
δ=1.39/1.53 (2H, m, H-8), 1.46/1.64 (2H, m, H-7), 1.66/1.82 (2H, m, H-3), 3.12/3.26 (2H, m, H-2), 3.15/3.26 (2H, m, H-9), 3.19/3.27 (2H, m, H-6), 3.34/3.39 (2H, m, H-4), 5.88/5.93 (d
j=12.5 Hz, H-8
' '), 6.32/6.35 (m,
j=15.5 Hz, H-8
' ' '), 6.39/6.44 (m,
j=15.5 Hz, H-8
'), 6.49/6.54 (m,
j=12.5 Hz, H-7
' '), 6.73 (m,
j=8.5 Hz, H-3
' ', 5
' '), 6.77 (m,
j=8.5 Hz, H-3
', 3
' ' ', 5
', 5
' ' '), 7.19 (d,
j=8.5 Hz, H-2
' ', 6
' '), 7.30 (d,
j=8.5 Hz, H-2
', 6
'), 7.38 (d,
j=8.5 Hz, H-2
' ' ', 6
' ' '), 7.42/7.46 (d,
j=15.5 Hz, H-7
', 7
' ' ');
13c-NMR (CD
3oD, 400 MHz)
δ=25.3/26.7 (C-7), 27.3/27.7 (C-8), 28.0/29.5 (C-3), 37.5/37.9 (C-2), 39.4/39.7 (C-9), 44.9/46.6 (C-4), 47.2/48.7 (C-6), 115.7 (C-3
' ', 5
' '), 115.9 (C-3
', 3
' ' ', 5
', 5
' ' '), 117.9/118.0 (C-8
', 8
' ' '), 120.3/120.4 (C-8
' '), 127.2 (C-1
', 1
' ' '), 127.9 (C-1
' '), 129.7 (C-2
', 6
', 2
' ' ', 6
' ' '), 130.5 (C-2
' ', 6
' '), 134.5/134.8 (C-7
' '), 141.9/142.0 (C-7
', 7
' ' '), 158.2 (C-4
' '), 159.7/159.8 (C-4
', 4
' ' '), 169.4/169.5 (C-9
', 9
' ' '), 171.9/172.0 (C-9
' '). determine that this compound is
n 1(E)-
n 5-(Z)-
n 10-(E)-tri-
p-coumaroylspermidine (EZE).As shown in Figure 8.
Peak 3:ESI-MS:
m/z584 [M+H]
+;
1h-NMR (CD
3oD, 400 MHz)
δ=1.34/1.43 (2H, m, H-8), 1.38/1.50 (2H, m, H-7), 1.61/1.74 (2H, m, H-3), 3.11/3.28 (4H, m, H-2,9), 3.31/3.33 (2H, m, H-6), 3.36/3.37 (2H, m, H-4), 5.72/5.75 (d
j=12.5 Hz, H-8
'), 5.88/5.91 (d,
j=12.5 Hz, H-8
' '), 6.33/5.35 (m,
j=15.5 Hz, H-8
' ' '), 6.41/6.42 (m,
j=12.5 Hz, H-7
' '), 6.45/6.46 (m,
j=12.5 Hz, H-7
'), 6.68 (m,
j=8.5 Hz, H-3
', 3
' ', 5
', 5
' '), 6.73 (m,
j=8.5 Hz, H-3
' ' ', 5
' ' '), 7.18 (d,
j=8.5 Hz, H-2
' ', 6
' '), 7.26/7.29 (m,
j=15.5 Hz, H-7
' ' '), 7.35 (d,
j=8.5 Hz, H-2
' ' ', 6
' ' '), 7.53 (d,
j=8.5 Hz, H-2
', 6
');
13c-NMR (CD
3oD, 400 MHz)
δ=25.4/26.8 (C-7), 27.5/27.8 (C-8), 28.1/29.5 (C-3), 37.4/37.7 (C-2), 39.2/39.5 (C-9), 44.8/46.6 (C-4), 47.5/48.8 (C-6), 115.9 (C-3
' ', 5
' '), 116.2 (C-3
', 5
'), 116.7 (C-3
' ' ', 5
' ' '), 117.9/118.1 (C-8
' ' '), 120.5 (C-8
' '), 121.2 (C-8
'), 127.2 (C-1
' ' '), 127.7 (C-1
', 1
' '), 129.7 (C-2
' ' ', 6
' ' '), 130.4/130.5 (C-2
' ', 6
' '), 131.5/131.6 (C-2
', 6
'), 134.5/134.8 (C-7
' '), 137.4/137.5 (C-7
'), 141.0/142.1 (C-7
' ' '), 158.3/158.4 (C-4
', 4
' '), 159.8/159.9 (C-4
' ' '), 169.4/169.5 (C-9
' ' '), 170.6/170.7 (C-9
'), 171.7/171.8 (C-9
' '). determine that this compound is
n 1(Z)-
n 5-(Z)-
n 10-(E)-tri-
p-coumaroylspermidine (ZZE).As shown in Figure 9.
Peak 4:ESI-MS:
m/z584 [M+H]
+;
1h-NMR (CD
3oD, 400 MHz)
δ=1.40/1.52 (2H, m, H-8), 1.41/1.55 (2H, m, H-7), 1.65/1.78 (2H, m, H-3), 3.12/3.29 (4H, m, H-2,9), 3.32/3.35 (2H, m, H-6), 3.35/3.36 (2H, m, H-4), 5.77/5.80 (d
j=12.5 Hz, H-8
' ' '), 5.83/5.86 (d,
j=12.5 Hz, H-8
'), 5.89/5.93 (d,
j=12.5 Hz, H-8
' '), 6.50/6.52 (m,
j=12.5 Hz, H-7
' '), 6.56/6.59 (m,
j=12.5 Hz, H-7
'), 6.61/6.64 (m,
j=12.5 Hz, H-7
' ' '), 6.72 (m,
j=8.5 Hz, H-3
', 3
' ', 3
' ' ', 5
', 5
' ', 5
' ' '), 7.18 (d,
j=8.5 Hz, H-2
' ', 6
' '), 7.39 (d,
j=8.5 Hz, H-2
', 2
' ' ', 6
', 6
' ' ');
13c-NMR (CD
3oD, 400 MHz)
δ=25.3/26.7 (C-7), 27.1/27.6 (C-8), 28.3/29.7 (C-3), 37.6/38.1 (C-2), 39.5/39.9 (C-9), 44.5/46.2 (C-4), 47.3/48.6 (C-6), 116.3/116.4 (C-3
', 3
' ', 5
', 5
' '), 116.7 (C-3
' ', 5
' '), 120.5 (C-8
' '), 121.2/121.3 (C-8
', 8
' ' '), 127.6/127.7 (C-1
', 1
' ' '), 127.8/127.9 (C-1
' '), 130.4 (C-2
', 6
', 2
' ', 6
' '), 131.5/131.6 (C-2
' ' ', 6
' ' '), 134.5/134.8 (C-7
' '), 137.2/137.3 (C-7
' ' '), 137.8/138.1 (C-7
'), 158.5/158.6 (C-4
', 4
' ', 4
' ' '), 169.9/170.1 (C-9
', 9
' ' '), 17 (C-9
' '). determine that this compound is
n 1,
n 5,
n 10-(Z)-tri-
p-coumaroylspermidine (ZZZ).As shown in figure 10.
1, safflower spermidine crude extract preparation
The preparation of safflower spermidine crude extract is with embodiment 1.
2, spermidine is prepared in high-speed counter-current separation
In separating funnel, prepare chloroform-methanol-water (4:5:4, v/v) two-phase solvent system, hold over night after shake well.Lower is stationary phase mutually, and upper is moving phase mutually, difference ultrasonic degas 30 min before using.Get 100 mg sample powder to be separated, concussion is dissolved under 10 mL in phase solution completely, standby.
With 15 mL/min flow velocitys, will under solvent systems, pump into mutually main frame and be full of separated solenoid coil, ON cycle water-bath be 25 ℃ by Temperature Setting.Open host power supply and be rotated in the forward with 850 r/min, after stabilization of speed, with 1.8 mL/min flow pump, enter moving phase, until moving phase, from tubing string outlet outflow and baseline stability, sample solution is injected by sample introduction circle.Tubing string exit effluent liquid is continuous detecting under 280 nm wavelength, according to color atlas as shown in Figure 5, manually collects each chromatographic peak component, from 100 mg sample to be separated, disposable separation prepares 1.5 mg compounds 1,4.8 mg compound 2,7.9 mg compound 3,12.4 mg compounds 4.HPLC detects purity and is respectively 95.8%, 98.5%, 97.6% and 96.8%.As shown in Fig. 7-Figure 10.
1, safflower spermidine crude extract preparation
The preparation of safflower spermidine crude extract is with embodiment 1.
2, spermidine is prepared in high-speed counter-current separation
In separating funnel, prepare chloroform-methanol-water (1:1:1, v/v) two-phase solvent system, hold over night after shake well.Lower is stationary phase mutually, and upper is moving phase mutually, difference ultrasonic degas 30 min before using.Get 120 mg sample powder to be separated, concussion is dissolved under 10 mL in phase solution completely, standby.
With 15 mL/min flow velocitys, will under solvent systems, pump into mutually main frame and be full of separated solenoid coil, ON cycle water-bath be 25 ℃ by Temperature Setting.Open host power supply and be rotated in the forward with 850 r/min, after stabilization of speed, with 1.5 mL/min flow pump, enter moving phase, until moving phase, from tubing string outlet outflow and baseline stability, sample solution is injected by sample introduction circle.Tubing string exit effluent liquid is continuous detecting under 260 nm wavelength, according to color atlas as shown in Figure 5, manually collects each chromatographic peak component, from 120 mg sample to be separated, disposable separation prepares 1.9 mg compounds 1,5.7mg compound 2,8.4 mg compound 3,13.1 mg compounds 4.HPLC detects purity and is respectively 95.6%, 98.3%, 97.1% and 96.5%.As shown in Fig. 7-Figure 10.
1, safflower spermidine crude extract preparation
Getting safflower 800 g adds 60% alcohol in 50 ℃ of refluxing extraction 4 times by liquid ratio 10:1, each 1.5 h, united extraction liquid is also evaporated to dryly, by 300 mL deionized water dissolvings for the medicinal extract of evaporate to dryness, then by isopyknic sherwood oil and ethyl acetate, extracts respectively 4 times.Get 600 g AB-8 macroporous resins, pack the chromatography column of diameter 20 cm into, by water loading, 25% and 45% ethanol is wash-out successively.45% ethanol eluate is evaporated to dry, is stored in refrigerator and treats that further HSCCC is separated.
2, spermidine is prepared in high-speed counter-current separation
In separating funnel, prepare chloroform-methanol-water (5:4:4, v/v) two-phase solvent system, hold over night after shake well.Lower is stationary phase mutually, and upper is moving phase mutually, difference ultrasonic degas 30 min before using.Get 100 mg sample powder to be separated, concussion is dissolved under 10 mL in phase solution completely, standby.
With 15 mL/min flow velocitys, will under solvent systems, pump into mutually main frame and be full of separated solenoid coil, ON cycle water-bath be 25 ℃ by Temperature Setting.Open host power supply and be rotated in the forward with 850 r/min, after stabilization of speed, with 2.0 mL/min flow pump, enter moving phase, until moving phase, from tubing string outlet outflow and baseline stability, sample solution is injected by sample introduction circle.Tubing string exit effluent liquid is continuous detecting under 280 nm wavelength, according to color atlas as shown in Figure 5, manually collects each chromatographic peak component, from 100 mg sample to be separated, disposable separation prepares 1.2 mg compounds 1,4.3 mg compound 2,7.5 mg compound 3,11.1 mg compounds 4.HPLC detects purity and is respectively 95.9%, 97.1%, 96.7% and 96.3%.As shown in Fig. 7-Figure 10.
1, safflower spermidine crude extract preparation
Getting safflower 1 kg adds 80% alcohol in 60 ℃ of refluxing extraction 4 times by liquid ratio 20:1, each 2.5 h, united extraction liquid is also evaporated to dryly, by 500 mL deionized water dissolvings for the medicinal extract of evaporate to dryness, then by isopyknic sherwood oil and ethyl acetate, extracts respectively 5 times.Get 1 kg AB-8 macroporous adsorbent resin, pack the chromatography column of diameter 30 cm into, by water loading, 40% and 55% ethanol is wash-out successively.55% ethanol eluate is evaporated to dry, is stored in refrigerator and treats that further HSCCC is separated.
2, spermidine is prepared in high-speed counter-current separation
In separating funnel, prepare chloroform-methanol-water (5:4:5, v/v) two-phase solvent system, hold over night after shake well.Lower is stationary phase mutually, and upper is moving phase mutually, difference ultrasonic degas 30 min before using.Get 100 mg sample powder to be separated, concussion is dissolved under 10 mL in phase solution completely, standby.
With 15 mL/min flow velocitys, will under solvent systems, pump into mutually main frame and be full of separated solenoid coil, ON cycle water-bath be 25 ℃ by Temperature Setting.Open host power supply and be rotated in the forward with 850 r/min, after stabilization of speed, with 2.0 mL/min flow pump, enter moving phase, until moving phase, from tubing string outlet outflow and baseline stability, sample solution is injected by sample introduction circle.Tubing string exit effluent liquid is continuous detecting under 280 nm wavelength, according to color atlas as shown in Figure 5, manually collects each chromatographic peak component, from 100 mg sample to be separated, disposable separation prepares 1.2 mg compounds 1,4.3 mg compound 2,7.0 mg compound 3,11.3 mg compounds 4.HPLC detects purity and is respectively 95.1%, 97.5%, 97.3% and 95.9%.As shown in Fig. 7-Figure 10.
Claims (6)
1. use high speed adverse current chromatogram separated method of preparing spermidine from safflower, it is characterized in that described method comprises the following steps:
1. chloroform, methyl alcohol and aqueous solution are become to mixed solvent, layering;
2. safflower spermidine crude extract is dissolved in step ① lower floor;
3. get 1. mixed solvent layering of step, lower to stationary phase, upper as moving phase, stationary phase and moving phase are successively pumped in high speed adverse current chromatogram, by step 2. solution through sampling valve, enter chromatographic column, after sample introduction, continue to pump into moving phase;
4. according to detector spectrogram, collect respectively isolate, concentrated, obtain 1,2,3 and 4 four spermidine monomer.
2. the method for four spermidines in the separated safflower of high speed adverse current chromatogram as claimed in claim 1, is characterized in that: the chloroform of described step in 1., the volume ratio of first alcohol and water are 3～5: 4: 3～5.
3. the method for four spermidines in the separated safflower of high speed adverse current chromatogram as claimed in claim 1, is characterized in that: the described step concentration that 2. middle safflower crude extract is dissolved in after mixed solvent is 6～15 mg/mL.
4. the method for four spermidines in the separated safflower of the high speed adverse current chromatogram as described in claim 1, it is characterized in that: described safflower spermidine crude extract is prepared by following method: by liquid ratio 10～20:1, add 60～80% alcohol in 45～65 ℃ of refluxing extraction 2～4 times safflower dried floral, each 1.5～2.5 hours, united extraction liquid is also evaporated to dry, by the medicinal extract deionized water dissolving of evaporate to dryness, then by isopyknic sherwood oil and ethyl acetate, extract respectively 3～6 times; By macroporous adsorbent resin on water, first use 25～35% ethanol elution, then use 45～55% ethanol elutions; 45～55% ethanol eluates are evaporated to dry, obtain.
5. the method for four spermidines in the separated safflower of the high speed adverse current chromatogram as described in claim 1, is characterized in that: described high-speed counter-current chromatograph flow rates is 0.5～2 mL/min, and speed range is 600～900 r/min.
6. the method for four spermidines in the separated safflower of high speed adverse current chromatogram as claimed in claim 1, is characterized in that: described detection wavelength is 210～360nm.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105158355A (en) * | 2015-08-12 | 2015-12-16 | 山西大学 | Method for rapidly measuring content of four spermidine ingredients in carthamus tinctorius simultaneously |
| CN110279738A (en) * | 2019-07-03 | 2019-09-27 | 山西大学 | A kind of extracting method of antidepressant spermidine active component and the purposes of spermidine effective part extract |
| CN114436888A (en) * | 2022-03-29 | 2022-05-06 | 中日友好医院(中日友好临床医学研究所) | Spermidine alkaloid compound, and preparation method, pharmaceutical composition and application thereof |
| CN116143651A (en) * | 2023-03-08 | 2023-05-23 | 西北农林科技大学 | Efficient separation and purification method of peony stamen tri-tonkinensis spermidine |
-
2013
- 2013-09-04 CN CN201310396939.XA patent/CN103508912A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| WEN-CONG LI,ET AL.: "Preparative separation and purification of four cis-trans isomers of coumaroylspermidine analogs from safflower by high-speed counter-current chromatography", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| 扈晓佳，等: "红花的化学成分及其药理活性研究进展", 《药学实践杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105158355A (en) * | 2015-08-12 | 2015-12-16 | 山西大学 | Method for rapidly measuring content of four spermidine ingredients in carthamus tinctorius simultaneously |
| CN105158355B (en) * | 2015-08-12 | 2017-01-18 | 山西大学 | Method for rapidly measuring content of four spermidine ingredients in carthamus tinctorius simultaneously |
| CN110279738A (en) * | 2019-07-03 | 2019-09-27 | 山西大学 | A kind of extracting method of antidepressant spermidine active component and the purposes of spermidine effective part extract |
| CN114436888A (en) * | 2022-03-29 | 2022-05-06 | 中日友好医院(中日友好临床医学研究所) | Spermidine alkaloid compound, and preparation method, pharmaceutical composition and application thereof |
| CN116143651A (en) * | 2023-03-08 | 2023-05-23 | 西北农林科技大学 | Efficient separation and purification method of peony stamen tri-tonkinensis spermidine |
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