CN108220240A - Application of the rehmanin in terms of CIK cell in vitro proliferation is promoted - Google Patents
Application of the rehmanin in terms of CIK cell in vitro proliferation is promoted Download PDFInfo
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- CN108220240A CN108220240A CN201810249755.3A CN201810249755A CN108220240A CN 108220240 A CN108220240 A CN 108220240A CN 201810249755 A CN201810249755 A CN 201810249755A CN 108220240 A CN108220240 A CN 108220240A
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- 210000004405 cytokine-induced killer cell Anatomy 0.000 title claims abstract description 27
- 230000035755 proliferation Effects 0.000 title claims abstract description 20
- 238000000338 in vitro Methods 0.000 title claims abstract description 19
- 239000001963 growth medium Substances 0.000 claims abstract description 8
- 230000001737 promoting effect Effects 0.000 claims abstract description 6
- 239000012980 RPMI-1640 medium Substances 0.000 claims abstract description 5
- 239000000126 substance Substances 0.000 claims description 5
- 239000000571 coke Substances 0.000 abstract description 18
- 230000000694 effects Effects 0.000 abstract description 7
- 229910052799 carbon Inorganic materials 0.000 abstract description 6
- 125000004432 carbon atom Chemical group C* 0.000 abstract description 5
- 230000004663 cell proliferation Effects 0.000 abstract description 3
- -1 carbon atom flavin compound Chemical class 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 36
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 27
- 239000000284 extract Substances 0.000 description 22
- 241000405414 Rehmannia Species 0.000 description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 239000000243 solution Substances 0.000 description 16
- 239000011347 resin Substances 0.000 description 14
- 229920005989 resin Polymers 0.000 description 14
- 238000004128 high performance liquid chromatography Methods 0.000 description 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 12
- 229910052708 sodium Inorganic materials 0.000 description 12
- 239000011734 sodium Substances 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 10
- 239000007788 liquid Substances 0.000 description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000002027 dichloromethane extract Substances 0.000 description 6
- 239000003480 eluent Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000000741 silica gel Substances 0.000 description 6
- 229910002027 silica gel Inorganic materials 0.000 description 6
- 229960001866 silicon dioxide Drugs 0.000 description 6
- 239000012535 impurity Substances 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 230000002745 absorbent Effects 0.000 description 4
- 239000002250 absorbent Substances 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- 238000004262 preparative liquid chromatography Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- KKAJSJJFBSOMGS-UHFFFAOYSA-N 3,6-diamino-10-methylacridinium chloride Chemical compound [Cl-].C1=C(N)C=C2[N+](C)=C(C=C(N)C=C3)C3=CC2=C1 KKAJSJJFBSOMGS-UHFFFAOYSA-N 0.000 description 2
- 206010013786 Dry skin Diseases 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
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- 238000010828 elution Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000010812 external standard method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000005556 structure-activity relationship Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
- C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
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Abstract
The invention discloses application of the rehmanin in terms of CIK cell in vitro proliferation is promoted.It is a discovery of the invention that rehmanin B, rehmanin D and coke rehmanin A can significantly improve CIK cell in vitro proliferation, and burnt rehmanin B does not have this effect.For burnt rehmanin B compared with burnt rehmanin A, the carbon atom configurations of only Cl connections is different, and the carbon atom configuration is identical with coke rehmanin A in rehmanin B and rehmanin D, it was demonstrated that the promotion CIK cell proliferation activity of the configuration of carbon atom flavin compound over the ground is most important.Therefore, rehmanin B, rehmanin D and burnt rehmanin A can be used for preparing the culture medium for promoting CIK cell in vitro proliferation, for example can be prepared by adding rehmanin B, rehmanin D or coke rehmanin A in RPMI1640 culture solutions.
Description
Technical field
The invention belongs to field of medicaments, and in particular to application of the rehmanin in terms of CIK cell in vitro proliferation is promoted.
Background technology
Rehmanin B, rehmanin D, coke rehmanin A and burnt rehmanin B are natural products isolated from glutinous rehmannia, chemical
Structure is closely similar, and chemical information is as shown in the table.
Have not yet to see above-mentioned four kinds of rehmanins and with four kinds of rehmanin extracts as main component in CIK cell body
Research report in terms of outer proliferation.
Invention content
Present invention aims at provide application of the rehmanin in terms of CIK cell in vitro proliferation is promoted.
Above-mentioned purpose is achieved through the following technical solutions：
Application of the rehmanin of following chemical constitution in terms of CIK cell in vitro proliferation is promoted；
Application of the above-mentioned rehmanin in terms of the culture medium for promoting CIK cell in vitro proliferation is prepared.
A kind of culture medium for promoting CIK cell in vitro proliferation, contains above-mentioned rehmanin.
Preferably, above-mentioned culture medium is prepared by adding above-mentioned rehmanin in RPMI1640 culture solutions.
The technology of the present invention advantage：
Present invention discover that rehmanin B, rehmanin D, coke rehmanin A can promote CIK cell in vitro to be proliferated, and then can use
In the culture medium for being prepared into promotion CIK cell in vitro proliferation.
Description of the drawings
Fig. 1 is the HPLC collection of illustrative plates of glutinous rehmannia extract prepared by embodiment 2；
Fig. 2 does not add the extract obtained HPLC collection of illustrative plates of isopropanol for 2 step S4 silica gel column chromatographies eluant, eluent of embodiment；
Fig. 3 is 3 preparative liquid chromatography figure of embodiment；
Fig. 4-6 is respectively the HPLC collection of illustrative plates of 3 gained rehmanin B of embodiment, rehmanin D, burnt rehmanin A；
Fig. 7 is that if step S3 substitutes XDA-1B macroporous absorbent resins, step using XDA-1 macroporous absorbent resins in embodiment 3
The HPLC collection of illustrative plates of glutinous rehmannia extract obtained by rapid S4；
Fig. 8 is the HPLC chromatogram of the mixed mark solution of embodiment 4；
Fig. 9 is the separation that embodiment 4 marks solution using the mobile phase for not adding 16 phosphinylidynes of N--Serine sodium to mixing
Figure.
Specific embodiment
The essentiality content of the present invention is specifically introduced with reference to embodiment, but it will be appreciated by those skilled in the art that not
Protection scope of the present invention should be confined to these specific embodiments.
Embodiment 1：The influence that rehmanin B, rehmanin D, coke rehmanin A and coke rehmanin B are proliferated CIK cell in vitro
First, experiment material
Rehmanin B, rehmanin D, coke rehmanin A and burnt rehmanin B self-controls, purity is more than 95%.
2nd, experimental method
1st, individual cells are detached
PBMC is isolated from healthy volunteer's peripheral blood with Ficoll-Hypaque density-gradient centrifugation methods, adds 4mL PBS
Dilute 4mL whole blood (ratios 1:1) diluted blood, is taken out to be slowly added to, equipped in 4mL Ficoll-Hypaque centrifuge tubes, pay attention to
Liquid level interface is not upset, 2000r/min centrifugation 20min, in gentle aspiration mononuclearcell to another centrifuge tube.Add in life
Brine 5mL is managed, gently blows even and fine born of the same parents, centrifuge washing 2 times.
2nd, the culture of CIK cell
In the PBMC to suspend in physiological saline plus CIK initial incubation liquid, adjustment cell concentration are 5 × 105/ mL, plant in
In 24 orifice plates, per 200 μ L of hole, the RPMI1640 culture solutions of the IFN-γ containing 1000U/mL are firstly added, are placed in 5%CO2、37℃
It is cultivated in constant incubator；Add 100ng/mL AntiCD3 McAbs, 500U/mL IL-2 afterwards for 24 hours；Cultivate after 4d adjustment cell concentration for 1 ×
106/ mL adds 500U/mL IL-2 every 2d.Method is expanded to the 10th day harvest CIK cell like this.
3rd, CCK-8 methods detection rehmanin B, rehmanin D, coke rehmanin A and coke rehmanin B are to CIK cell proliferative effect
The CIK cell of collection culture 10d, 1 × 105/ mL cell numbers are inoculated in 96 orifice plates, and 200 μ L/ holes are separately added into 20 μ
M rehmanins B, rehmanin D, coke rehmanin A or burnt rehmanins B (control group adds isometric solvent), after being incubated 48h, add in
CCK8 after incubation, detects each hole light absorption value (OD) at microplate reader 450nm.
4th, statistical method
Using 19.0 statistical softwares of SPSS.Measurement data represents that comparison among groups are examined using t with means standard deviation.P
＜ 0.05 is statistically significant for difference.
3rd, experimental result
Compared with the control group, rehmanin B groups, rehmanin D groups, burnt rehmanin A group 48h proliferation rates significantly increase (P ＜
0.05), burnt rehmanin B group proliferation rates no significant difference (P ＞ 0.05).As a result such as following table.
The above results show rehmanin B, rehmanin D and coke rehmanin A can significantly improve CIK cell in vitro proliferation, and
Burnt rehmanin B does not have this effect.Burnt rehmanin B is compared with burnt rehmanin A, and the carbon atom configuration of only Cl connections is different, ground
The carbon atom configuration is identical with burnt rehmanin A in flavine B and rehmanin D, it was demonstrated that the configuration of carbon atom flavin chemical combination over the ground
The promotion CIK cell proliferation activity of object is most important, and this structure-activity relationship is found for the first time.
Therefore, rehmanin B, rehmanin D and coke rehmanin A can be used for preparing the culture for promoting CIK cell in vitro proliferation
Base, for example can be prepared by adding rehmanin B, rehmanin D or coke rehmanin A in RPMI1640 culture solutions.
Embodiment 2：With rehmanin B, rehmanin D, the preparation of coke rehmanin A glutinous rehmannia extracts as main component and activity
Preparation method includes the following steps：
Fresh rehmannia root is cleaned slice by step S1, and 55 DEG C of dryings obtain fresh rehmannia root piece；By 95% ethanol water of fresh rehmannia root piece
Solution circumfluence distillation 3 times, each 2h, solid-to-liquid ratio 1:20, merge extracting solution, medicinal extract is concentrated under reduced pressure to obtain；
Step S2 by the appropriate water dissolution of medicinal extract, first with 3 removal of impurities of petroleum ether equal-volume extraction, then is extracted with dichloromethane
It takes 3 times, combined dichloromethane extract liquor is concentrated and dried to obtain dichloromethane extract；
Step S3 dissolves 30% ethanol water of dichloromethane extract, mixes resin and is splined on the suction of XDA-1B macropores
Attached resin column, resin blade diameter length ratio are 1:10, sample resin accounts for resin total amount 1/10 is mixed, is first removed with 30% ethanol elution of 12BV
It is miscellaneous, then eluted with 75% ethyl alcohol with the flow velocity of 10BV/h, 8-10BV eluents are collected, it is thick that concentrate drying obtains glutinous rehmannia extract
Product；
Glutinous rehmannia extract crude product is splined on normal phase silicagel column by step S4, and silica gel blade diameter length ratio is 1:10, it mixes sample silica gel and accounts for silicon
The 1/10 of glue total amount；It is 10 with volume ratio:1:0.2 methylene chloride/methanol/isopropyl alcohol mixed solvent is washed with the speed of 10BV/h
It is de-, 6-7BV eluents are collected, is concentrated and dried and obtains the glutinous rehmannia extract for being rich in rehmanin B, rehmanin D, burnt rehmanin A.
Rich in rehmanin B, rehmanin D, burnt rehmanin A glutinous rehmannia extract HPLC analyses：
Chromatograph：Shimadzu LC-20AT high performance liquid chromatographs；
Chromatographic column：Shim-pack VP-ODS (4.6mm × 250mm, 5 μm)；
Mobile phase A phase：Acetonitrile/water is according to volume ratio 1:9 mixed liquor, 16 phosphinylidynes of N- containing 5ppm-Serine sodium；
Mobile phase B phase：Acetonitrile/water is according to volume ratio 9:1 mixed liquor, 16 phosphinylidynes of N- containing 5ppm-Serine sodium；
Elution program：0-5min, 15%B phase；5-15min, 15% → 55%B phase；15-30min, 55% → 65%B phase；
Flow velocity：1.0mL/min；
Column temperature：30℃；
Detection wavelength：207nm；
Sample size：10μL.
Fig. 1 is the HPLC collection of illustrative plates of above-mentioned glutinous rehmannia extract, and main component is rehmanin B, rehmanin D and burnt rehmanin A, is passed through
Quantified by external standard method, rehmanin B, rehmanin D and coke rehmanin A account for the 90.2% of extract gross weight.Since burnt rehmanin B does not have
Promote the activity of CIK cell proliferation, and it is very close with burnt rehmanin A structures, in order to which burnt rehmanin B exclusions are carried above-mentioned
It takes except object, a small amount of isopropanol is increased in the eluant, eluent of step S4 silica gel column chromatographies.If mobile phase does not add isopropanol, other
Constant, extract obtained HPLC collection of illustrative plates is operated as shown in Fig. 2, containing more multifocal rehmanin B and other impurities.
Method according to embodiment 1 tests the promotion activity that above-mentioned glutinous rehmannia extract is proliferated CIK cell in vitro, 5 μ g/mL
Proliferation rate after glutinous rehmannia extract intervention 48h is (26.18 ± 1.07) %, is significantly better than control group.
Embodiment 3：It is prepared by the separation of rehmanin B, rehmanin D, burnt rehmanin A monomers
Method for separating and preparing includes the following steps：
Fresh rehmannia root is cleaned slice by step S1, and 55 DEG C of dryings obtain fresh rehmannia root piece；By 95% ethanol water of fresh rehmannia root piece
Solution circumfluence distillation 3 times, each 2h, solid-to-liquid ratio 1:20, merge extracting solution, medicinal extract is concentrated under reduced pressure to obtain；
Step S2 by the appropriate water dissolution of medicinal extract, first with 3 removal of impurities of petroleum ether equal-volume extraction, then is extracted with dichloromethane
It takes 3 times, combined dichloromethane extract liquor is concentrated and dried to obtain dichloromethane extract；
Step S3 dissolves 30% ethanol water of dichloromethane extract, mixes resin and is splined on the suction of XDA-1B macropores
Attached resin column, resin blade diameter length ratio are 1:10, sample resin accounts for resin total amount 1/10 is mixed, is first removed with 30% ethanol elution of 12BV
It is miscellaneous, then eluted with 75% ethyl alcohol with the flow velocity of 10BV/h, 8-10BV eluents are collected, it is thick that concentrate drying obtains glutinous rehmannia extract
Product；
Glutinous rehmannia extract crude product is splined on normal phase silicagel column by step S4, and silica gel blade diameter length ratio is 1:10, it mixes sample silica gel and accounts for silicon
The 1/10 of glue total amount；It is 10 with volume ratio:1:0.2 methylene chloride/methanol/isopropyl alcohol mixed solvent is washed with the speed of 10BV/h
It is de-, 6-7BV eluents are collected, is concentrated and dried and obtains the glutinous rehmannia extract for being rich in rehmanin B, rehmanin D, burnt rehmanin A；
Step S5 prepares liquid phase separation：Preparative liquid chromatography parameter is as follows, collects ground according to chromatogram is (as shown in Figure 3)
Flavine B, rehmanin D, the corresponding fractions of burnt rehmanin A；
Preparative liquid chromatography parameter：
Chromatographic system：150 preparative liquid chromatography systems of Waters Prep
Chromatographic column：XBridge Prep C18 columns (19mm × 250mm, 5 μm)
Mobile phase A phase：Acetonitrile/water is according to volume ratio 1:9 mixed liquor, 16 phosphinylidynes of N- containing 5ppm-Serine sodium；
Mobile phase B phase：Acetonitrile/water is according to volume ratio 9:1 mixed liquor, 16 phosphinylidynes of N- containing 5ppm-Serine sodium；
Elution program：0-5min, 15%B phase；5-15min, 15% → 55%B phase；15-30min, 55% → 65%B phase；
Flow velocity：16.0mL/min；
Column temperature：30℃；
Detection wavelength：207nm；
Sample size：100μL；
Step S6, desalination：Methanol is used after rehmanin B, rehmanin D, the corresponding fractions of burnt rehmanin A are concentrated to dryness respectively
Ultrasound, 16 phosphinylidynes of N--Serine sodium do not dissolve in methanol, be collected by filtration filtrate be concentrated and dried up to rehmanin B, rehmanin D,
Burnt rehmanin A monomers.
The HPLC analyses of rehmanin B, rehmanin D, burnt rehmanin A monomers：
Chromatograph：Shimadzu LC-20AT high performance liquid chromatographs；
Chromatographic column：Shim-pack VP-ODS (4.6mm × 250mm, 5 μm)；
Mobile phase A phase：Acetonitrile/water is according to volume ratio 1:9 mixed liquor, 16 phosphinylidynes of N- containing 5ppm-Serine sodium；
Mobile phase B phase：Acetonitrile/water is according to volume ratio 9:1 mixed liquor, 16 phosphinylidynes of N- containing 5ppm-Serine sodium；
Elution program：0-5min, 15%B phase；5-15min, 15% → 55%B phase；15-30min, 55% → 65%B phase；
Flow velocity：1.0mL/min；
Column temperature：30℃；
Detection wavelength：207nm；
Sample size：10μL.
Fig. 4-6 is respectively the HPLC collection of illustrative plates of the rehmanin B being prepared, rehmanin D, burnt rehmanin A, it is seen that its purity is equal
It is higher, through quantified by external standard method more than 95%.
In above-mentioned preparation method, if step S3 substitutes XDA-1B macroporous absorbent resins, step using XDA-1 macroporous absorbent resins
In glutinous rehmannia extract obtained by rapid S4 between impurity component apparent more (as shown in Figure 7), especially rehmanin B and burnt rehmanin A
Impurity for rehmanin B and burnt rehmanin A monomers isolate and purify influence it is very big.
Embodiment 4：The method of burnt rehmanin B in the burnt rehmanin A of detection
HPLC analytical parameters：
Chromatograph：Shimadzu LC-20AT high performance liquid chromatographs；
Chromatographic column：Shim-pack VP-ODS (4.6mm × 250mm, 5 μm)；
Mobile phase A phase：Acetonitrile/water is according to volume ratio 1:9 mixed liquor, 16 phosphinylidynes of N- containing 5ppm-Serine sodium；
Mobile phase B phase：Acetonitrile/water is according to volume ratio 9:1 mixed liquor, 16 phosphinylidynes of N- containing 5ppm-Serine sodium；
Elution program：0-5min, 15%B phase；5-15min, 15% → 55%B phase；15-30min, 55% → 65%B phase；
Flow velocity：1.0mL/min；
Column temperature：30℃；
Detection wavelength：207nm；
Sample size：10μL.
Singly mark solution：The burnt rehmanin A of a concentration of 0.1mg/mL, burnt rehmanin B methanol solutions is respectively configured.
Mixed mark solution：The burnt rehmanin A of configuration, burnt rehmanin B concentration are respectively the methanol solution of 0.1mg/mL.
Fig. 8 is the HPLC chromatogram of mixed mark solution.It can be seen that burnt rehmanin A, coke rehmanin B can be under above-mentioned chromatographic condition
It efficiently separates, realizes baseline separation, separating degree is more than 2.0.
If use conventional acetonitrile/water as mobile phase (mobile phase does not add 16 phosphinylidynes of N--Serine sodium, other
Parameter constant), the HPLC chromatogram of mark solution is mixed as shown in figure 9, burnt rehmanin A, burnt rehmanin B separating difficulties are big.This shows
N- 16 phosphinylidynes-Serine sodium is added in mobile phase helps to increase burnt rehmanin A, coke rehmanin B this chiral isomers
Discrimination on reverse-phase chromatographic column.
Claims (4)
1. application of the rehmanin of following chemical constitution in terms of CIK cell in vitro proliferation is promoted；
2. application of the rehmanin described in claim 1 in terms of the culture medium for promoting CIK cell in vitro proliferation is prepared.
3. a kind of culture medium for promoting CIK cell in vitro proliferation, it is characterised in that：Contain rehmanin described in claim 1.
4. culture medium according to claim 3, it is characterised in that：Claim 1 institute is added in RPMI1640 culture solutions
The rehmanin stated is prepared.
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| CN201810249755.3A CN108220240B (en) | 2018-03-26 | 2018-03-26 | Application of the rehmanin in terms of promoting CIK cell in vitro proliferation |
| PCT/CN2018/100123 WO2019184194A1 (en) | 2018-03-26 | 2018-08-11 | Preparation, analysis and cellular immunity therapeutic use of rehmannin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019184194A1 (en) * | 2018-03-26 | 2019-10-03 | 马海周 | Preparation, analysis and cellular immunity therapeutic use of rehmannin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019184194A1 (en) * | 2018-03-26 | 2019-10-03 | 马海周 | Preparation, analysis and cellular immunity therapeutic use of rehmannin |
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