CN101468061B - Extract of immature exocarp of Juglans mandshurica Maxim., preparation method and medical use - Google Patents
Extract of immature exocarp of Juglans mandshurica Maxim., preparation method and medical use Download PDFInfo
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Abstract
The invention provides a north Qinglongyi extract, preparation method and medicinal use. The active ingredient of the existing North Qinglongyi extract is not clear lacks of quantitative index, therefore the mechanism of pharmacological effects is difficulty to expatiate. The anti-tumor active ingredient of the existing North Qinglongyi extract is prepared using alcohol extraction method combinedwith macroporous resin and gel chromatography technique. The north Qinglongyi extract comprises compound of general formula (I) with more than 70 percent, the compound of structural formula is described. This invention is applied to the medical field.
Description
Technical field:
the present invention relates to isolating noval chemical compound QINGLONGYISU A in the immature exocarp of Juglans mandshurica Maxim is master's preparation method of extract, and the purposes in the preparation antitumor drug.
Background technology:
cancer is the serious disease that threatens the human life, and therefore, the research of cancer therapy drug is the focus of World Focusing always.The Chinese medicine antitumor has distinctive feature at the chemicals that compares aspect raising immunity and the reduction mutagenesis toxicity; Therefore; From motherland's medicine treasure-house, seek the antitumor Chinese medicine of determined curative effect; Clear and definite its effective substance, and then develop the new drug of high-efficiency low-toxicity, become the focus that tcm field is studied for many years.
Immature exocarp of Juglans mandshurica Maxim be the juglandaceae plant Juglans mandshurica (
Juglans MandshuricaMAXIM.) immature fruit abounds with in each county, outskirts of a town, northeast, at analgesic of treating various cancers and dermatosis and various pain commonly used among the people.Immature exocarp of Juglans mandshurica Maxim recorded the medical material standard in the Heilongjiang Province and became a kind of Chinese medicine and be applied to clinical and produce calendar year 2001.Alimentary tract cancers such as the crude extract preparation for treating gastric cancer of immature exocarp of Juglans mandshurica Maxim, the esophageal carcinoma, have the case of two more than ten years treatment observation to prove (Li Zhongyuan. the 120 routine clinical observations of Exocarpium Juglandis Immaturum treatment carcinoma of esophago cardia.
Chinese medicine information, 1988, (3): 3), its antitumor action is unquestionable.Yet its effective substance research does not deeply launch; Bibliographical information rests on the pharmacological action of its crude extract more and (Ji Yubin is gone up in the antitumor action research that lacks its effective ingredient; The horse grand plan, Yang Bo draws the cutting edge of a knife or a sword in morning. the antitumor action research of the different extract parts of immature exocarp of Juglans mandshurica Maxim.
Chinese herbal medicine, 2004,35 (10): 1145-1147), and existing extract of immature exocarp of Juglans mandshurica Maxim effective ingredient is indeterminate, and the lacking quantitative index causes pharmacological mechanism to be difficult to further elaboration.The inventor has separated a noval chemical compound from immature exocarp of Juglans mandshurica Maxim, called after QINGLONGYISU A (qinglongyitin A).This chemical compound is to people HL-60 leukaemia, people Kato-III stomach cancer cell and people A
549
Lung carcinoma cell has significant inhibitory effect, and the scale-model investigation of HCC transplanted tumor in nude mice has also proved the antitumaous effect of this chemical compound.The present invention adopts ethanol extraction to combine macroporous resin and gel chromatography technology, has prepared the extract of immature exocarp of Juglans mandshurica Maxim antitumor effective ingredient, utilizes the medicine of this technology preparation can make the content of such chemical compound be higher than 70%.Pharmacodynamic study is the result show; With this compounds serves as that the main extract of forming has good antitumor action; Its antitumaous effect obviously is superior to other crude extract, can process the antitumor drug of the effective ingredient in Chinese that is suitable for clinical use with drug excipient, and is stronger than the crude extract preparation active function of present bibliographical information; Effective ingredient is clearer and more definite, also is more prone to control the stable and reliable of its quality.
Summary of the invention:
the purpose of this invention is to provide a kind of new chemical compound and preparation method of extract thereof with anti-tumor activity.
Above-mentioned purpose realizes through following technical scheme:
Extract of immature exocarp of Juglans mandshurica Maxim, the compounds content that contains general formula (I) is greater than 70%, and the structural formula of this chemical compound is:
（I）。
The preparation of
described a kind of extract of immature exocarp of Juglans mandshurica Maxim is got fresh immature exocarp of Juglans mandshurica Maxim and is extracted with soak with ethanol, and extracting solution concentrates; Concentrated solution adds aqueous dispersion, gets supernatant after centrifugal, and supernatant passes through macroporous adsorbent resin; Adsorbent resin is used ethanol elution, inject polydextran gel behind the eluent thickening filtration, collect 40% ethanol elution; Be concentrated into dried, in the dry thing QINGLONGYISU A content greater than 70%.
The preparation of
described a kind of extract of immature exocarp of Juglans mandshurica Maxim is describedly got fresh immature exocarp of Juglans mandshurica Maxim and is extracted with soak with ethanol, be with fresh immature exocarp of Juglans mandshurica Maxim with 8～10 times of amount one weeks of 75%～95% soak with ethanol, extract three times.
The preparation of described a kind of extract of immature exocarp of Juglans mandshurica Maxim, described extracting solution concentrate, and concentrated solution adds aqueous dispersion, and is centrifugal, are meant behind the merge extractive liquid, concentrating under reduced pressure and are concentrated into relative density 1.10-1.14 (50
0
C), concentrated solution adds aqueous dispersion, and is centrifugal, gets supernatant after centrifugal, and supernatant is used ethanol elution through macroporous adsorbent resin with adsorbent resin.
The preparation of
described a kind of extract of immature exocarp of Juglans mandshurica Maxim, described add aqueous dispersion centrifugal after, get supernatant; Filter through absorption with macroporous adsorbent resin, be meant centrifugal after, get supernatant; Inject the Diaion HP-20 macroporous adsorbent resin of having handled well, the ratio of medical material and resin is 1:20～1:50, flow velocity 10ml/min; Wash with the washing of 3～5 times of bed volumes, 30% ethanol of 5 times of bed volumes respectively, collect 30% ethanol elution, the eluent evaporated under reduced pressure obtains residue; Residue filters with a small amount of 40% dissolve with ethanol, and filtrating is injected polydextran gel; Collect 40% ethanol elution, be concentrated into dried, in the dry thing QINGLONGYISU A content greater than 70%.
The preparation of
described a kind of extract of immature exocarp of Juglans mandshurica Maxim; Described filtrating is injected polydextran gel, collects 40% ethanol elution and is meant on the injection sephadex lh-20 post column diameter 2.5cm; Column length 1.5m; Applied sample amount is that the 5g residue/at every turn, flow velocity 1.0ml/min is with 40% ethanol elution of 3 times of bed volumes.
The preparation of
extract of immature exocarp of Juglans mandshurica Maxim, described 40% ethanol elution with sephadex lh-20 is concentrated into dried, and QINGLONGYISU A content is greater than 70% in the dry thing.
The application of extract in the preparation antitumor drug of the chemical compound of general formula (I) contained in
.
This technical scheme has following beneficial effect:
1. the invention provides a kind of chemical compound of not reporting with antitumor action; Through to cancerous cell inhibition test and pharmacodynamics test; Proved that this chemical compound has good active anticancer, can process the antitumor drug that is suitable for clinical use with drug excipient.
from the Chinese medicine immature exocarp of Juglans mandshurica Maxim separation and purification this chemical compound, purity reaches more than 98%.This chemical compound is an extraction separation from natural plants; Have novel structure, the unique advantage of effect; Can process the antitumor drug that is suitable for clinical use with drug excipient on the one hand, also can be used as lead compound on the other hand, its structure activity relationship is further studied; Synthetic a series of cancer therapy drug and drug regimen; More can be used as the index components of the immature exocarp of Juglans mandshurica Maxim medical material and the quality of the pharmaceutical preparations thereof control, realize the stable and controlled of the immature exocarp of Juglans mandshurica Maxim medical material and the quality of the pharmaceutical preparations thereof, break through the bottleneck of present immature exocarp of Juglans mandshurica Maxim research and development.
Chinese medicine and the quality of the pharmaceutical preparations thereof stable and controlled be the bottleneck of the modernization of Chinese medicine always; This chemical compound is one of effective ingredient of Chinese medicine immature exocarp of Juglans mandshurica Maxim; Can be used as the index of the immature exocarp of Juglans mandshurica Maxim medical material and the quality of the pharmaceutical preparations thereof control; Setting up with this composition on this basis is the discriminating of index and the quality control standard of assay project, realizes the stable and controlled of the immature exocarp of Juglans mandshurica Maxim medical material and the quality of the pharmaceutical preparations thereof, and this chemical compound will be widely used as the Chinese medicine reference substance.
Extract thing that contains QINGLONGYISU A provided by the invention and compound recipe Exocarpium Juglandis Immaturum tincture are relatively to H
22
Growth has the obvious suppression effect, and can obviously prolong the mice life span, has good antitumor action.This shows, the extract of immature exocarp of Juglans mandshurica Maxim effective site provided by the present invention, it is higher to have an active constituent content; The characteristics that pharmacological action is stronger; Not only solved the indefinite difficult problem of present immature exocarp of Juglans mandshurica Maxim effective substance, and the extract that is provided can cooperate with various medical adjuvant (excipient, diluent, flavouring agent, lubricant etc.), process the medicine of multiple dosage form; Like freeze-dried powder, injection, tablet, capsule, granule and oral liquid etc., be widely used in clinical.
Description of drawings:
Accompanying drawing 1 is a The compounds of this invention (I)
1
H-
1
Observed structure chart in the H COSY spectrum.
accompanying drawing 2 is an observed structure chart during the HMBC of The compounds of this invention (I) composes.
The specific embodiment of the present invention:
Embodiment 1:
Extract of immature exocarp of Juglans mandshurica Maxim, the compounds content that contains general formula (I) is greater than 70%, and the structural formula of this chemical compound is:
（I）。
The preparation of
described a kind of extract of immature exocarp of Juglans mandshurica Maxim is got fresh immature exocarp of Juglans mandshurica Maxim and is extracted with soak with ethanol, and extracting solution concentrates; Concentrated solution adds aqueous dispersion, gets supernatant after centrifugal, and supernatant passes through macroporous adsorbent resin; Adsorbent resin is used ethanol elution, inject polydextran gel behind the eluent thickening filtration, collect 40% ethanol elution; Be concentrated into dried, in the dry thing QINGLONGYISU A content greater than 70%.
The preparation of
described a kind of extract of immature exocarp of Juglans mandshurica Maxim is describedly got fresh immature exocarp of Juglans mandshurica Maxim and is extracted with soak with ethanol, be with fresh immature exocarp of Juglans mandshurica Maxim with 8～10 times of amount one weeks of 75%～95% soak with ethanol, extract three times.
The preparation of described a kind of extract of immature exocarp of Juglans mandshurica Maxim, described extracting solution concentrate, and concentrated solution adds aqueous dispersion, and is centrifugal, are meant behind the merge extractive liquid, concentrating under reduced pressure and are concentrated into relative density 1.10-1.14 (50
0
C), concentrated solution adds aqueous dispersion, and is centrifugal, gets supernatant after centrifugal, and supernatant is used ethanol elution through macroporous adsorbent resin with adsorbent resin.
The preparation of
described a kind of extract of immature exocarp of Juglans mandshurica Maxim, described add aqueous dispersion centrifugal after, get supernatant; Filter through absorption with macroporous adsorbent resin, be meant centrifugal after, get supernatant; Inject the Diaion HP-20 macroporous adsorbent resin of having handled well, the ratio of medical material and resin is 1:20～1:50, flow velocity 10ml/min; Wash with the washing of 3～5 times of bed volumes, 30% ethanol of 5 times of bed volumes respectively, collect 30% ethanol elution, the eluent evaporated under reduced pressure obtains residue; Residue filters with a small amount of 40% dissolve with ethanol, and filtrating is injected polydextran gel; Collect 40% ethanol elution, be concentrated into dried, in the dry thing QINGLONGYISU A content greater than 70%.
The preparation of
described a kind of extract of immature exocarp of Juglans mandshurica Maxim; Described filtrating is injected polydextran gel, collects 40% ethanol elution and is meant on the injection sephadex lh-20 post column diameter 2.5cm; Column length 1.5m; Applied sample amount is that the 5g residue/at every turn, flow velocity 1.0ml/min is with 40% ethanol elution of 3 times of bed volumes.
The preparation of
extract of immature exocarp of Juglans mandshurica Maxim, described 40% ethanol elution with sephadex lh-20 is concentrated into dried, and QINGLONGYISU A content is greater than 70% in the dry thing.
The application of extract in the preparation antitumor drug of the chemical compound of general formula (I) contained in
.
Embodiment 2:
The preparation of chemical compound (I)
Material source: immature exocarp of Juglans mandshurica Maxim be the juglandaceae plant Juglans mandshurica (
Juglans MandshuricaMAXIM.) immature fruit was adopted in 5 constant virtues, Heilongjiang Province county mid-July, and the plant specimen sample is concealed in Chinese herbal medicine specimen shop, institute for drug control, Heilongjiang Province.
Extract and separate: fresh immature exocarp of Juglans mandshurica Maxim with one week of 75% soak with ethanol, is extracted three times altogether, merge extractive liquid,, concentrating under reduced pressure is concentrated into relative density 1.10-1.14 (50
0
C) concentrated solution, concentrated solution add 10 times of water gagings and disperse, and be centrifugal, gets supernatant; Inject the macroporous adsorbent resin of having handled well (ratio of concentrated solution and resin is 1:50), flow velocity 10ml/min washes with the washing of 3 times of bed volumes, 30% ethanol of 5 times of bed volumes respectively, collects 30% ethanol elution; The eluent evaporated under reduced pressure obtains residue, and residue dissolves with small amount of methanol, filters, and filtrating is injected (column diameter 2cm on the sephadex lh-20 post; Column length 2m, applied sample amount are 1g residue/each), with methanol-eluted fractions; Eluent carries out thin layer chromatography on silica gel plate, developing solvent is chloroform-methanol (84:16), merges R
f
Value is 0.3 one-component, must this chemical compound with recrystallizing methanol, and purity is more than 98%.
Embodiment 3:
The structure determination of chemical compound (I)
Structure determination
With Shimadzu UV-160 spectrophotometric determination ultraviolet spectra; With JASCO FT/IR-300E (KBr tabletting) spectrophotometric determination infrared spectrum; Measure optical rotation with JASCO DIP-370 digital polarimeter; Measure ESI-MS with LCQ mass analyzer, measure HR-FAB-MS with JEOL Mstation spectrometer
1
H-with
13
The C-NMR spectrum is marked in TMS does with JEOL ECP-500 spectrometer.
The physicochemical property of chemical compound (I):
chemical compound (I) is an amorphous powder.IR（KBr） 
max? ：3414,?2924,?2857,?2372,?1618,?1456,?1391,?1263,?1116,?618。UV（MeOH） 
max （log ）：209nm?(3.52),?263nm?(3.92),?344nm?(4.41)。[
] D 25 -21°?(
c?=?0.1,?MeOH)。
1 H-NMR (500 MHz, CD 3 OD) and 13 C-NMR (125MHz, CD 3 OD) the spectrum data are seen table 1.ESI-MS (positive)
m/
z[379.1 M+Na]; HR-FAB-MS (positive)
m/
z[379.1002 M+Na] (value of calculation C
19 H 16 O 7 Na 379.1005).
Chemical compound (I) 1 Show five fragrant protons in the H-NMR spectrum, 
6.67 (1H, dd,
J=8.2,1.1),
7.42 (1H, dd,
J=8.2,7.8),
7.29 (1H, dd,
J=7.8,1.1) be a cover ABC coupling system,
6.53 (1H, s),
7.06 (1H is the substituted aroma system of a cover para-position hydrogen s), at high field region
2.50 (1H, dd,
J=12.4,4.2),
2.57 (1H, dd,
J=12.4,1.8) be that a methylene and a methine exist
3.15 (1H, d,
J=4.2,1.8) ortho position coupling, its excess-three proton exists respectively
3.31 (1H, m) with
2.68 (2H, m) ortho position coupling, these 1 H- 1 Obtain conclusive evidence (seeing accompanying drawing 1) in the H COSY spectrum. 13 The C-NMR spectrum combines the HMQC spectrum to know 
208.7 be carbonyl carbon,
48.9 with
36.7 be respectively a methylene and a methine,
70.9 are for being connected with the tertiary carbon of hydroxyl.In the HMBC spectrum 
2.50 (H-3),
2.57 methylene (H-3) with
208.7 (C-1) carbonyl is relevant,
3.15 methine (H-2) with
70.9 (C-4) relevant, can infer and contain the 1,2,3,4-Tetrahydrooxonaphthalene structure in the molecule.In the HMBC spectrum
6.67 (H-5) with 70.9 (C-4) relevantly also proved this point, and shown that the phenyl ring in the 1,2,3,4-Tetrahydrooxonaphthalene is an ABC coupling system.An other cover aroma system is to be connected on
70.9 (C-4), and this can observe the relevant peaks of
7.06 (H-5') and
70.9 (C-4) in the HMBC spectrum.
3.31 (H-7') are relevant with
116.1 (C-2') in the HMBC spectrum;
6.53 (H-2'); Relevant with
40.3 (C-7'), show that
40.3 (C-7') and
45.6 (C-8') are connected on another phenyl ring.Wherein
2.68 (H-8') are long-range relevant with 48.9 (C-2);
3.31 (H-7') are long-range relevant with
208.7 (C-1); Show that C-7' is connected on the C-2; These can be through
3.31 (H-7') and
36.7 (C-3);
2.50 (H-3),
2.57 (H-3) prove conclusively with long-range relevant the obtaining of
40.3 (C-7').Substituent group from phenyl ring sees that two hydroxyls are arranged on the 1,2,3,4-Tetrahydrooxonaphthalene, lays respectively at C-4 and C-8, and two vicinal hydroxyl groups are arranged on another phenyl ring, and two oxygen atoms are still arranged in the molecular formula, in conjunction with 13 The C-NMR spectrum ( 
176.9) data, showing has a carboxyl to exist, and it can only be connected on the C-8'.Therefore, chemical compound (I) 1 H-NMR with 13 The C-NMR data are arranged like table 1.From HMBC spectrum (accompanying drawing 2), the structure of this chemical compound has obtained conclusive evidence.
Table 1 chemical compound (I)
1
H-NMR with
13
The C-NMR data
Embodiment 4:
a kind of above-mentioned chemical compound application in the medicament preparation of antitumor field.
Suppress people HL-60 leukaemia, people Kato-III stomach cancer cell and people A
549
The activity experiment of lung carcinoma cell:
Cell culture
People HL-60 leukaemia, people Kato-III stomach cancer cell and people A 549 Lung carcinoma cell is incubated at RPMI 1640 culture medium, adds 10% FBS, L-glutamine, 100 U/ml penicillins and 100 
The g/ml streptomycin.The take the logarithm cell counting of trophophase is with 3
10 4 The concentration of individual/ml is inoculated in 96 orifice plates, every hole 180 
L is 37 0 C, 5% CO 2 Incubator in cultivated 24 hours.
Cell toxicant is measured
Variable concentrations (1-100 
M) medicine is with EtOH-H 2 O (1:9) is a dissolution with solvents to 20 
L, blank control group adds 20
The EtOH-H of l 2 O (1:9) solution, cell continue to cultivate 72 hours, adopt mtt assay to measure then, and the cell after promptly cultivating adds 10 
L MTT (5 mg/ml) phosphate buffer, 37 0 C, 5% CO 2 Cultivated 4 hours in the incubator, centrifugal, get supernatant 150 L discards, and adds 175
L DMSO, jolting was measured light absorption value with ELIASA at 550 nm after 10 minutes.Calculate according to following formula, draw IC 50
Suppression ratio (%)=
(control wells A value-blank well A value)-(test hole A value-blank well A value)* 100%
Control wells A value-blank well A value
Control wells: only add cell and do not add medicine
Blank well: only solubilizer does not add cell and medicine
Experimental port: add cell and add medicine again
Result of the test
Table 2 is seen in
.The result shows that chemical compound (I) all has inhibitory action in various degree to three kinds of tumor cells.
Table 2. chemical compound (I) suppresses the IC of three kinds of tumor cells
50
(
M) value
| Sample | HL-60 | KATO III | A 549 |
| CDDP | 0.3 ± 0.06 | 3.3 ± 0.81 | 2.7 ± 0.71 |
| Chemical compound (I) | 18.0 ± 8.4 | 12.8 ± 9.3 | 32.5 ± 16.4 |
Annotate: each numerical value is the meansigma methods ± standard deviation of three independent experiments
Embodiment 5:
Pharmacodynamics test
experimental animal: Kunming mouse, body constitution amount 18g-22g, male and female dual-purpose.
Medicine and reagent: injection normal saline (blank), cyclophosphamide (positive control), chemical compound (I) tumor strain: rat liver cancer H
22
Test method:
1, mouse tumor is heavily reached the exponential influence of immune organ: mice random packet, male and female half and half.Be respectively chemical compound (I) group, cyclophosphamide group, normal saline group.Each organizes mice in the subcutaneous conventional inoculation H of right front axil
22
Tumor cell suspension 0.2ml (about 1 * 10
6
-2 * 10
6
Individual cell), respectively organizes the ip administration after the inoculation, continuously 7d.Put to death mice after the last administration, weigh, claim tumor piece, thymus and spleen quality, calculate tumour inhibiting rate, thymus index and spleen index.
, to H
22
The influence of mice increase in life span: mouse peritoneal inoculation H
22
Tumor cell suspension 0.2ml observes and record dead mouse situation every day.
The result sees the following form:
Annotate: compare * P< with the normal saline group; 0.05; Compare with the cyclophosphamide group,
P<0.05.
Chemical compound provided by the invention (I) is to H
22
Growth has the obvious suppression effect, and can obviously prolong the mice life span, has good antitumor action
Embodiment 6:
The preparation of extract of immature exocarp of Juglans mandshurica Maxim:
Fresh immature exocarp of Juglans mandshurica Maxim with 8 times of amount one weeks of 75% soak with ethanol, is extracted three times; Merge extractive liquid,, concentrating under reduced pressure is concentrated into relative density 1.10-1.14 (50
0
C); Concentrated solution adds aqueous dispersion, and is centrifugal, gets supernatant; Inject the Diaion HP-20 macroporous adsorbent resin of having handled well (ratio of medical material and resin is 1:50); Flow velocity 10ml/min washes with the washing of 3 times of bed volumes, 30% ethanol of 5 times of bed volumes respectively, collects 30% ethanol elution; The eluent evaporated under reduced pressure obtains residue, and residue is with a small amount of 40% dissolve with ethanol, filters, and filtrating is injected on the sephadex lh-20 post (column diameter 2.5cm, column length 1.5m, applied sample amount are 5g residue/each), with 40% ethanol elution of 3 times of bed volumes; Eluent is concentrated into dried, and QINGLONGYISU A content is greater than 70% in the dry thing.
Embodiment 7:
The preparation of extract of immature exocarp of Juglans mandshurica Maxim:
Fresh immature exocarp of Juglans mandshurica Maxim with 10 times of amount one weeks of 80% soak with ethanol, is extracted three times; Merge extractive liquid,, concentrating under reduced pressure is concentrated into relative density 1.10-1.14 (50
0
C); Concentrated solution adds aqueous dispersion, and is centrifugal, gets supernatant; Inject the Diaion HP-20 macroporous adsorbent resin of having handled well (ratio of medical material and resin is 1:30); Flow velocity 10ml/min washes with the washing of 5 times of bed volumes, 30% ethanol of 5 times of bed volumes respectively, collects 30% ethanol elution; The eluent evaporated under reduced pressure obtains residue, and residue filters with a small amount of 40% dissolve with ethanol; Filtrating is injected on the sephadex lh-20 post (column diameter 2.5cm, column length 1.5m, applied sample amount are 5g residue/each); Flow velocity 1.0ml/min is with 40% ethanol elution of 3 times of bed volumes; Eluent is concentrated into dried, and QINGLONGYISU A content is greater than 70% in the dry thing.
Embodiment 8:
The preparation of extract of immature exocarp of Juglans mandshurica Maxim:
Fresh immature exocarp of Juglans mandshurica Maxim with 10 times of amount one weeks of 95% soak with ethanol, is extracted three times; Merge extractive liquid,, concentrating under reduced pressure is concentrated into relative density 1.10-1.14 (50
0
C); Concentrated solution adds aqueous dispersion, and is centrifugal, gets supernatant; Inject the Diaion HP-20 macroporous adsorbent resin of having handled well (ratio of medical material and resin is 1:20); Flow velocity 10ml/min washes with the washing of 5 times of bed volumes, 30% ethanol of 5 times of bed volumes respectively, collects 30% ethanol elution; The eluent evaporated under reduced pressure obtains residue, and residue filters with a small amount of 40% dissolve with ethanol; Filtrating is injected on the sephadex lh-20 post (column diameter 2.5cm, column length 1.5m, applied sample amount are 5g residue/each); Flow velocity 1.0ml/min is with 40% ethanol elution of 3 times of bed volumes; Eluent is concentrated into dried, and QINGLONGYISU A content is greater than 70% in the dry thing.
Embodiment 9:
The pharmacodynamics test of extract of immature exocarp of Juglans mandshurica Maxim
experimental animal: Kunming mouse, body constitution amount 18g-22g, male and female dual-purpose.
medicine and reagent: injection normal saline (blank), extract of immature exocarp of Juglans mandshurica Maxim (QINGLONGYISU A content is greater than 70%), commercially available compound recipe Exocarpium Juglandis Immaturum tincture.
Tumor strain: rat liver cancer H
22
Test method:
1 pair of mouse tumor heavily reaches the exponential influence of immune organ: mice random packet, male and female half and half.Be respectively the extract of immature exocarp of Juglans mandshurica Maxim group, compound recipe Exocarpium Juglandis Immaturum tincture group, normal saline group.Each organizes mice in the subcutaneous conventional inoculation H of right front axil
22
Tumor cell suspension 0.2ml (about 1 * 10
6
-2 * 10
6
Individual cell), respectively organizes the ip administration after the inoculation, continuously 7d.Put to death mice after the last administration, weigh, claim tumor piece, thymus and spleen quality, calculate tumour inhibiting rate, thymus index and spleen index.
To H
22
The influence of mice increase in life span: mouse peritoneal inoculation H
22
Tumor cell suspension 0.2ml observes and record dead mouse situation every day.
The result sees the following form:
Annotate: compare * P< with the normal saline group; 
0.05; Compare with compound recipe Exocarpium Juglandis Immaturum tincture group,
P<0.05.
Claims (3)
1.
A kind of extract of immature exocarp of Juglans mandshurica Maxim is characterized in that: the compounds content that contains general formula I is greater than 70%, and the structural formula of this chemical compound is:
?
I。
2. the method for preparing of
described a kind of extract of immature exocarp of Juglans mandshurica Maxim of a kind of claim 1 is characterized in that: get fresh immature exocarp of Juglans mandshurica Maxim with 8～10 times of 75%～95% alcoholic acid one weeks of aqueous solution soaking of amount, extract three times; Concentrating under reduced pressure and be concentrated into the relative density 1.10-1.14 under 50 ℃ behind the merge extractive liquid,, concentrated solution adds aqueous dispersion, and is centrifugal; After centrifugal, get supernatant, inject the Diaion HP-20 macroporous adsorbent resin of having handled well; The ratio of medical material and resin is 1:20～1:50, and flow velocity 10ml/min washes with the washing of 3～5 times of bed volumes, 30% ethanol of 5 times of bed volumes respectively; Collect 30% ethanol elution, the eluent evaporated under reduced pressure obtains residue, and residue is with a small amount of 40% dissolve with ethanol; Filter, filtrating is injected polydextran gel, collects 40% ethanol elution; Be concentrated into dried, dry thing be extract of immature exocarp of Juglans mandshurica Maxim in the claim 1.
3. the method for preparing of
a kind of extract of immature exocarp of Juglans mandshurica Maxim according to claim 2; It is characterized in that: described filtrating is injected polydextran gel, collects 40% ethanol elution and is meant on the injection sephadex lh-20 post column diameter 2.5cm; Column length 1.5m; Applied sample amount is that the 5g residue/at every turn, flow velocity 1.0ml/min is with 40% ethanol elution of 3 times of bed volumes.
4. a claim 1 extract of immature exocarp of Juglans mandshurica Maxim or utilize the application of extract of immature exocarp of Juglans mandshurica Maxim in the preparation antitumor drug of the method preparation of claim 2-3.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200710144961XA CN101468061B (en) | 2007-12-28 | 2007-12-28 | Extract of immature exocarp of Juglans mandshurica Maxim., preparation method and medical use |
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| CN102370695B (en) * | 2010-08-19 | 2014-04-16 | 黑龙江省中医研究院 | Qinglongyi active extract, its preparation method and its application |
| CN102784195A (en) * | 2011-05-20 | 2012-11-21 | 中国科学院兰州化学物理研究所 | Method for extracting anti-tumor active component from traditional Chinese medicine pericarpium juglantis |
| CN103217491B (en) * | 2013-03-21 | 2014-07-23 | 黑龙江大学 | Method for establishing high performance liquid chromatography fingerprint spectrum of Exocarpium Juglandis Immaturum antioxidant active extract, its standard fingerprint spectrum and use |
| RU2653880C1 (en) * | 2017-08-07 | 2018-05-15 | Федеральное государственное автономное образовательное учреждение высшего образования "Дальневосточный федеральный университет" (ДВФУ) | Dessert |
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| Title |
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| Jun xi liu et al..Two new diarylheptanoids from the pericarps of Juglans regia L..《Chinese Chemical Letters》.2007,第18卷(第08期),第943-946页. * |
| 刘全宇.青龙衣化学成分的研究.《黑龙江中医药大学》.2007,(第4期),第14-21页. * |
| 王晓泊等.青龙衣药用研究现状.《上海医药》.2007,第28卷(第03期),第120-122页. * |
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