CN101468061A - 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 combined with 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 above. This invention is applied to the medical field.
Description
Technical field:
The present invention relates to preparation method of extract based on isolating noval chemical compound QINGLONGYISU A in the immature exocarp of Juglans mandshurica Maxim, 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 is the immature fruit of juglandaceae plant Juglans mandshurica (Juglans Mandshurica MAXIM.), abounds with in each county, outskirts of a town, northeast, at the analgesic that is commonly used for the various cancers of treatment and dermatosis and various pain among the people.Immature exocarp of Juglans mandshurica Maxim recorded and became a kind of Chinese medicine in Heilongjiang Province's medical material standard 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. and Chinese herbal medicine, 2004,35 (10): 1145-1147), and existing extract of immature exocarp of Juglans mandshurica Maxim effective ingredient is indeterminate, lacks quantizating 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
549Lung carcinoma cell has significant inhibitory effect, and the scale-model investigation of hepatoma carcinoma cell transplanted tumor in nude mice has also proved the antitumaous effect of this chemical compound.The present invention adopts ethanol extraction in conjunction with 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 better than other crude extract, can make the antitumor drug of the effective ingredient in Chinese that is suitable for clinical use with drug excipient, crude extract preparation active function than present bibliographical information is stronger, 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 by 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:
The preparation of described a kind of extract of immature exocarp of Juglans mandshurica Maxim, getting fresh immature exocarp of Juglans mandshurica Maxim extracts with soak with ethanol, extracting solution concentrates, and concentrated solution adds aqueous dispersion, gets supernatant after centrifugal, supernatant passes through macroporous adsorbent resin, with the adsorbent resin 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 concentrates, concentrated solution adds aqueous dispersion, centrifugal, be meant behind the merge extractive liquid, concentrating under reduced pressure and be concentrated into relative density 1.10-1.14 (50 ℃) that concentrated solution adds aqueous dispersion, centrifugal, get supernatant after centrifugal, supernatant is by macroporous adsorbent resin, with the adsorbent resin ethanol elution.
The preparation of described a kind of extract of immature exocarp of Juglans mandshurica Maxim, described add aqueous dispersion centrifugal after, get supernatant, filter by 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, and flow velocity 10ml/min is respectively with the washing of 3～5 times of bed volumes, 30% ethanol of 5 times of bed volumes is washed, collect 30% ethanol elution, the eluent evaporated under reduced pressure obtains residue, and residue filters with a small amount of 40% dissolve with ethanol, filtrate 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 filtrate is injected polydextran gel, collecting 40% ethanol elution 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.
Contain the application of extract in the preparation antitumor drug of the chemical compound of general formula (I).
This technical scheme has following beneficial effect:
1. the invention provides a kind of chemical compound of not reporting with antitumor action, by to cancerous cell inhibition test and pharmacodynamics test, proved that this chemical compound has good active anticancer, can make 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 make 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.
The 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, realize the stable and controlled of the immature exocarp of Juglans mandshurica Maxim medical material and the quality of the pharmaceutical preparations thereof, this chemical compound will be extensive use of as the Chinese medicine reference substance.
4. extract thing that contains QINGLONGYISU A provided by the invention and compound recipe Exocarpium Juglandis Immaturum tincture are relatively to H
22Growth 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 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 multiple medical accessory (excipient, diluent, flavouring agent, lubricant etc.), make the medicine of multiple dosage form, as 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)
1H-
1Observed structure chart in the H COSY spectrum.
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:
The preparation of described a kind of extract of immature exocarp of Juglans mandshurica Maxim, getting fresh immature exocarp of Juglans mandshurica Maxim extracts with soak with ethanol, extracting solution concentrates, and concentrated solution adds aqueous dispersion, gets supernatant after centrifugal, supernatant passes through macroporous adsorbent resin, with the adsorbent resin 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 concentrates, concentrated solution adds aqueous dispersion, centrifugal, be meant behind the merge extractive liquid, concentrating under reduced pressure and be concentrated into relative density 1.10-1.14 (50 ℃) that concentrated solution adds aqueous dispersion, centrifugal, get supernatant after centrifugal, supernatant is by macroporous adsorbent resin, with the adsorbent resin ethanol elution.
The preparation of described a kind of extract of immature exocarp of Juglans mandshurica Maxim, described add aqueous dispersion centrifugal after, get supernatant, filter by 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, and flow velocity 10ml/min is respectively with the washing of 3～5 times of bed volumes, 30% ethanol of 5 times of bed volumes is washed, collect 30% ethanol elution, the eluent evaporated under reduced pressure obtains residue, and residue filters with a small amount of 40% dissolve with ethanol, filtrate 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 filtrate is injected polydextran gel, collecting 40% ethanol elution 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.
Contain the application of extract in the preparation antitumor drug of the chemical compound of general formula (I).
Embodiment 2:
The preparation of chemical compound (I)
Material source: immature exocarp of Juglans mandshurica Maxim is the immature fruit of juglandaceae plant Juglans mandshurica (Juglans MandshuricaMAXIM.), adopts 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: with fresh immature exocarp of Juglans mandshurica Maxim with one week of 75% soak with ethanol, extract altogether three times, merge extractive liquid,, concentrating under reduced pressure is concentrated into the concentrated solution of relative density 1.10-1.14 (50 ℃), and concentrated solution adds 10 times of water gagings and disperses, centrifugal, get supernatant, inject the macroporous adsorbent resin of having handled well (ratio of concentrated solution and resin is 1:50), flow velocity 10ml/min, respectively with the washing of 3 times of bed volumes, 30% ethanol of 5 times of bed volumes is washed, collect 30% ethanol elution, the eluent evaporated under reduced pressure obtains residue, and residue dissolves with small amount of methanol, filter, filtrate is injected on the sephadex lh-20 post (column diameter 2cm, 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
fValue 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 JASCOFT/IR-300E (KBr tabletting) spectrophotometric determination infrared spectrum, measure optical rotation with JASCO DIP-370digital polarimeter, measure ESI-MS with LCQ mass analyzer, measure HR-FAB-MS with JEOL Mstation spectrometer
1H-and
13The 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)ν
max：3414，2924，2857，2372，1618，1456，1391，1263，1116，618。UV(MeOH)λ
max(logε)：209nm(3.52)，263nm(3.92)，344nm(4.41)。[α]
D 25-21°(c＝0.1，MeOH)。
1H-NMR (500MHz, CD
3OD) and
13C-NMR (125MHz, CD
3OD) the spectrum data see 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
19H
16O
7Na 379.1005).
Chemical compound (I)
1Show five fragrant protons in the H-NMR spectrum, at δ 6.67 (1H, dd, J=8.2,1.1), δ 7.42 (1H, dd, J=8.2,7.8), δ 7.29 (1H, dd, J=7.8,1.1) be a cover ABC coupling system, δ 6.53 (1H, s), (1H s) is the aroma system that a cover para-position hydrogen replaces to δ 7.06, at high field region δ 2.50 (1H, dd, J=12.4,4.2), δ 2.57 (1H, dd, J=12.4,1.8) be that a methylene and a methine are at δ 3.15 (1H, d, J=4.2,1.8) the ortho position coupling, its excess-three proton is respectively at δ 3.31 (1H, m) and δ 2.68 (2H, m) ortho position coupling, these
1H-
1Obtain conclusive evidence (seeing accompanying drawing 1) in the H COSY spectrum.
13C-NMR spectrum composes as can be known in conjunction with HMQC that δ 208.7 is carbonyl carbon, is respectively a methylene and a methine at δ 48.9 and δ 36.7, and δ 70.9 is for being connected with the tertiary carbon of hydroxyl.δ 2.50 (H-3) in the HMBC spectrum, the methylene of δ 2.57 (H-3) is relevant with δ 208.7 (C-1) carbonyl, and the methine of δ 3.15 (H-2) is relevant with δ 70.9 (C-4), can infer and contain the 1,2,3,4-Tetrahydrooxonaphthalene structure in the molecule.δ 6.67 (H-5) is relevant with δ 70.9 (C-4) in the HMBC spectrum has also proved this point, and shows 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 the δ 70.9 (C-4), and this can observe the relevant peaks of δ 7.06 (H-5 ') and δ 70.9 (C-4) in HMBC spectrum.δ 3.31 in HMBC spectrum (H-7 ') and δ 116.1 (C-2 ') are relevant, and δ 6.53 (H-2 ') is relevant with δ 40.3 (C-7 '), show that δ 40.3 (C-7 ') and δ 45.6 (C-8 ') are connected on another phenyl ring.Wherein δ 2.68 (H-8 ') is long-range relevant with 48.9 (C-2), δ 3.31 (H-7 ') long-range relevant with δ 208.7 (C-1), show that C-7 ' is connected on the C-2, this can pass through δ 3.31 (H-7 ') and δ 36.7 (C-3), δ 2.50 (H-3), the long-range relevant conclusive evidence that obtains of δ 2.57 (H-3) and δ 40.3 (C-7 ').Substituent group from the phenyl ring has two hydroxyls on the 1,2,3,4-Tetrahydrooxonaphthalene, lay 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
13C-NMR composes (δ 176.9) data, and showing has a carboxyl to exist, and it can only be connected on the C-8 '.Therefore, chemical compound (I)
1H-NMR and
13The C-NMR data are arranged as table 1.From HMBC spectrum (accompanying drawing 2), the structure of this chemical compound has obtained conclusive evidence.
Table 1 chemical compound (I)
1H-NMR and
13The C-NMR data
Embodiment 4:
A kind of above-mentioned application of chemical compound in the medicament preparation of antitumor field.
Suppress people HL-60 leukaemia, people Kato-III stomach cancer cell and people A
549The activity experiment of lung carcinoma cell:
Cell culture
People HL-60 leukaemia, people Kato-III stomach cancer cell and people A
549Lung carcinoma cell is incubated at RPMI 1640 culture medium, adds 10% FBS, L-glutamine, 100U/ml penicillin and 100 μ g/ml streptomycins.The take the logarithm cell counting of trophophase is with 3 * 10
4The concentration of individual/ml is inoculated in 96 orifice plates, and every hole 180 μ l are at 37 ℃, 5% CO
2Incubator in cultivated 24 hours.
Cell toxicant is measured
The medicine of variable concentrations (1-100 μ M) is with EtOH-H
2O (1:9) is dissolution with solvents to 20 μ l, and blank group adds the EtOH-H of 20 μ l
2O (1:9) solution, cell continue to cultivate 72 hours, adopt mtt assay to measure then, and the cell after promptly cultivating adds 10 μ l MTT (5mg/ml) phosphate buffers, 37 ℃, 5% CO
2Cultivated 4 hours in the incubator, centrifugal, get supernatant 150 μ l and discard, add 175 μ l DMSO, jolting was measured light absorption value with microplate reader at 550nm after 10 minutes.Calculate according to following formula, draw IC
50
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
See Table 2.The result shows, chemical compound (I) all has in various degree inhibitory action to three kinds of tumor cells.
Table 2. chemical compound (I) suppresses the IC of three kinds of tumor cells
50(μ M) value
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
22Tumor cell suspension 0.2ml (about 1 * 10
6-2 * 10
6Individual 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.
2, to H
22The influence of mice increase in life span: mouse peritoneal inoculation H
22Tumor cell suspension 0.2ml observes and record dead mouse situation every day.
The results are shown in following table:
Annotate: compare * P＜0.05 with the normal saline group; Compare P＜0.05 with the cyclophosphamide group.
Chemical compound provided by the invention (I) is to H
22Growth 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 ℃); 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 filters with a small amount of 40% dissolve with ethanol, and filtrate 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 ℃); 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, filtrate 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 ℃); 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, filtrate 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
22Tumor cell suspension 0.2ml (about 1 * 10
6-2 * 10
6Individual 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.
2 couples of H
22The influence of mice increase in life span: mouse peritoneal inoculation H
22Tumor cell suspension 0.2ml observes and record dead mouse situation every day.
The results are shown in following table:
Annotate: compare * P＜0.05 with the normal saline group; Compare P＜0.05 with compound recipe Exocarpium Juglandis Immaturum tincture group.
Claims (8)
1. extract of immature exocarp of Juglans mandshurica Maxim, it 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:
2, the preparation of the described a kind of extract of immature exocarp of Juglans mandshurica Maxim of a kind of claim 1, it is characterized in that: get fresh immature exocarp of Juglans mandshurica Maxim and extract with soak with ethanol, extracting solution concentrates, and concentrated solution adds aqueous dispersion, gets supernatant after centrifugal, supernatant passes through macroporous adsorbent resin, with the adsorbent resin 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%.
3. the preparation of a kind of extract of immature exocarp of Juglans mandshurica Maxim according to claim 2 is characterized in that: describedly get fresh immature exocarp of Juglans mandshurica Maxim and extract 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.
4. according to the preparation of claim 2 or 3 described a kind of extract of immature exocarp of Juglans mandshurica Maxim, it is characterized in that: described extracting solution concentrates, concentrated solution adds aqueous dispersion, centrifugal, be meant behind the merge extractive liquid, concentrating under reduced pressure and be concentrated into relative density 1.10-1.14 (50 ℃) that concentrated solution adds aqueous dispersion, centrifugal, get supernatant after centrifugal, supernatant is by macroporous adsorbent resin, with the adsorbent resin ethanol elution.
5. the preparation of a kind of extract of immature exocarp of Juglans mandshurica Maxim according to claim 4, it is characterized in that: described add aqueous dispersion centrifugal after, get supernatant, filter by 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, and flow velocity 10ml/min is respectively with the washing of 3～5 times of bed volumes, 30% ethanol of 5 times of bed volumes is washed, collect 30% ethanol elution, the eluent evaporated under reduced pressure obtains residue, and residue filters with a small amount of 40% dissolve with ethanol, filtrate is injected polydextran gel, collect 40% ethanol elution, be concentrated into dried, in the dry thing QINGLONGYISU A content greater than 70%.
6. the preparation of a kind of extract of immature exocarp of Juglans mandshurica Maxim according to claim 5, it is characterized in that: described filtrate is injected polydextran gel, collecting 40% ethanol elution 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.
7. according to the preparation of claim 2 or 3 or 4 or 5 or 6 described a kind of extract of immature exocarp of Juglans mandshurica Maxim, it is characterized in that: described 40% ethanol elution with sephadex lh-20 is concentrated into dried, and QINGLONGYISU A content is greater than 70% in the dry thing.
8, a kind of application of extract in the preparation antitumor drug that contains the chemical compound of general formula (I).
Priority Applications (1)
| 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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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102370695A (en) * | 2010-08-19 | 2012-03-14 | 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 |
| CN103217491A (en) * | 2013-03-21 | 2013-07-24 | 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100548957C (en) * | 2006-10-11 | 2009-10-14 | 东北林业大学 | A kind of method of extracting regianin |
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2007
- 2007-12-28 CN CN200710144961XA patent/CN101468061B/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102370695A (en) * | 2010-08-19 | 2012-03-14 | 黑龙江省中医研究院 | Qinglongyi active extract, its preparation method and its application |
| 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 |
| CN103217491A (en) * | 2013-03-21 | 2013-07-24 | 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 |
| 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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| CN101468061B (en) | 2012-04-04 |
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