CN102349943A - Juglans mandshurica husk extractive and preparation method thereof - Google Patents
Juglans mandshurica husk extractive and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a juglans mandshurica husk extractive which contains the main medical active ingredients of 8-hydroxyl-9,10-anthraquinone-1-carboxylic acid, REG IOLONE, 4,5,8- trihydroxy-alpha-naphthyl ketone-3-O-beta-D-glucopyranoside, 4,5,8- trihydroxy-alpha- naphthyl ketone-3-O-(6'-O- nutgall acyl)-beta-D- glucopyranoside, 1-(4'- hydroxyl phenyl)-7-(3''-methoxy-4''-hydroxyl phenyl) heptane-3-alcohol, 4-hydroxy-2,6- syringol-1-O-beta-D-glucopyranoside, 4- hydroxyl-2,6- syringol-1-O-(6'-O- nutgall acyl)-beta-D- glucopyranoside, vanillic acid-4-O-(6'-O- nutgall acyl)-beta-D-glucopyranoside, vanillic aldehyde, vanillic acid, gallic acid, syringic acid, kaempferol, quercetin glycoside, myrica rubra and quercetin. The juglans mandshurica husk extractive has good effect in curing hepatopathy and restraining the growth of tumor and especially has the effect of directly killing the hepatocarcinoma cells.
Description
Technical field
The present invention relates to a kind of Chinese medicine, specifically relate to a kind of manchurian walnut bark extract-Cortex Juglandis mandshuricae element and preparation method thereof.
Background technology
Juglans mandshurica (Juglans mandshurica Maxim) is a Juglandaceae Juglans deciduous tree, is the important associated species of northeast leaved Korean Pine woods, also is famous three big hard one of wealthy in northeast, mainly is distributed in China the Northeast.Juglans mandshurica is that protective plant is treasured by China.
In recent years, the hepatopathy sickness rate accounts for first of the legal report of various infectious disease.The number that hepatitis B virus is carried in the whole world surpasses 2.8 hundred million.China is the hepatopathy district occurred frequently, and hepatitis B virus carriers surpasses 0.93 hundred million, and patient's number surpasses 1,000 ten thousand, wherein develops into liver cirrhosis or hepatocarcinoma more than the 25-40%, and World Health Organization (WHO) classifies hepatitis, cancer, AIDS as three big " killers " that threaten human survival.
At present, the method for western medical treatment hepatopathy mainly contains two kinds: a kind of medicine that can suppress virus replication that is to use, like interferon, lamivudine etc.; These suiting the medicine to the illness property of thing medicine are strong; Therapeutic effect is obvious, but is prone to recurrence after the drug withdrawal, even transfers irreversible serious consequence sometimes to.Another kind method is performed the operation exactly, and this mainly is to the cancer patient.Postoperative chemotherapy is bigger to patient's injury.Because western medicine and medical practitioners does not have better method in this respect, people invest Chinese medicine to sight.
Summary of the invention
The object of the invention provides a kind of manchurian walnut bark extract-Cortex Juglandis mandshuricae element that is used to treat hepatopathy, particularly hepatocarcinoma.
Another object of the present invention provides the plain production method of a kind of manchurian walnut bark extract Cortex Juglandis mandshuricae.
Technical scheme is:
Medicinal active component mainly contains in the manchurian walnut bark extract: 8-hydroxyl-9; 10-anthraquinone-1-carboxylic acid, regiolone, 4; 5; 8-trihydroxy-α-naphthalenone-3-O-β-D-pyranglucoside, 4; 5; 8-trihydroxy-α-naphthalenone-3-O-(6 '-O-galloyl)-β-D-pyranglucoside, 1-(4 '-hydroxy phenyl)-7-(3 "-methoxyl group-4 "-hydroxy phenyl) heptane-3-alcohol, 4-hydroxyl-2,6-syringol-1-O-β-D-pyranglucoside, 4-hydroxyl-2,6-syringol-1-O-(6 '-O-galloyl)-β-D-pyranglucoside, vanillic acid-4-O-(6 '-O-galloyl)-β-D-pyranglucoside, vanillin, vanillic acid, gallic acid, syringic acid, kaempferol, Quercetin, Fructus Myricae rubrae is general and Quercitroside.
The purposes of manchurian walnut bark extract:
To boil water the drinking of Cortex Juglandis mandshuricae commonly used among the people is in order to treatment breast side of body overflow, symptoms such as bitter taste in the mouth and dry throat.On this basis, we study the extract of Cortex Juglandis mandshuricae, confirm chemical analysis and pharmacological evaluation.
Manchurian walnut bark extract is carried out zoopery to be shown: compare with model control group, medicine (15mgkg participates in the experiment
-1) to mice S
180The growth of sarcoma has significant inhibitory effect, and suppression ratio reaches more than 40%, and administration group tumor amount all has and alleviates, and compares with model control group that there were significant differences (P＜0.05 or P＜0.01).Through long-time repeated multiple times experiment, not only have the inhibitory action of tumor, prompting is compared with the chemotherapeutics xeloda, and CLI finds that the dosage of performance tumor-inhibiting action has appreciable impact to the periphery leukocyte count in the whole animal inhibition test.During through pharmacological evaluation primary study manchurian walnut bark extract, hepatocarcinoma H
22The tumor body of tumor-bearing mice increases has significant inhibitory effect.In vitro tests shows that this medicine has direct killing effect to HCC, and to not damage of normal cell.
Manchurian walnut bark extract can be made into capsule, tablet, pill, injection and injectable powder thereof, and is easy to use, treatment hepatopathy and cancer thereof, determined curative effect.
Instructions of taking:
Oral, every day twice, each 2 slices// ball (the each 1 pin/bottle of injection) contains the plain 7-15mg of Cortex Juglandis mandshuricae.
The method for preparing of manchurian walnut bark extract is to get Cortex Juglandis mandshuricae 100kg powder to become coarse powder (or being broken into fritter), adds 6-8 and doubly measures solvent: ethanol (or water); The ethanol mass concentration is 70-95% (water is drinking water), and reflux three times (water is carried and is heating extraction), heating-up temperature are 80-90 ℃ (water is carried 100 ℃); Each 1 hour (water is carried 3 hours); Filter, 50-60 ℃ is evaporated to the mother solution that every 1ml contains medical material 0.5-1g, after the macroporous adsorbent resin separation and purification; Get pale brown color grease 200-300mg, yield is 2 ‰-3 ‰.
The specific embodiment
Embodiment one
The method for preparing of manchurian walnut bark extract is to get Cortex Juglandis mandshuricae 100kg to wear into 50-60 order coarse powder (or being broken into fritter), adds 6-8 times of drinking water; Heating extraction three times, temperature are 100 ℃, each 3 hours; Filter, 50-60 ℃ is evaporated to the mother solution that every 1ml contains medical material 0.5-1g, after the macroporous adsorbent resin separation and purification; Get pale brown color manchurian walnut bark extract grease 200mg, yield is 2 ‰.
Embodiment two
The method for preparing of manchurian walnut bark extract is to get Cortex Juglandis mandshuricae 100kg to wear into 40-50 order coarse powder (or being broken into fritter), adds 6 times of amount ethanol; Concentration of alcohol is 70%, and reflux three times, heating-up temperature are 80-90 ℃; Each 1 hour, filter, 50-60 ℃ is evaporated to the mother solution that every 1ml contains medical material 0.5-1g; After the macroporous adsorbent resin separation and purification, get pale brown color manchurian walnut bark extract grease 200mg, yield is 2 ‰.
Embodiment three
The method for preparing of manchurian walnut bark extract is to get Cortex Juglandis mandshuricae 100kg to wear into coarse powder (or being broken into fritter), adds 7 times of amount ethanol; Concentration of alcohol is 80%, and reflux three times, heating-up temperature are 80-90 ℃; Each 1 hour, filter, 50-60 ℃ is evaporated to the mother solution that every 1ml contains medical material 0.5-1g; After the macroporous adsorbent resin separation and purification, get pale brown color manchurian walnut bark extract grease 250mg, yield is 2.5 ‰.
Embodiment four
The method for preparing of manchurian walnut bark extract is to get Cortex Juglandis mandshuricae 100kg powder to become coarse powder (or being broken into fritter), adds 8 times of amount ethanol; Concentration of alcohol is 95%, and reflux three times, heating-up temperature are 80-90 ℃; Each 1 hour, filter, 50-60 ℃ is evaporated to the mother solution that every 1ml contains medical material 0.5-1g; After the macroporous adsorbent resin separation and purification, get pale brown color manchurian walnut bark extract grease 300mg, yield is 3 ‰.
The manchurian walnut bark extract chemical constituent is formed
Through the solvent fractional extraction; Silica gel column chromatography repeatedly; Polyamide column chromatography, ODS, Sephadex LH-20; The preparation thin layer and repeatedly means such as recrystallization obtain 18 chemical compounds; Measure the structure of identifying 16 chemical compounds wherein with means such as spectroscopic data analysis through physicochemical constant, be respectively: (1) 8-hydroxyl-9, the 10-anthracene is awake-1-carboxylic acid, (2) regiolone, (3) 4; 5; 8-trihydroxy-α-naphthalenone-3-O-β-D-pyranglucoside, (4) 4,5,8-trihydroxy-α-naphthalenone-3-O-(6 '-O-galloyl)-β-D-pyranglucoside, (5) 1-(4 '-hydroxy phenyl)-7-(3 "-methoxyl group-4 "-hydroxy phenyl) heptane-3-alcohol, (6) 4-hydroxyl-2; 6-syringol-1-O-β-D-pyranglucoside, (7) 4-hydroxyl-2,6-syringol-1-O-(6 '-O-galloyl)-β-D-pyranglucoside, (8) vanillic acid-4-O-(6 '-O-galloyl)-β-D-pyranglucoside, (9) vanillin, (10) vanillic acid, (11) gallic acid, (12) syringic acid, (13) kaempferol, (14) Quercetin, (15) Fructus Myricae rubrae is general and (16) Quercitroside.Wherein chemical compound 1 is a new natural product, and wherein chemical compound 6,7 obtains for separating first in belonging to 8, and 2,9,12,13 and 14 is to separate first in this plant to obtain.The discriminating situation is following:
Chemical compound 1,8-hydroxy-9,10-anthraquinone-1-carboxylic acid
Yellow needle (EtOH-H
2O),
1H-NMR with
13C-NMR.
Chemical compound 2, regiolone
White crystals, 10% sulphuric acid-ethanol colour developing is green speckle (thin layer silica gel), 365nm is tangible green fluorescence down.UVλ
max(MeOH，nm)：222，260，340。UVλ
max(MeCN，nm)：222，260，340。CD，Δε(λ?
max，0.1mg/ml，MeCN)：Δε＝+1600(219nm)，Δε＝+400(260nm)，Δε＝+50(340nm)。
1H-NMR(300MHz，DMSO-d
6)，δ：12.42(1H，s，OH-8)，7.55(1H，t，H-6)，7.08(1H，d，J＝7.5Hz，H-5)，6.85(1H，d，J＝8.2Hz，H-7)，5.64(1H，d，J＝5.8Hz，OH-4)，4.76(1H，m，H-4)，2.76?(2H，m，CH
2-2)，2.2(1H，dddd，H-3β)，2.0(1H，dddd，H-3α)。
13C-NMR，δ205.4(C-1)，161.6(C-8)，148.8(C-4a)，136.9(C-6)，117.5(C-5)，115.9(C-7)，115.1(C-8a)，66.1(C-4)，35.4(C-2)，31.6(C-3)。
Chemical compound 3,4,5,8-trihydroxy-α-tetralone-5-O-β-D-glucopyranoside
Be glassy yellow fluorescence under the amorphous powder, 365nm, 10% sulphuric acid-ethanol colour developing is green speckle (thin layer silica gel).
ESI-MS：354.9[M-H]
-，391.0[M+Cl]
-，379.1[M+Na]
+。UVλ
max(MeOH，nm)：230，263，356。CD，Δε(λ
max，0.01mg/ml，MeOH)：Δε＝-25(225nm)，Δε＝-5(260nm)，Δε＝-3(355nm)。
1H-NMR with
13C-NMR.
Chemical compound 4,4,5,8-trihydroxy-α-tetralone-5-O-β-D-(6 '-O-galloyl) glucopyranoside
Be glassy yellow fluorescence under the amorphous powder, 365nm, 10% sulphuric acid-ethanol colour developing is green speckle (thin layer silica gel).
ESI-MS：507.0[M-H]
-，542.9[M+Cl]
-，531.1[M+Na]
+。
1H-NMR with
13C-NMR.
Chemical compound 5,1-(4 '-hydroxyphenyl)-7-(3 "-methoxy-4 "-hydroxyphenyl) hepta-3-ol
The yellow oily solid, ESI-MS:329.0 [M-H]
-, 353.2 [M+Na]
+, 354.2 [M+Na+H]
+[329.0 M-H]
-The secondary anion: 313.9,286.9,220.8,206.8,192.9,134.8; [353.2 M+Na]
+The secondary cation: 337.2,313.2,219.1,189.1,137.0,121.0.
1H-NMR with
13C-NMR.。
Chemical compound 6,4-hydroxy-2,6-dimethoxyphenol1-O-β-D-glucopyranoside
Colourless crystallization (MeOH-H
2O), ESI-MS:330.9 [M-H]
-, 366.9 [M+Cl]
-, 355.1 [M+Na]
+ 1H-NMR with
13C-NMR.
Chemical compound 7,4-hydroxy-2,6-dimethoxyphenol-1-β-D-(6 '-O-galloyl) glucopyranoside
Colourless needle (MeOH-H
2O), ESI-MS:483.0 [M-H]
-, 518.9 [M+Cl]
-, 485.1 [M+H]
+, 507.1 [M+Na]
+ 1H-NMR with
13C-NMR.
Chemical compound 8, Vanillic acid-4-O-β-D-(6 '-O-galloyl) glucopyranoside
Colourless crystallization (MeOH), ESI-MS:481.0 [M-H]
-, 505.1 [M+Na]
+ 1H-NMR with
13C-NMR.
Chemical compound 9, Vanillin
White needle (MeOH), ferric chloride-potassium ferricyanide reacting positive.
1H-NMR，δ10.23(1H，br.s，OH-4)，9.77(1H，s，CHO)，7.43(1H，dd，J＝8.0，1.8Hz，H-6)，7.38(1H，d，J＝1.8Hz，H-5)，6.95(1H，d，J＝8.0Hz，H-2)，3.83(3H，s，OCH
3).
13C-NMR，δ191.1(CHO)，153.1(C-4)，148.2(C-3)，128.7(C-1)，126.2(C-2)，115.5(C-6)，110.7(C-5)，55.7(OCH
3)。
Chemical compound 10, Vanillic acid
White needle (MeOH).Ferric chloride-potassium ferricyanide reacting positive, the bromophenol blue reacting positive.
1H-NMR(DMSO-d6)δ：12.50(1H，brs，COOH)，9.85(1H，brs，OH-4)，7.44(1H，dd，J＝8.7Hz，1.8Hz，H-6)，7.43(1H，d，J＝1.8Hz，H-2)，6.84(1H，d，J＝8.7Hz，H-5)，3.81(3H，s，OCH
3-3)。
Chemical compound 11, Gallic acid
Colourless needle, ferric chloride-potassium ferricyanide reacting positive, bromocresol green reacting positive.
1H-NMR(DMSO-d6)δ：12.30(1H，br.s，COOH)，9.20(3H，br.s，OH-3，4，5)，6.91(2H，s，H-2，6)。Chemical compound 12, Syringic acid
Colourless needle, ferric chloride-potassium ferricyanide reacting positive, bromophenol blue reacting positive.
1H-NMR(DMSO-d6)δ：12.40(1H，br.s，COOH)，9.12(1H，br.s，OH-4)，7.20(2H，s，H-2，6)，3.80(6H，s，OCH
3-3，5)。
13C-NMR，δ168.0(COOH)，122.6(C-1)，106.8(C-2，6)，147.3(C-3，5)，139.5(C-4)，55.9(OCH
3-3，5)。
Chemical compound 13, Kaempferol
Pale yellow powder, ferric chloride-potassium ferricyanide reacting positive, hydrochloric acid magnesium powder reacting positive.
1H-NMR，δ12.49(1H，br.s，OH-5)，10.70(1H，br.s，OH-7)，10.11(1H，br.s，OH-4’)，9.42(1H，br.s，OH-3)，8.05(2H，d，J＝8.7Hz，H-2’，6’)，6.92(2H，d，J＝8.7Hz，H-3’，5’)，6.44(1H，s，H-8)，6.19(1H，s，H-6)。
Chemical compound 14, Quercetin
The yellow green powder, ferric chloride-potassium ferricyanide reacting positive, hydrochloric acid magnesium powder reacting positive.
1H-NMR(DMSO-d?
6)δ：12.51(1H，brs，OH-5)，10.09(1H，brs，OH-7)，9.37(3H，brs，OH-3，3′，4′)，7.68(1H，d，J＝1.9Hz，H-2′)，7.55(1H，dd，J＝8.5Hz，1.9Hz，H-6′)，6.92(1H，d，J＝8.5Hz，H-5′)，6.41(1H，d，J＝1.7Hz，H-8)，6.19(1H，d，J＝1.7Hz，H-6)。
Chemical compound 15, Myricitrin
Pale yellow powder, ferric chloride-potassium ferricyanide reacting positive, aluminum chloride are reflected at fluorescence reinforcement under the ultraviolet.
1H-NMR，δ12.67(1H，s，OH-5)，8-10(several?protons，p-OH)，6.96(2H，s，H-2’，6’)，6.36(1H，d，J＝1.9Hz，H-8)，6.20(1H，d，J＝1.9Hz，H-6)，5.19(1H，s，H-1”)，3-5(several?protons，H-2”-4”)，0.84(3H，d，J＝6Hz，CH
3-5”)。
13C-NMR，δ157.5(C-2)，134.3(C-3)，177.8(C-4)，161.4(C-5)，98.7(C-6)，164.3(C-7)，93.5(C-8)，156.5(C-9)，104.0(C-10)，119.6(C-1’)，107.9(C-2’)，145.8(C-3’)，136.5(C-4’)，145.8(C-5’)，107.9(C-6’)，102.0(C-1”)，70.5(C-2”)，70.6(C-3”)，71.3(C-4”)，70.1(C-5”)，17.6(C-6”)。
Chemical compound 16, Quercitrin
Pale yellow powder, ferric chloride-potassium ferricyanide reacting positive, aluminum chloride are reflected at fluorescence reinforcement under the ultraviolet.
1H-NMR，δ12.65(1H，s，OH-5)，7.26(1H，br.s，H-2′)，7.22(1H，br.d，J＝8.0Hz，H-6′)，6.82(1H，br.d，J＝8.0Hz)，6.41(1H，br.d，H-8)，6.19(1H，br.d，H-6)，5.25(1H，s，H-1”)，3-5(several?protons，H-2”-4”)，0.81(3H，d，J＝5.7Hz，CH
3-5”)。
13C-NMR，δ156.5(C-2)，133.8(C-3)，177.0(C-4)，161.1(C-5)，99.3(C-6)，164.0(C-7)，93.8(C-8)，156.6(C-9)，104.0(C-10)，120.9(C-1’)，115.2(C-2’)，146.0(C-3’)，115.2(C-4’)，149.0(C-5’)，120.9(C-6’)，101.7(C-1”)，70.3(C-2”)，70.4(C-3”)，71.2(C-4”)，70.0(C-5”)，17.4(C-6”)。
The plain pharmacodynamics of manchurian walnut bark extract Cortex Juglandis mandshuricae
Through preparation murine sarcoma S
180, hepatocarcinoma H
22, hepatocarcinoma Hca-F tumor strain bearing mouse model, investigate the tumor-inhibiting action of the plain various dose of Cortex Juglandis mandshuricae respectively to tumor-bearing mice, the result shows: plain 7.5, the 15mgkg of Cortex Juglandis mandshuricae
-1Continuously gastric infusion 7 days is to murine sarcoma S
180, rat liver cancer Hca-F tumor-bearing mice all has remarkable tumor-inhibiting action; And tumor-inhibiting action presents dose-effect relationship.The plain 15mgkg of Cortex Juglandis mandshuricae
-1To rat liver cancer H
22Tumor-bearing mice has significant tumor-inhibiting action, and 3.75,7.5mgkg
-1It there is certain tumor-inhibiting action trend, but not statistically significant.Through finding with the cyclophosphamide combined medication, during with the plain medication separately of Cortex Juglandis mandshuricae relatively, plain 3.75,7.5, the 15mgkg of Cortex Juglandis mandshuricae
-1When continuous gastric infusion 7 days and CTX Combined application, to sarcoma S
180Model mice tumour inhibiting rate all improve effect and 15mgkg
-1During with the CTX Combined application, press down tumor and present summation action.Through external preparation Hela cell (human cervical carcinoma cell), 7402BEL (human liver cancer cell) and Hca-F cell (Murine Ascitic Hepatoma Cells), use the MTT detection method suppresses the tumor cell ability respectively to drug group to be measured, positive drug control group and normal control group (RPMI-1640) observation.Result of the test shows: in selected dosage range, the Cortex Juglandis mandshuricae element all has remarkable inhibitory action to Hela cell, 7402BEL cell and Hca-F cell.Research is also found, the plain 15mgkg of Cortex Juglandis mandshuricae
-1Continuously gastric infusion 7 days is to murine sarcoma S
180The tumor-bearing mice peripheral white blood cell has remarkable rising effect, and 3.75,7.5mgkg
-1Certain rising effect trend is arranged, but not statistically significant.Prompting Cortex Juglandis mandshuricae element continuous use in selected dosage range does not find that tumor-bearing mice bone marrow is had inhibitory action.The plain 15mgkg of Cortex Juglandis mandshuricae
-1Gastric infusion can significantly improve cyclophosphamide in 7 days and cause immunologic hypofunction mouse spleen organ coefficient and carbon clearance index continuously; This shows; The Cortex Juglandis mandshuricae element is the antineoplastic while in vivo, and is less to the phagocytic function influence of reticuloendothelial system, not the nonspecific immunity function of injured mice.
One, antitumor action
(1) whole animal test
Test objective is investigated manchurian walnut bark extract Cortex Juglandis mandshuricae plain anti-tumor in vivo effect and the medication of combined chemotherapy medicine and whether is produced the synergistic antitumor effect.
Test method and result
1. to murine sarcoma S
180Inhibitory action
Test method
External recovery murine sarcoma S
180Tumor strain cell, the recovery three generations dilutes with physiological saline solution; Choose healthy mice, every the above-mentioned cell suspension 0.2ml of usefulness lumbar injection, about week back inoculation mouse web portion obviously rises greatly; Extract ascites, in sterile test tube, using physiological saline solution dilution oncocyte to count concentration is 2 * 10
7Individual/ml.Inoculate buying Kunming mouse with above-mentioned ascites diluent, every subcutaneous vaccination 0.2ml behind axillary fossa place sterilization skin sets up murine sarcoma S
180Lotus tumor model
[1] [2] [3]
With the tumor-bearing mice random packet, 10 every group, be respectively model control group, the plain basic, normal, high administration group of Cortex Juglandis mandshuricae (3.75,7.5,15mgkg
-1), join a Capsules group (10mgkg
-1), compound recipe wood chicken mixture group (10mlkg
-1), xeloda group (1000mgkg
-1).From modeling continuous gastric infusion 7 days after 24 hours, every day 1 time, administration volume 20mlkg
-1 [4] [5]
Behind this administration 24h, the cervical vertebra dislocation method is put to death animal, dissects and peels off the tumor piece, and electronic balance claims that tumor is heavy.Calculate tumour inhibiting rate according to following formula:
Tumour inhibiting rate=(the average tumor of the average tumor weight/matched group of 1-administration group is heavy) * 100%
Experimental data is represented with mean+SD
.
Result of the test
Compare Cortex Juglandis mandshuricae element 7.5,15mgkg with model control group
-1Continuously gastric infusion 7 days is to murine sarcoma S
180Tumor-bearing mice has significant tumor-inhibiting action: the plain 3.75mgkg of Cortex Juglandis mandshuricae
-1Certain tumor-inhibiting action trend is arranged, but not statistically significant.Experiment is done 3 times altogether, and the high dose group suppression ratio all reaches more than 30%.The result sees table 1.
Table 1 Cortex Juglandis mandshuricae element is to murine sarcoma S
180Inhibitory action
Compare with model group
*P＜0.05,
*P＜0.01
2. to the inhibitory action of rat liver cancer Hca-F
Test method
External conventional recovery rat liver cancer Hca-F tumor strain cell, other is the same.
Result of the test
Compare Cortex Juglandis mandshuricae element 7.5,15mgkg with model control group
-1Continuously gastric infusion 7 days has significant tumor-inhibiting action to rat liver cancer Hca-F tumor-bearing mice; The plain 3.75mgkg of Cortex Juglandis mandshuricae
-1Certain tumor-inhibiting action trend is arranged, but not statistically significant.Experiment is done 3 times altogether, and the high dose group tumour inhibiting rate all reaches more than 30%.The result sees table 2
Table 2 Cortex Juglandis mandshuricae element is to the inhibitory action of rat liver cancer Hca-F
Compare with model group
*P＜0.05,
*P＜0.01
3. to rat liver cancer H
22Inhibitory action
Test method
External conventional recovery rat liver cancer H
22Tumor strain cell, other is the same.
Result of the test
Compare Cortex Juglandis mandshuricae element 7.5,15mgkg with model control group
-1Continuously gastric infusion 7 days is to rat liver cancer H
22Tumor-bearing mice has significant tumor-inhibiting action; 3.75mgkg
-1Certain tumor-inhibiting action trend is arranged, but not statistically significant.Experiment is done 3 times altogether, and the high dose group tumour inhibiting rate all reaches more than 30%.The result sees table 3.
Table 3 Cortex Juglandis mandshuricae element is to rat liver cancer H
22Inhibitory action
Compare with model group
*P＜0.05,
*P＜0.01
With chemotherapeutics cyclophosphamide (CTX) Combined application to murine sarcoma S
180The tumor-inhibiting action test method of tumor-bearing mice
To inoculate murine sarcoma S behind the 24h
180Tumor-bearing mice is divided into 8 groups at random, 10 every group.Be model control group, CTX (10mgkg
-1), the plain basic, normal, high dose groups of Cortex Juglandis mandshuricae (3.75,7.5,15mgkg
-1), the Cortex Juglandis mandshuricae element is low+CTX, in+CTX, height+CTX dose groups, dosage is respectively (3.75+10,7.5+10,15+10mgkg
-1).Wherein, CTX organizes the 1st, 3,5,7 day intraperitoneal injection after inoculating back 24 hours, and the administration volume is 0.1ml/10g, administration every day 1 time, and administration is 4 times altogether.The Cortex Juglandis mandshuricae element is inoculated back 24 hours beginning gastric infusions, administration every day 1 time, successive administration 7 days in the tumor strain.Plain each dosage merging CTX of Cortex Juglandis mandshuricae organizes in inoculating back 24 hours and gives each dosage Cortex Juglandis mandshuricae plain and lumbar injection CTX by above-mentioned medication filling stomach.Put to death animal on the 8th day, and stripped the tumor piece and weigh, calculate tumour inhibiting rate, Q-value respectively according to formula (1), (2).Judge the drug combination curative effect according to Q-value, Q＜0.85 is an antagonism, and Q is addition between 0.85～1.5, and Q＞1.15 are for collaborative
[6] [7]
(1) tumour inhibiting rate=(the average tumor of the average tumor weight/matched group of 1 one administration groups is heavy) * 100%
(2) Q-value=E
(A+B)/ [E
A+ E
B(1-E
A)], (E wherein
A, E
B, E
(A+B)Expression is tried the tumour inhibiting rate of drug group, CTX group and drug combination group respectively)
[7]
Result of the test
Relatively, when plain each dosage of Cortex Juglandis mandshuricae and CTX Combined application, Combined application is to sarcoma S during with the plain medication separately of Cortex Juglandis mandshuricae
180The effect that all improves of tumor-bearing mice tumour inhibiting rate, and when high dose and CTX drug combination, Q=0.86 presses down tumor and is summation action.The result sees table 4
Table 4 Cortex Juglandis mandshuricae element and cyclophosphamide (CTX) drug combination are to murine sarcoma S
180The tumor-inhibiting action of tumor-bearing mice
Compare with model group
*P＜0.05
*P＜0.01
Q
Low=0.65, Q
In=0.76, Q
High=0.86
(2) in vitro tests
Test objective is investigated and is tried thing in external influence to Hela cell, 7402BEL cell and Hca-F cell proliferation.
Test method and result:
1. divide into groups and cell preparation:
1.1 divide into groups
Plain (dosage: 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, 3.125 μ g/ml, 1.56 μ g/ml) the positive drug group (Gan Dike) of Cortex Juglandis mandshuricae (dosage: 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml,
3.125μg/ml、1.56μg/ml)
Dimethyl sulfoxide group (50%, 25%, 12.5%, 6.25%, 3.125%, 1.56%)
1.2 cell strain preparation:
1.2.1Hela the preparation of cell and 7402BEL cell (human liver cancer cell)
1) take the logarithm the Hela cell and the 7402BEL cell of trophophase, 0.125% trypsinization.With culture fluid adjustment cell concentration to 2 * 10
5/ ml.
2) get 96 well culture plates, every hole adds 100 μ l cell suspension, puts 37 ℃, 5%CO
2Hatch 24h.
1.2.2Hca-F the preparation of cell (Murine Ascitic Hepatoma Cells)
1) aseptic extraction mouse ascites, the normal saline washing is centrifugal.With culture fluid adjustment cell concentration to 1 * 10
6/ ml.
2) get 96 well culture plates, every hole adds 100 μ l cell suspension, puts 37 ℃, 5%CO
2Hatch 24h.
2. detection method and interpretation of result:
2.1MTT method
1) doubling dilution testing sample and positive drug.Cells and supernatant is gone in suction, and every hole adds 100 μ l testing sample and positive drug respectively, respectively establishes 3 multiple holes, and control wells adds 1640 growth-promoting medias, and establishes the blank group, and 37 ℃, 5%CO
2Hatch 24h, see table 5
Table 5
| The plain μ g/ml of Cortex Juglandis mandshuricae | Positive drug μ g/ml | Dimethyl sulfoxide % |
| 100 | 100 | 50 |
| 50 | 50 | 25 |
| 25 | 25 | 12.5 |
| 12.5 | 12.5 | 6.25 |
| 6.25 | 6.25 | 3.125 |
| 3.125 | 3.125 | 1.56 |
| 1.56 | 1.56 | 0.78 |
| 1640 contrasts | ? | Blank |
Annotate: each concentration is established 3 multiple holes.
After cultivating 20h, every hole adds MYY10 μ l (MTT concentration, 5mg/ml prepares with PBS), continues to cultivate 4h.The centrifugal 10min of 1000r/min abandons supernatant, and every hole adds dimethyl sulfoxide 10 μ l, and the 10min that on micro-oscillator, slowly vibrates is with the OD value at ELIASA mensuration wavelength 570mm place.
2.2 date processing and interpretation of result:
Tumour inhibiting rate=(control wells OD value-experimental port OD value)/control wells OD value tumor cell is seen table 6-8 (being one of three identical result of the tests) to the inhibition sensitivity of medicine.
Table 6 medicine to be measured is to the effect of Hela cell inhibiting
Annotate: cell number is 2 * 10
5/ ml, each concentration is established 3 multiple holes.
Table 7 medicine to be measured is to the effect of Hca-F cell inhibiting
Annotate: cell number is 1 * 10
6/ ml, each concentration is established 3 multiple holes.
Table 8 medicine to be measured is to the effect of 7402BEL cell inhibiting
Annotate: cell number is 2 * 10
5/ ml, each concentration is established 3 multiple holes.
Conclusion (of pressure testing):
The Cortex Juglandis mandshuricae element has certain inhibitory action (medium sensitivity) to the Hela cell when 100 μ g/ml, 50 μ g/ml dosage; When 1.56 μ g/ml dosage, the 7402BEL cell had certain inhibitory action (medium sensitivity); When 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, 3.125 μ g/ml, 1.56 μ g/ml dosage, the Hca-F cell had significant inhibitory effect (sensitivity).
Two, leukogenic effect
Test objective is investigated manchurian walnut bark extract Cortex Juglandis mandshuricae element at the performance therapeutical effect simultaneously, to the influence of tumor-bearing mice peripheral white blood cell.
Test method
Murine sarcoma S
180Recover, inoculate ditto, with murine sarcoma S
180Tumor-bearing mice divides 8 groups at random, 10 every group.Be respectively normal control group, model control group, the plain basic, normal, high dose groups of Cortex Juglandis mandshuricae (3.75,7.5,15mgkg
-1), join a Capsules group (10mgkg
-1), compound recipe wood chicken mixture group (10mlkg
-1), xeloda group (1000mgkg
-1).Wherein, the xeloda group is administration in the 1st, 3,5,7 day behind modeling 24h, all the other successive administrations 7 days behind modeling 24h, once a day.Route of administration is gastric infusion.Behind last administration 24h, respectively organize the mouse orbit rear vein beard 20 μ l that take a blood sample, count with conventional blood leukocytes counting method at the OLYMPLS-BH2 microscopically
[8] [9]
Result of the test
Compare with model group: the plain 15mgkg of Cortex Juglandis mandshuricae
-1Gastric infusion is 7 days continuously, to murine sarcoma S
180The effect of being significantly increased of tumor-bearing mice peripheral white blood cell; 3.75,7.5mgkg
-1Peripheral white blood cell there is certain rising effect trend, but not statistically significant.The result sees table 9.
Table 9 Cortex Juglandis mandshuricae element is to murine sarcoma S
180The influence of tumor-bearing mice PBL
Compare with normal group
#P＜0.05,
##P＜0.01,
###P＜0.001
Compare with model group
*P＜0.05,
*P＜0.01,
* *P＜0.001
Three, to the influence of body non-specific immunity
Test objective is investigated the influence of manchurian walnut bark extract Cortex Juglandis mandshuricae element to the body non-specific immunity.
Test method
Choose 70 of healthy Kunming mouses, be divided into 7 groups at random, 10 every group.Be respectively the normal control group, model control group, the plain basic, normal, high dosed administration group of Cortex Juglandis mandshuricae (3.75,7.5,15mgkg
-1), join a Capsules group (10mgkg
-1), compound recipe wood chicken mixture group (10m1kg
-1).Inoculate second day and begin except that the normal control group, each organizes continuous gastric infusion 7 days.In administration beginning in the 5th day, all the other respectively organize tumor-bearing mice intraperitoneal injection of cyclophosphamide (CTX50mgkg
-1), once a day, successive administration 3 days.1h after the last administration, every caudal vein injection india ink (normal saline dilution in 1: 4) 0.1ml/10g, respectively at behind 2min, the 12min behind the injection prepared Chinese ink in the eye socket rear vein beard 20 μ l that take a blood sample, be blown into 0.1%Na at once
2CO
3Among the solution 2ml, shake up, 680nm surveys absorbance.Animal is put to death in dislocation, gets thymus, liver and spleen and weighs, and calculates organ index (mg/g), is calculated as follows and cleans up index K and phagocytic index α:
K＝(logA
1-logA
2)/(T
2-T
1)
α=
3√ K * body weight/(liver weight+spleen is heavy)
(A wherein
1Be the absorbance of the 1st blood sampling, A
2Be the absorbance of the 2nd blood sampling, T
1Be the time of the 1st blood sampling, T
2It is the time of the 2nd blood sampling)
Result of the test
The result shows, model control group and normal control group relatively, clear index K in corridor and phagocytic index α significantly descend, thymus, spleen coefficient significantly reduce, and show cell nonspecific immunity level reduction after the modeling.Compare Cortex Juglandis mandshuricae element 7.5,15mgkg with model control group
-1Dose groups spleen coefficient significantly raises, and the thymus coefficient presents certain rising effect trend.Prompting is compared with chemotherapeutics, and the Cortex Juglandis mandshuricae element is the antineoplastic while in vivo, and is less to the phagocytic function influence of reticuloendothelial system, not the cell nonspecific immunity function of injured mice.The result sees table 10.
Table 10 Cortex Juglandis mandshuricae element is to immunologic hypofunction mice carbon clearance and the exponential influence of immune organ
Compare with normal group
△P＜0.05,
△ △ △P＜0.001
Compare with model group
*P＜0.05,
*P＜0.01
Conclusion (of pressure testing)
1 with model group relatively, plain 7.5, the 15mgkg of Cortex Juglandis mandshuricae
-1The continuous irrigation stomach is given 7 days to murine sarcoma S
180, rat liver cancer Hca-F tumor-bearing mice all has remarkable tumor-inhibiting action; And tumor-inhibiting action presents dose-effect relationship.3.75kg
-1Certain inhibitory action trend is arranged, but not statistically significant.
2. compare 15mgkg with model group
-1Continuously gastric infusion 7 days is to rat liver cancer H
22Tumor-bearing mice has remarkable tumor-inhibiting action; 3.75,7.5mgkg
-1Certain inhibitory action trend is arranged, but not statistically significant.
3. compare with solvent control group, Cortex Juglandis mandshuricae plain (dosage is: 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, 3.125 μ g/ml, 1.56 μ g/ml) all has remarkable inhibitory action external to Hela cell, 7402BEL cell and Hca-F cell proliferation.
4. during with the plain medication separately of Cortex Juglandis mandshuricae relatively, plain 3.75,7.5, the 15mgkg of Cortex Juglandis mandshuricae
-1When continuous gastric infusion 7 days and CTX Combined application, to sarcoma S
180The effect that all improves of model mice tumour inhibiting rate, and high dose group presses down tumor and presents summation action.
5. compare the plain 15mgkg of Cortex Juglandis mandshuricae with model group
-1Continuously gastric infusion 7 days is to murine sarcoma S
L80The tumor-bearing mice peripheral white blood cell has remarkable rising effect, and prompting Cortex Juglandis mandshuricae element continuous use in selected dosage range does not find that tumor-bearing mice bone marrow is had inhibitory action.
6. plain 7.5, the 15mgkg of Cortex Juglandis mandshuricae
-1Continuously gastric infusion can significantly improve cyclophosphamide in 7 days and cause immunologic hypofunction mouse spleen coefficient, and significantly improved carbon and clean up index; The thymus coefficient there is certain rising effect trend, but not statistically significant.
The pharmacodynamics comparative study shows: to the tumor animal model; Various kinds of cell tumor strain model carries out the inside and outside antitumor imitates research; Tumour inhibiting rate between 30%-42.7, anticancer effect than the wooden chicken mixture of compound recipe and in the market PTS to join a capsule all high, suitable with anticancer chemical medicine xeloda effect.
※ coarse powder: refer to and to be no more than 40% powder (No. two sieve 24 orders, No. four sieve 65 orders) all through No. two sieves through No. four sieves but be mixed with.
Claims (1)
1. the application of manchurian walnut bark extract in the medicine of the anti-hepatocarcinoma of preparation, wherein said manchurian walnut bark extract method for preparing is following: get Cortex Juglandis mandshuricae 100kg and wear into coarse powder or be broken into fritter, add 6-8 and doubly measure solvent; Solvent is ethanol or water, and when solvent adopted ethanol, the ethanol mass concentration was 70-95%; Reflux three times, heating-up temperature are 80-90 ℃, each 1 hour; When solvent adopted water, heating extraction three times, heating-up temperature were 100 ℃; Each water is carried 2-3 hour, and with ethanol extraction or water extraction gained medical filtration, 50-60 ℃ is evaporated to the mother solution that every ml contains medical material 0.5-1g; After the macroporous adsorbent resin separation and purification, get pale brown color grease 200-300g.
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| CN112546054A (en) * | 2020-06-11 | 2021-03-26 | 广东盛普生命科技有限公司 | Application of galloyl-BETA-D-glucopyranose in preparing anti-coronavirus medicine |
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| CN103145677A (en) * | 2013-02-27 | 2013-06-12 | 中山火炬职业技术学院 | Method for separating active ingredients from aquilaria sinensis lamina by utilizing high-speed countercurrent chromatography |
| CN103145677B (en) * | 2013-02-27 | 2014-12-10 | 中山火炬职业技术学院 | Method for separating active ingredients from aquilaria sinensis lamina by utilizing high-speed countercurrent chromatography |
| CN112546054A (en) * | 2020-06-11 | 2021-03-26 | 广东盛普生命科技有限公司 | Application of galloyl-BETA-D-glucopyranose in preparing anti-coronavirus medicine |
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