CN101711790A - Wild Juglans mandshurica bark water extract used for curing liver cancer - Google Patents
Wild Juglans mandshurica bark water extract used for curing liver cancer Download PDFInfo
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- CN101711790A CN101711790A CN200810218437A CN200810218437A CN101711790A CN 101711790 A CN101711790 A CN 101711790A CN 200810218437 A CN200810218437 A CN 200810218437A CN 200810218437 A CN200810218437 A CN 200810218437A CN 101711790 A CN101711790 A CN 101711790A
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Abstract
The invention discloses a novel wild Juglans mandshurica bark water extract used for curing liver cancer. The water extract is prepared according to the following steps: soaking the Juglans mandshurica bark into water for 24h, wherein the weight of water is 5 times of that of the Juglans mandshurica bark, heating up to 100 DEG C and lasting for 30min, filtering, repeatedly extracting the filter residue once, mixing the filter liquor of the two times, and heating and concentrating. The invention also discloses an application of the wild Juglans mandshurica bark water extract in preparing medicines for curing liver cancer. The Juglans mandshurica bark in China has rich resource and wide source; and the technical scheme provided by the invention fully utilizes the natural medicine material advantage of our country and gives plays to the culture essence of traditional Chinese medical science and traditional Chinese medicine.
Description
Technical field
The present invention relates to a kind of Wild Juglans mandshurica bark water extract that is used for the treatment of hepatocarcinoma.
Background technology
Malignant tumor has become one of principal element that threatens the human life, chemicals is still one of main means of current anticancer therapy, but nearly all anticarcinogen all exists the comparison serious adverse effects, in many side effect, especially the most common to feel sick, to vomit, other also have alopecia, and inappetence etc. bring very big misery to the user.Different medicines, the degree of untoward reaction performance has bigger difference, but chemical generally medicine is even more serious than the untoward reaction of Chinese herbal medicine, however it is also less to be used for the antineoplastic Chinese herbal medicine at present, can not satisfy existing antitumor needs.
Summary of the invention
Technical problem to be solved by this invention provides a kind of novel Wild Juglans mandshurica bark water extract that is used for the treatment of hepatocarcinoma.
Another object of the present invention provides the application of above-mentioned Wild Juglans mandshurica bark water extract in the medicine of preparation treatment hepatocarcinoma.
The present invention is achieved through the following technical solutions above-mentioned purpose:
A kind of novel Wild Juglans mandshurica bark water extract that is used for the treatment of hepatocarcinoma is after Cortex Juglandis mandshuricae is added the water logging bubble 24h of 5 times of weight, to be heated to 100 ℃ of lasting 30min, filters, and filtering residue repeats to extract once, twice filtrate is mixed and heated concentrating promptly.
The application of above-mentioned Wild Juglans mandshurica bark water extract in preparation treatment liver-cancer medicine.
Described application is that preparation anti-hepatocarcinoma oral formulations, ejection preparation and other can be used for the pharmaceutical preparation of human body.
Compared with prior art, the present invention has following beneficial effect:
In malignant tumor class disease, the pathogenic characteristic of hepatocarcinoma is state of an illness concealment, the grade of malignancy height, and clinical manifestation rises and falls hurried, and the treatment that the patient is used for this disease need drop into more manpower financial capacity.Wild Juglans mandshurica bark water extract is saved overspending for the patient, eases one's family burden.The exploitation and the industrialization of achievements such as wild Cortex Juglandis mandshuricae water decoction of while will be subjected to the more favor of extensive patients, and its market potential is very huge, so its economic benefit is huge.In addition on the one hand, China's Cortex Juglandis mandshuricae aboundresources, wide material sources, technical scheme provided by the invention has made full use of the crude drug material advantage of China, has brought into play the cultural quintessence of China's Chinese medicine.The promotion and application clinically in future of wild Cortex Juglandis mandshuricae water decoction, help to finish the early prevention of hepatocarcinoma, preceding early treatment and improve survival rate, thereby can promote social population's health well, improve the health of the people, strengthen social productive forces, its social benefit is huge equally.
Description of drawings
Fig. 1 is that the cell of embodiment 2 adds the photo that the form behind the Cortex Juglandis mandshuricae water decoction changes.
Fig. 2 is the cell growth curve of embodiment 2.
Fig. 3 is the cell survival rate curve of embodiment 2.
Fig. 4 is the MTT experiment suppression ratio curve of embodiment 2.
The specific embodiment
Ga Shan has purchased this kind wild type Cortex Juglandis mandshuricae in her from the Heilongjiang Province, is the natural product after drying.Take by weighing after Cortex Juglandis mandshuricae 100g adds water 500g and soak 24h, heat 100 ℃ of 30min, filter, filtering residue is added 500g water again, heat 100 ℃ of 30min, filter, with twice filtrate mixing and heat and being concentrated into 200ml.The Cortex Juglandis mandshuricae water decoction is carried out silica gel column chromatography repeatedly.Carry out gradient elution (1: 0-0: 1), chloroform extract is carried out silica gel column chromatography equally with chloroform-dehydrated alcohol.Through gas chromatograph/mass spectrograph method measure, analyze the Cortex Juglandis mandshuricae water decoction medicinal ingredient.Measure the crude drug content of Cortex Juglandis mandshuricae water decoction, and with 0.22 μ m micropore filter filtration sterilization, the Juglans mandshurica bark water extract crude drug content that is obtained after the filter membrane degerming is 0.5g/ml, it is standby to put into 4 ℃ of preservations of refrigerator.
Embodiment 1 Cortex Juglandis mandshuricae water decoction anti-liver cancer drug is imitated and Study on mechanism
1. the cultivation of human hepatoma cell strain SMMC-7721 is gone down to posterity.
With people's hepatocarcinoma SMMC-7721 cell inoculation in the RPMI1640 culture fluid that contains 10% newborn calf serum, 100U/ml green grass or young crops/streptomycin, putting into 37 ℃ of 5%CO2 incubators cultivates, cell covers with the back with the cultivation of going down to posterity of 0.25% trypsinization, goes down to posterity once in every 3-5 days.
2. morphological observation.
To be inoculated in behind the cell dissociation in the 6 porocyte culture plates, put into and add Chinese walnut severe edema due to hypofunction of the spleen decoct after 37 ℃ of 5%CO2 incubators are cultivated 4h, 4 drug study groups and 1 PBS negative control group, a blank group are set, the medicine final concentration of experimental group is respectively 34mg/L, 17mg/L, 8.5mg/L, 1.7mg/L, negative control group adds 10 μ l PBS, the blank group does not add any component, and the variation of observation of cell form under inverted phase contrast microscope is taken pictures behind the continuation cultivation 24h.
3.MTT method is observed the Cortex Juglandis mandshuricae water decoction to the effect of human liver cancer cell SMMC-7721 inhibition of proliferation.
Cell dissociation is transferred cell concentration to 5 * 10 after making single cell suspension
7/ L, and be inoculated in each holes, centre of 96 orifice plates, the every hole 190 μ l of experimental group, the every hole 200 μ l of matched group, the zeroing group only adds 200 μ l RPMI1640 culture fluid, experimental group added the Cortex Juglandis mandshuricae water decoction after 37 ℃ of 5%CO2 incubators were cultivated 24h, medicine is by 2 times of dilutions, 8 drug study groups are set, every group 3 multiple hole, each 6 multiple hole of blank group and zeroing group are continued cultivation and are sopped up supernatant after 24 hours, add fresh RPMI1640 culture fluid of 80 μ l and 20 μ l MTT, lucifuge is outwelled supernatant after cultivating 4h, add 150 μ l DMSO, vibration makes behind the resolution of precipitate on microplate reader 490nm place detect absorbance, calculating suppression ratio=1-medicine group OD value/matched group OD value gently.
4. the flow cytometer method detects cell cycle.
Transfer cell concentration to 1 * 107/L behind the SMMC-7721 cell dissociation, be inoculated in 6 orifice plates, every hole 2ml, cultivate experimental group adding Cortex Juglandis mandshuricae water decoction after 24 hours, 4 experimental grouies and 1 matched group are set, and collecting cell after 24 hours is continued to cultivate in every group 3 multiple hole, wash cell one time with PBS, outwell supernatant, it is resuspended to add 1ml PBS, and 4 ℃ of 70% ice precooled ethanol are fixedly more than the 1h, centrifugal, it is resuspended to add 500 μ l PBS, adds the RNA enzyme 5 μ l of 1mg/ml, 37 ℃ hatch 10min after, the PI dyestuff 2 μ l that add 1mg/ml, lucifuge dyeing 30min.Back sample liquid is crossed 300 order nylon wires to remove impurity, 488nm excitation wavelength working sample, flow cytometry analysis cell cycle and detection apoptosis peak.
5. experimental result: in the MTT experiment, not medication group cell OD value is 0.6458 ± 0.0915, and 0.025g/ml medicine group OD value is 0.5963 ± 0.0639, cell inhibitory rate is 7.65%, 0.1g/ml medicine group OD value is 0.5788 ± 0.0942, cell inhibitory rate is 10.37%, when drug level reaches 3.2g/ml, the OD value is 0.2013 ± 0.1704, and cell inhibitory rate is 68.86%.The visible cell proliferation inhibition rate increases along with the rising of drug level, and the median lethal concentration IC50 of medicine is 1.56mg/ml as calculated.
Embodiment 2 Cortex Juglandis mandshuricae water decoctions are to BALB/c mouse liver cancer model tumor growth suppresses, tumor generates therapeutical effect and excite the research of immunization
1. the external Mus transplanted tumor of Cortex Juglandis mandshuricae water decoction suppresses experiment.
Get 50 female Mus of SPF level BALB/c, raise in the SPF environment.The SMMC-7721 cell culture gets 2 * 10 during to exponential phase
7Cell inoculation begins administration when tumor tissue grows to about 1 * 1cm under the right back butt of experimental mouse.Experiment is divided into negative control group (normal saline), Cortex Juglandis mandshuricae water decoction various dose group and positive controls (cyclophosphamide) at random with the BALB/c Mus.Injectable drug in the tumor body, 1 time every other day.Respectively at before the treatment, treatment back 1 week, 2 weeks, 3 weeks, 4 weeks peeled off the subcutaneous tumors piece, measure tumor body average external volume, it is heavy to measure tumor, calculates tumour inhibiting rate (%).
2. the Cortex Juglandis mandshuricae water decoction is to the research of liver cancer model experimental mouse hemogram systematic influence.
In the treatment experiment of BALB/c Mus transplanted tumor, respectively with administration before, 1 week, 2 weeks, 3 weeks are gathered experimental mouse tail blood after the administration, and with administration after 4 weeks put to death animals, pluck eyeball and get blood.Prepare the mixing anticoagulation that adds heparin after getting blood, carry out hemogram indexs such as leukocyte, erythrocyte, hemoglobin, lymphsystem respectively and detect.
3. the Cortex Juglandis mandshuricae water decoction is to the research of liver cancer model experimental mouse Immune Function.
1. prepare liver cancer model experimental mouse splenocyte and thymus cell suspension, be adjusted to desired concn and carry out that lymphocyte transformation, thymocyte cell are spontaneous mixes experiment.
2. experimental mouse prepares no anticoagulation after three, 2 methods are got blood, and puts into 4 ℃ of refrigerators and left standstill 24 hours, after operation preparation serum routinely.Get serum and carry out the active detection of lymphocyte factor (IL-2, IL-10), the active detection of IFN-, NK cytoactive mensuration, its determination techniques adopts the standard method of indirect elisa method to carry out.
Experimental result: it is as follows to detect wild type Cortex Juglandis mandshuricae antihepatocarcinoma effect result of the test through molecular biology method:
(1) morphocytology changes: observe visible experimental group cell shrinkage under inverted phase contrast microscope, cell volume diminishes, density is lower than matched group, the cell of experimental group death is more, therefore can see a lot of cell debriss, this phenomenon is seen shown in Figure 1 along with the increase of drug level is more and more obvious.
(2) cell growth curve: growth curve and cell survival rate that cell adds behind the Cortex Juglandis mandshuricae water decoction are seen Fig. 2 and Fig. 3 respectively.By Fig. 2 and Fig. 3 as can be seen: the cell proliferation rate of experimental group is slow than matched group, and the experimental group cell concentration of same time point is low than cellular control unit concentration, and experimental group was delayed than the matched group time during same concentration.The cell survival rate of experimental group is low than matched group, and these trend are along with the increase of drug level is more obvious.
(3) cell inhibitory effect experiment: variable concentrations medicine group OD value and suppression ratio see Table 1 and Fig. 4 respectively, and the increase OD value along with drug level reduces as seen from Figure 4, the suppression ratio increase.Calculating IC50 (medicine median lethal concentration) with formula is 1.56mg/ml.
Table 1MTT experimental result (X ± SD)
(4) Flow cytometry cell cycle result: the cell cycle different phase proportion that each experimental group and matched group record sees Table 2, and table 2 is respectively to organize cell cycle distribution situation data are made chi-square criterion of the SPSS11.5 statistical software result:
Table 2 cell cycle distribution situation
Compare with matched group,
*Be p＜0.05,
*Be p＜0.01,
* *Be p＜0.001
The result as seen, people's hepatocarcinoma SMMC-7721 cell volume dwindles, transparency reduces, the cell membrane shrinkage; Cell growth curve moves down to the right; The median lethal concentration IC50 of medicine is 1.56mg/ml; High concentration medicine can promote apoptosis, and cell cycle arrest is in S → G2 phase.The zoopery of hepatocarcinoma transplanted tumor BALB/c Mus shows that the Cortex Juglandis mandshuricae water decoction can suppress tumor growth, excites the immune system function and reach the purpose for the treatment of tumor.So the Cortex Juglandis mandshuricae water decoction has clear and definite antihepatocarcinoma effect.
Claims (3)
1. Wild Juglans mandshurica bark water extract that is used for the treatment of hepatocarcinoma, it is characterized in that described water extract is after Cortex Juglandis mandshuricae is added the water logging bubble 24h of 5 times of weight, to be heated to 100 ℃ of lasting 30min, filters, filtering residue repeats to extract once, twice filtrate is mixed and heated to concentrate making.
2. the application of the described Wild Juglans mandshurica bark water extract of claim 1 in preparation treatment liver-cancer medicine.
3. application as claimed in claim 2 is characterized in that being applied to prepare anti-hepatocarcinoma oral formulations, ejection preparation and other can be used for the pharmaceutical preparation of human body.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102274279A (en) * | 2011-06-29 | 2011-12-14 | 广东药学院 | Juglans mandshurica bark extract and application thereof in preparing anticancer drugs |
| CN102626113A (en) * | 2012-03-26 | 2012-08-08 | 王福 | Method for extracting pesticide from juglans mandshurica fruit green peels and leaves |
| CN115089627A (en) * | 2022-05-24 | 2022-09-23 | 吉林农业大学 | Walnut mountain ash flavone extraction and purification process |
-
2008
- 2008-10-17 CN CN200810218437A patent/CN101711790A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102274279A (en) * | 2011-06-29 | 2011-12-14 | 广东药学院 | Juglans mandshurica bark extract and application thereof in preparing anticancer drugs |
| CN102626113A (en) * | 2012-03-26 | 2012-08-08 | 王福 | Method for extracting pesticide from juglans mandshurica fruit green peels and leaves |
| CN115089627A (en) * | 2022-05-24 | 2022-09-23 | 吉林农业大学 | Walnut mountain ash flavone extraction and purification process |
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Open date: 20100526 |