CN103989745A - A paper mulberry fruit extract product with anti-hepatoma activity and an extraction method thereof - Google Patents
A paper mulberry fruit extract product with anti-hepatoma activity and an extraction method thereof Download PDFInfo
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- CN103989745A CN103989745A CN201410199454.6A CN201410199454A CN103989745A CN 103989745 A CN103989745 A CN 103989745A CN 201410199454 A CN201410199454 A CN 201410199454A CN 103989745 A CN103989745 A CN 103989745A
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Abstract
A paper mulberry fruit extract product with anti-hepatoma activity is disclosed. An extraction method of the extract product includes: drying paper mulberry fruit in a drying oven, grinding the dried paper mulberry fruit into powder, adding petroleum ether according to a ratio of 1:10 (g/g), vibrating for 5 h. performing suction filtration, putting the filter residue in a fuming cupboard to remove the residue petroleum ether, collecting the degreased paper mulberry fruit, extracting the degreases paper mulberry fruit with eight times and six times of water for twice, performing suction filtration, collecting the filtrate, performing vacuum filtration for 5 times, combining the filtrates of the 5 times of filtration, performing vacuum concentration until the concentration is 1 g crude drug per mL, after the drug liquid is cooled naturally, slowly adding ethanol under stirring until the final concentration is 70%, sealing and refrigerating for 48 h, filtering, recovering the ethanol from the filtrate to obtain a purified liquid, and performing centrifugation to obtain the extract product. In-vitro and in-vivo pharmacological experiments prove that: the extract product can inhibit mouse transplanted hepatoma H22, can prolong the lifetime of a mouse with EAC ascitic tumor, and can inhibit the growth of the human hepatoma carcinoma cell HePG2.
Description
One, technical field
The present invention relates to field of Chinese herbal medicine technology, specifically relate to a kind of Fructus Broussonetiae extract and extracting method thereof with resisting liver cancer activity, it is a kind of active substance extracting from Fructus Broussonetiae, confirms to have resisting liver cancer activity by interior animal experiment and In vitro cell experiment.
Two, background technology
Primary hepatocarcinoma (HCC) is one of common malignant tumor of China, annual death rate occupies malignant tumor second, IARC (IARC) estimates global onset of liver cancer 56.4 ten thousand in 2000, PLC mortality 54.9 ten thousand, wherein Chinese onset of liver cancer 30.6 ten thousand, dead 30.0 ten thousand.There is data to show, liver cancer patient occurred after clinical symptoms about life cycle in average June, surgery alone expense is 20,000 yuan of left and right, also have postoperative infection, complication processing etc., expense is difficult to determine, chemotherapy or intervene operation 1 time are 10,000 yuan of left and right, if there are side reaction, complication, secondary disease etc., follow-up expense is huge.Although costly, curative effect is still very low, and within 5 years, survival rate is lower than 12%.Chinese medicine hepatocarcinoma has long-term clinical practice basis, by determination for the treatment of based on pathogenesis obtained through differentiation of symptoms and signs, improves on the whole patient's clinical symptoms, improves its quality of life, and from the Chinese medicine of natural plants, medicine source abundance, relative price is cheap, toxic and side effects is little, and patient compliance is good, has good DEVELOPMENT PROSPECT.
The inventor is engaged in the anti-liver cancer research of Chinese medicine for a long time, the clinical experience of the anti-hepatocarcinoma of development based on inheritance distinguished veteran doctors of TCM, the clinical of the effective proved recipe of multinomial anti-hepatocarcinoma and experiment subject study are carried out, and obtained patent of invention " a kind of dampness removing cancer therapy drug and preparation method and preparing the application in cancer therapy drug " in 2007, No. 358825th, certificate number, patent No. ZL200410041407.5.On the basis of early-stage Study, the inventor extracts a kind of active substance with resisting liver cancer activity from Chinese medicine Fructus Broussonetiae, and has passed through preliminary animal and cell experiment checking.
Chinese medicine Fructus Broussonetiae (FructusBroussonetiae) beginning is loaded in Han dynasty " Mingyi Bielu ", have another name called for Fructus Broussonetiae, Grains Shi, Grains, paper mulberry Fructus Persicae, for the dry mature fruit of moraceae plants Broussonetia papyrifera Broussonetia papyrifera (L.) Vent., main product in Henan, the ground such as Hubei, Shanxi, Gansu.Fructus Broussonetiae sweet in the mouth is cold in nature nontoxic, has the effects such as nourishing kidney tonifying YIN, liver heat removing and eyesight improving, inducing diuresis to remove edema, is usually used in treating the hepatic and renal YIN deficiency, osteopyrexia and fever, night sweat nocturnal emission, Light-headedness, blurred vision cataracta, edema distension, the diseases such as dysuria.Liu Hanji " property of medicine general examination " medium cloud: " Fructus Broussonetiae, impotence can be strong, edema can be moved back, and fills skin, helps waist knee joint, physical strength profiting, tonify deficiency labor, improving healthy complexion, strengthening bone and muscle, improving eyesight ".Fructus Broussonetiae fruit is containing alkaloid, Saponin (0.51%), vitamin B and oils and fats.Seed oil-containing 31.7%, contains unsaponifiable matter 2.67%, satisfied fatty acid 9.0%, oleic acid 15.0%, linoleic acid 76.0% in oil.In Fructus Broussonetiae, contain 17 seed amino acids, the highest with glutamic acid, arginine, aspartate content.Calculate with 100g drying sample powder, Fructus Broussonetiae total amino acids content is 12.44g, and essential amino acid total amount is 3.92g.Pharmacological research demonstration, Fructus Broussonetiae has the effect of antioxidation, anti-senile dementia, slow down aging, and Fructus broussonetiae oil can significantly reduce blood fat, prevention and cure of cardiovascular disease.Some pharmacological evaluation show, the Related Component of Fructus Broussonetiae has the effect of significant inhibition tumor cell growth, when Fructus Broussonetiae total alkaloids concentration 50 μ g/L, medicine is more obvious to the inhibitory action of growth of tumour cell, mtt assay records suppression ratio more than 20%, and colony forming method records suppression ratio more than 30%.And in the time that drug level reaches 100 μ g/L, Fructus Broussonetiae total alkaloids demonstrates comparatively significant inhibiting tumour cells effect, the inhibitory rate of cell growth of two kinds of methods is respectively more than 50% and 60%.
Three, summary of the invention
1, goal of the invention: a kind of Fructus Broussonetiae extract and extracting method thereof with resisting liver cancer activity is provided.
2, the present invention seeks to realize by following technical scheme:
There is a Fructus Broussonetiae extract for resisting liver cancer activity, it is characterized in that drying by Chinese medicine Fructus Broussonetiae the extract of pulverizing rear water extract-alcohol precipitation and obtain;
The extracting method of Fructus Broussonetiae extract is as follows:
(1) Fructus Broussonetiae is placed in to baking oven and dries, and by the Fructus Broussonetiae grind into powder after drying;
(2) Papermulberry Fruit is added to petroleum ether concussion 5h, then sucking filtration with the weight ratio of 1: 10;
(3) filtering residue is placed in fume hood and removes remaining petroleum ether, collect the Fructus Broussonetiae after defat;
(4) by defat Fructus Broussonetiae with 8 times, 6 times amount extracting in waters 2 times, then sucking filtration collect filtrate, leaching 5 times repeatedly, merges the filtrate of 5 acquisitions and is evaporated to 1g crude drug/mL;
(5), after medicinal liquid lets cool, slowly add while stirring ethanol, to final concentration be 70%, airtight cold preservation 48h, filter, filtrate recycling ethanol, obtains refined liquid, then row centrifugation obtain extract.
Described a kind of Fructus Broussonetiae extract with resisting liver cancer activity, is characterized in that this extract is a kind of polysaccharide component, can measure its content.
Described a kind of Fructus Broussonetiae extract with resisting liver cancer activity is in the application of preparing in Hepatoma therapy medicine.
3, beneficial effect:
The present invention is the extract obtaining from Chinese medicine Fructus Broussonetiae, for mouse bearing liver cancer H
22, its high, medium and low dosage is respectively 57.76%, 48.45%, 40.37% to the suppression ratio of tumor growth, for ehrlich carcinoma ascitic type mice, obviously extends life cycle of tumor-bearing mice with matched group phase specific energy.In vitro cell experiment studies show that, the high, medium and low dosage of this extract is to human hepatoma cell strain HepG
2suppression ratio be respectively 76.2%, 70.5%, 74.5%.
Four, detailed description of the invention
Embodiment 1: the extracting method of Fructus Broussonetiae extract
(1) Fructus Broussonetiae 2000g is placed in to baking oven and dries, and by the Fructus Broussonetiae grind into powder after drying;
(2) Papermulberry Fruit 2000g is added to petroleum ether 20Kg concussion 5h, then vacuum filtration with the weight ratio of 1: 10;
(3) filtering residue is placed in fume hood and removes remaining petroleum ether, collect the Fructus Broussonetiae after defat, recording weight is 1800g;
(4) 1800g defat Fructus Broussonetiae is added water 14.4L extracts after 1 time sucking filtration and collects filtrate, then the 10.8L that adds water extracts 1 time and collect filtrate, and leaching 5 times respectively, merges the filtrate of 5 acquisitions and be evaporated to 1g crude drug/mL;
(5), after medicinal liquid lets cool, slowly add while stirring ethanol, to final concentration be 70%, airtight cold preservation 48h, filter, filtrate recycling ethanol, obtains refined liquid, then row centrifugation obtain Fructus Broussonetiae extract.
Embodiment 2: determination of polysaccharide in Fructus Broussonetiae extract
1 preparation glucose standard reserving solution
The anhydrous glucose that accurately takes the dry constant weight of 1050C, reference substance 15mg, puts in 100ml volumetric flask, adds water to scale, shakes up and obtain glucose standard reserving solution, contains anhydrous glucose 0.15mg in every 1ml.
2 preparation standard curves
Precision measures reference substance solution 0.3,0.6,0.9,1.2,1.5ml, be placed in respectively 10ml volumetric flask, add deionized water 3.0ml, each precision adds 5% cresols solution 1ml, shakes up, precision adds sulphuric acid 5ml rapidly, shake up, place 10min, put in 400C water-bath and be incubated 15min, after taking-up, be cooled to rapidly room temperature, add and be settled to scale, shake up, taking corresponding actual in blank, according to ultraviolet visible spectrophotometry, measure absorbance at 490nm place, taking absorbance as vertical coordinate, concentration is abscissa, the standard curve of mapping to obtain, calculates regression equation.
3 calculate polyoses content
Get extract 0.2mg, accurately weighed, to put in 100ml volumetric flask, the jolting be diluted to scale of adding water, shakes up, and filters, and discards just filtrate.Precision measures filtrate 1ml, puts in 10ml volumetric flask, and the 3.0ml that adds water, measures absorbance according to front method, sample parallel assay 6 times, and substitution returns agenda, calculates polyoses content.
Embodiment 3: Fructus Broussonetiae extract is to mouse bearing liver cancer H
22the effect of growth
60 of Kunming kind healthy adult mices, male, body weight 18-22g, 10 of blank groups, with purgation modeling: by frozen ascites hepatoma cells strain Heps recovery, In vitro culture be passaged to carry out after exponential phase centrifugal, after PBS buffer rinses, 4 times of dilutions of normal saline, fully mix, subcutaneous injection 0.2ml under every mice right fore axillary fossa, has inoculated rear normal diet and has raised, after modeling, 24h is divided into model group, positive drug group (5-Fu, 20mg/kg), the high, medium and low dosage group of Fructus Broussonetiae extract by 50 mices according to table of random number method.Within after inoculation the 5th day, start administration, each group mice is with 0.1mL/10g body weight lumbar injection, 1 time every other day, administration 5 times altogether, model group gives normal saline, positive drug group gives 5-Fu by Mouse Weight 20mg/kg, and the high, medium and low dosage group of Fructus Broussonetiae extract dosage is respectively 100mg/kg, 200mg/kg, 400mg/kg.15d cervical vertebra dislocation method is put to death mice, plucks eyeball and gets blood.Detect Mouse Weight, tumor weight, tumour inhibiting rate, Serum ALT, AST, Urea, Crea, spleen and thymus index.
Before the medication of table 1 mice, medication mid-term, put to death before body weight change
unit: g)
The contrast of the each group of table 2 biochemical indicator
The each group of table 3 tumor weight, tumour inhibiting rate, spleen index and thymus index comparison
Embodiment 4: the impact of Fructus Broussonetiae extract on EAC ascites tumor survival time of mice
50 of Kunming kind healthy adult mices, are male, and average weight 18-22g, is divided into model group, positive drug group (5-Fu), the high, medium and low dosage group of Fructus Broussonetiae extract according to table of random number method.The strain of EAC tumor is gone down to posterity once for 7-10 days, after abdominal cavity is transmitted 3 times, starts formal experiment.Under aseptic condition, select the rear 7-10 days good tumor kind of the general status animals of inoculation, to lie on the back fixing, sterilization skin, extracts ascites, becomes the solution of 1: 3 with 0.9% normal saline dilution, and adjusting cell number is 2 × 10
6individual/mL, every mouse peritoneal injection 0.2ml.Modeling plays administration on the 2nd day, each group mice is with 0.1mL/10g body weight lumbar injection, 1 time every other day, administration 5 times altogether, model group gives normal saline, positive drug group gives 5-Fu by Mouse Weight 20mg/kg, and the high, medium and low dosage group of Fructus Broussonetiae extract dosage is respectively 100mg/kg, 200mg/kg, 400mg/kg.Observe the ordinary circumstance of each group of mice, timing is weighed, and records each group of mice survival day, calculates increase in life span, and detection of drugs is on the tumor animal impact of life cycle.
The each group of table 4 Mouse Weight changes contrast (g)
The contrast of the each group of table 5 survival time of mice
Embodiment 5: Fructus Broussonetiae extract is to human liver cancer cell HepG
2the impact of growth vigor
1 medicine and instrument
1.1 object cell: HepG
2; Culture medium: DMEM+10%FBS+1%P/S+1%L-Glntamine.
1.2 medicines: Fructus Broussonetiae extract, 5-FU (traditional Chinese medicines, article No.: 6500761).
1.3CCK-8 detection kit (dojindo, CK04).
1.4 microplate reader: Thermo, MuLTiSKANMK3.96 orifice plates (NEST, article No. 701001).
2 experimental techniques
2.1 cell culture
Hep G
2cell is placed in DMEM+10%FBS+1%P/S+1%L-Glntamine culture medium, 5%CO
2, in 37 DEG C of CO2 gas incubator, cultivate.
2.2 medicine preparations
2.2.1 positive control drug
5-FU: take 10mg, dissolve preparation with 1mlDMSO, get this solution 100 μ l, being diluted to concentration with PBS is that the mother solution of 1ug/ μ l is for subsequent use.
2.2. experimental drug
Take 0.24g Fructus Broussonetiae extract in 5mlDMEM minimal medium, after high pressure steam sterilization, centrifuging and taking supernatant, now concentration is 30mg/ml.
2.3 medicines are to human liver cancer cell HepG
2the impact of growth vigor
Cell suspension (cell number is about 70000/ml) is inoculated in to (7000 cells/well) in 96 culture plates, and 37 DEG C of 5%CO2 incubators are cultivated, cell Taking Pictures recording before dosing.Fructus Broussonetiae extract is added to Tissue Culture Plate, reach final concentration 0.3mg/ml, 0.75mg/ml, 3mg/ml.The concentration of 5-FU is 10 μ g/ml.Drug treating 24h, 48h, then Taking Pictures recording.After drug treating cell 24h, 48h, every hole adds 10ulCCK-8, mixes in rear incubator and hatches 3 hours, measures the light absorption value at 450nm place.Microplate reader reads testing sample and blank in the OD at 450nm place value, and the OD value of each sample to be tested is designated as to measured value, and the OD value of blank is designated as blank value, final value=measured value-blank value.
3 results
The high, medium and low dosage of Fructus Broussonetiae extract is respectively 51.39%, 40.58%, 36.77% to the suppression ratio 24h of HepG2 cell lines, and 48h is respectively 76.2%, 70.5%, 74.5%.
Human liver cancer cell HepG after table 6 drug treating
2growth inhibited
Claims (3)
1. there is a Fructus Broussonetiae extract for resisting liver cancer activity, it is characterized in that drying by Chinese medicine Fructus Broussonetiae the extract of pulverizing rear water extract-alcohol precipitation and obtain;
The extracting method of Fructus Broussonetiae extract is as follows:
(1) Fructus Broussonetiae is placed in to baking oven and dries, and by the Fructus Broussonetiae grind into powder after drying;
(2) add petroleum ether to shake 5 h, then vacuum filtration with the weight ratio of 1: 10 Papermulberry Fruit;
(3) filtering residue is placed in fume hood and removes remaining petroleum ether, collect the Fructus Broussonetiae after defat;
(4) by defat Fructus Broussonetiae with 8 times, 6 times amount extracting in waters 2 times, then sucking filtration collect filtrate, leaching 5 times repeatedly, merges the filtrate of 5 acquisitions and is evaporated to 1g crude drug/mL;
(5), after medicinal liquid lets cool, slowly add while stirring ethanol, to final concentration be 70%, airtight cold preservation 48h, filter, filtrate recycling ethanol, obtains refined liquid, then row centrifugation obtain extract.
2. a kind of Fructus Broussonetiae extract with resisting liver cancer activity according to claim 1, is characterized in that this extract is a kind of polysaccharide component, can measure its content.
As claimed in claim 1 a kind of Fructus Broussonetiae extract with resisting liver cancer activity in the application of preparing in Hepatoma therapy medicine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105079339A (en) * | 2015-09-21 | 2015-11-25 | 马根全 | Traditional Chinese medicinal composition for treating liver cancer |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102133334A (en) * | 2011-03-08 | 2011-07-27 | 山东大学威海分校 | Preparation method of compound fructus broussonetiae preparation and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102133334A (en) * | 2011-03-08 | 2011-07-27 | 山东大学威海分校 | Preparation method of compound fructus broussonetiae preparation and application thereof |
Non-Patent Citations (3)
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| 孙杰: "响应面法提取楮实子多糖研究", 《食品研究与开发》 * |
| 张静: "中药楮实子的研究现状与展望", 《中华中医药学刊》 * |
| 邵金华: "微波法提取楮实子多糖的工艺研究", 《氨基酸和生物资源》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105079339A (en) * | 2015-09-21 | 2015-11-25 | 马根全 | Traditional Chinese medicinal composition for treating liver cancer |
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Application publication date: 20140820 |