CN102697871B - Chinese herbal preparation with liver protecting and enzyme reducing effects and production method of same - Google Patents
Chinese herbal preparation with liver protecting and enzyme reducing effects and production method of same Download PDFInfo
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- CN102697871B CN102697871B CN201210183992.7A CN201210183992A CN102697871B CN 102697871 B CN102697871 B CN 102697871B CN 201210183992 A CN201210183992 A CN 201210183992A CN 102697871 B CN102697871 B CN 102697871B
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Abstract
The invention belongs to the field of traditional Chinese medicine and relates to Chinese herbal preparation with hepatitis B virus resisting, liver protecting and enzyme reducing effects, and particularly relates to a Chinese herbal preparation which is made of extractive of Chinese knotweed (herb of polygonum Chinense L); and research results show that the Chinese herbal preparation can inhibit the secretion of HBsAg and HBeAg, and can remarkably lower blood serum ALT and MDA of an acute liver injured rat caused by carbon tetrachloride, and favorable liver protecting and enzyme reducing effects are displayed.
Description
Technical field
The invention belongs to tcm field, relate to and take Herb Polygoni Chinensis's (herb of polygonaceae plant Herb Polygoni Chinensis Polygonum chinenseL.) and prepare the method for Chinese medicine preparation and the anti-hepatitis virus of this Chinese medicine preparation and the liver protecting and ALT lowering results of pharmacodynamic test as raw material.
Background technology
Herb Polygoni Chinensis is the herb of polygonaceae plant Herb Polygoni Chinensis Polygonum chinense L..Its acrid in the mouth, hardship, cool in nature, poisonous, there is clearing away heat-damp and promoting diuresis, removing pathogenic heat from blood and toxic substance from the body, suppressing the hyperactive liver improving eyesight, the effect of relaxing muscles and tendons to promote blood circulation; Cure mainly dysentery, have loose bowels, laryngopharynx swelling and pain, diphtheria, pertussis, hepatitis, leukorrhagia, carbuncle, eczema, corneal nebula, traumatic injury etc. [the herbal editorial board of State Administration of Traditional Chinese Medicine China. China's book on Chinese herbal medicine. the 2nd volume. Shanghai: Shanghai science tech publishing house, 1999:647-649].Herb Polygoni Chinensis's herb resource is abundant, and China central and east and southwestern each province all produce, and is one of conventional Chinese herbal medicine among the people.
Relevant Herb Polygoni Chinensis's pharmacological action, it is few that existing Research Literature is reported.Mainly contain: antibacterial action [Hu Haibin. the research of " Resistant strain " Chinese herbal medicine screening test. Guangdong College of Medical Pharmacy's journal, 1990,6 (1): 40-41], antitumor [screening study of the conventional Chinese herbal medicine in the .50 kind Guangxi such as Wei Jinyu, strong medicine antitumor action. Guangxi traditional Chinese medicine college journal, 2003,6 (4): 3-7] and antivirus action [experimentation of the .60 kind Counteracting Hepatitis B Virus by Chinese Herbal Drugs such as Zhang Zheng. journal of Beijing Medical University, 1988,3 (20): 211-213].
To on April 26th, 2012, published, relevant with Herb Polygoni Chinensis patent documentation amounted to 45, and wherein patent of invention is 44, is all Chinese traditional compound medicine.The Chinese medicine preparation that the Herb Polygoni Chinensis of take is folk prescription exploitation, there is not yet patent literature.
In sum, the present invention has sufficient novelty, creativeness and practicality.
Summary of the invention
Inventor prepares a kind of ethanol extraction position with function for protecting liver and reducing enzyme activity from Herb Polygoni Chinensis, and this effective site is prepared into Chinese medicine preparation, and this there is not yet bibliographical information mistake at home.
Technical scheme of the present invention addressed from Herb Polygoni Chinensis extract the effective site with function for protecting liver and reducing enzyme activity method, this effective site is prepared into the method for Chinese medicine preparation and this Chinese medicine preparation is carried out to the zooperal method of the liver protecting and ALT lowering and result.
The extracting method of Herb Polygoni Chinensis's the liver protecting and ALT lowering effective site, there is not yet bibliographical information.Inventor finds by scientific research, and the effective ingredient of Herb Polygoni Chinensis's the liver protecting and ALT lowering is dissolved in more than 65% ethanol, therefore, adopts 65%～95% ethanol water reflux, extract, in invention.Because containing a large amount of pigments and chlorophyll in alcohol extract, the present invention adopts D
301resin removes.The effective site that extraction obtains, can be prepared into for oral Chinese medicine preparation, and as tablet, oral liquid, granule etc., the method for making of the corresponding dosage form that its method for making and document are recorded is general.The part by weight scope of effective site and pharmaceutic adjuvant is as follows: in preparation, containing Herb Polygoni Chinensis's extract 0.1%～99.9%, surplus is medicament molding adjuvant.
Compared with prior art, the outstanding substantive distinguishing features of the present invention and significant progress are: disclose first from Herb Polygoni Chinensis, extract the liver protecting and ALT lowering effective site method, this effective site is prepared into the method for tablet, oral liquid, granule and this Chinese medicine preparation is carried out to the zooperal method of the liver protecting and ALT lowering and result.
The specific embodiment
The following example is used for illustrating the present invention, is not any restriction to protection domain of the present invention.
One, be the examples of implementation that extract the liver protecting and ALT lowering effective site below:
Extract embodiment mono-: Herb Polygoni Chinensis's herb, dry, break into coarse powder, with 12 times, measure 70% ethanol water reflux, extract, 2 times, each 2 hours; Merge alcohol extract, filter; Filtrate is passed through D
301macroporous resin column (resin demand is 3 times of medical material weight), collects effluent, be evaporated to rare cream (medical material: concentrated solution=2: 1, W/V).
Extract embodiment bis-: Herb Polygoni Chinensis's herb, dry, break into coarse powder, with 15 times, measure 85% ethanol water reflux, extract, 2 times, each 2 hours; Merge alcohol extract, filter; Filtrate is passed through D
301macroporous resin column (resin demand is 3 times of medical material weight), collects effluent, be evaporated to thick paste (medical material: concentrated solution=4: 1, W/V), thick paste is dried, and breaks into fine powder (60-80 order).
Two, be effective site to be prepared into the examples of implementation of medicine below:
Pharmaceutical preparation embodiment mono-: by the yield of effective site, calculate, get the dry cream fine powder of effective site that is equivalent to 1000g Herb Polygoni Chinensis's crude drug, nearly weigh 24g, add starch to total amount 100g, granulate, be pressed into tablet, the heavy 0.3g of plain sheet, every containing Herb Polygoni Chinensis's crude drug 3g.
Pharmaceutical preparation embodiment bis-: by the yield of effective site, calculate, get the dry cream fine powder of effective site that is equivalent to 1000g Herb Polygoni Chinensis's crude drug, nearly weigh 35g, with Icing Sugar, to total amount 500g, granulate, obtain granule, subpackage, every parcel 5g, every parcel is containing Herb Polygoni Chinensis's crude drug 10g.
Pharmaceutical preparation embodiment tri-: calculate by the yield of effective site, get the rare cream of effective site that is equivalent to 1000g Herb Polygoni Chinensis's crude drug, the about 500ml of volume, with white sugar 1200g, heating for dissolving, finally adds water to 2000ml, filters, and obtains oral liquid, subpackage, every 10ml, every containing Herb Polygoni Chinensis's crude drug 5g.
Three, be pharmacological action test method and result below:
The protective effect of a pair of acute liver damage of pharmacological evaluation
1.1 grouping and administrations
The tablet (hereinafter referred to as " Linesless charcoal master slice ") of getting " pharmaceutical preparation embodiment mono-" gained is experimental subject.
Get 25 of SPF level healthy SD rats, male and female dual-purpose, body weight is 200+20g, laboratory rearing is stablized after 2d, is divided at random totally 5 groups of blank group, model control group, positive controls, Linesless charcoal master slice high dose group and Linesless charcoal master slice low dose group, 5 every group.Each organizes equal gastric infusion, every day 1 time, continuously 7d.Linesless charcoal master slice high dose group and low dose group gavage dosage are respectively 2.0gkg
-1, 1.0gkg
-1(pressing crude drug amount calculates), positive controls bifendate dosage is 0.05gkg
-1, blank group and model group give equal-volume distilled water.
1.2 modelings and drawing materials
After last administration 1h, blank group lumbar injection equal-volume Oleum Arachidis hypogaeae semen, all the other each groups are the molten 5mLkg of subcutaneous injection 60% carbon tetrachloride Oleum Arachidis hypogaeae semen respectively
-1, fasting be can't help after water 24h, extracts eyeball and gets blood, and blood is placed 1h, 3000rmin
-1centrifugal 3min, gets upper serum, preserves, for the mensuration of the biochemical indicators such as ALT, AST, MDA, SOD in-4 ℃ of refrigerators; Get hepatomegaly leaf texture fixing in 4% neutral buffered formalin solution, for section simultaneously.
1.3 date processing and statistical analysis
Every experimental data all with "
" represent, to relatively carrying out independent sample t check between group, with Excel2003 statistical software, add up.
1.4 result
1.4.1 Biochemical Indices In Serum
Compare with blank group, model group serum alt, AST and MDA activity all significantly raise, and SOD is active significantly to be reduced, and illustrates that model is successfully established; Compare with model group, Linesless charcoal master slice high and low dose group Serum ALT and MDA activity all obviously reduce compared with model group, and SOD all obviously raises compared with model group, and AST is active in obviously changing.Biochemical Indices In Serum result shows, Linesless charcoal master slice has protective effect to carbon tetrachloride-induced liver injury in rats, and prompting Linesless charcoal master slice may be by free radical resisting to hepatocellular protection, suppresses lipid peroxidation mechanism and plays a role.In Table 1.
The impact of table 1 Linesless charcoal master slice on carbon tetrachloride-induced liver injury in rats Biochemical Indices In Serum
*: compare P < 0.05, * * with model group: compare P < 0.01 with model group.
1.4.2 Histopathological Studies
The micro-Microscopic observation of histopathologic slide is visible, blank group hepatic tissue lobules of liver clear in structure, and hepatic cords, centered by central vein, is radial to surrounding, and hepatocyte is polygon, and core is large, nuclear membrane is clear, kernel is obvious; Model group liver tissue injury is serious, and lobules of liver structural fuzzy is unclear, and in lobule, hepatocyte extensive edema, cavity shape, have point-like and piecemeal necrosis, the inflammatory cell infiltrations such as companion's neutrophilic granulocyte, mononuclear cell, and pathological changes is distributed as master with ring central vein; Positive controls is slight hepatic cell edema around central vein, only has a small amount of little cavity shape pathological changes; Linesless charcoal master slice high and low dose group edema degree is slight, has little cavity shape pathological changes, without obvious tissue necrosis.Histopathologic slide's result shows, Linesless charcoal master slice has larger improvement to the hepatic necrosis degree of carbon tetrachloride-induced liver injury in rats.(figure slightly)
1.5 conclusion
Herb Polygoni Chinensis's ethanol extract has good protection effect to acute liver injury of rats.
Pharmacological evaluation two effect on hepatitics B virus in vitros
The preparation of 2.1 reagents
Preparation containing Herb Polygoni Chinensis's extract test solution: get the dry cream 1g of " extracting embodiment bis-" gained, add dimethyl sulfoxide 50mL, configuration concentration is 0.02gmL
-1medicinal liquid, under gnotobasis, with the micropore filter of 0.22 μ m, filter, then with cell culture fluid be mixed with respectively 16,8,4,2,1,0.5mgmL
-1deng the medicinal liquid of 6 variable concentrations, be placed in 4 ℃ of refrigerators and preserve, stand-by.
The preparation of PBS phosphate buffered solution: get PBS phosphate-buffered salt, with ultra-pure water, be mixed with the buffer solution of 0.01N (pH=7.2～7.4), under gnotobasis, with the micropore filter that diameter is 0.22 μ m, filter, be placed in 4 ℃ of refrigerators and preserve, stand-by.
The preparation of Digestive system: be mixed with 0.25% trypsin solution by PBS phosphate buffered solution, the micropore filter with 0.22 μ m under gnotobasis filters, and is placed in 4 ℃ of refrigerators and preserves, stand-by.
The recovery of 2.2HepG2.2.15 cell strain and cultivation
The HepG2.2.15 cell that liquid nitrogen freezing is preserved sucks in centrifuge tube after 37 ℃ of water-baths are melted, and adds the culture fluid of 10 times of amounts, mixes 1000rmin
-1low-speed centrifugal, removes supernatant, with culture fluid, cleans once again.Then cell strain is seeded in culture bottle, puts in cell culture incubator at 37 ℃, 5%C0
2under environment, grow, the every 1～3d of visual cell growing state changes a culture fluid.
In covering with the culture bottle of HepG 2.2.15 cell, remove culture fluid, add 0.25% pancreatin, 37 ℃ of digestion 3～4min, add culture fluid and blow and beat into gently single cell suspension, go down to posterity at 1: 3.Digestion again after cell covers with, with after cell counting count board counting, adding culture fluid, to be diluted to concentration be 1 * 10
4individual mL
-1cell suspension, be inoculated in 96 porocyte culture plates every hole 180 μ L, 37 ℃, 5%C0
2cultivate 24h, cell attachment is tested after growing into monolayer.
2.3 medicines are to Cytotoxic detection
The mtt assay of setting up with reference to Mosmann
[13]the inhibitory action of detection of drugs to HepG2.2.15 Growth of Cells.Cell attachment grows into after monolayer, arranges and adds respectively medicinal liquid 20 μ L, makes in culture fluid hole that final concentration is 1.6,0.8,0.4,0.2,0.1,0.05,0.025mgL
-1deng 7 Concentraton gradient groups, establish the cell matched group that does not add medicine simultaneously, every group is repeated 5 holes.After drug effect 72h, each hole adds MTT 20 μ L and continues to cultivate 4h.The supernatant that inclines, adds PBS buffer solution to clean 2 times, is inverted in absorbent paper and pats dry gently, guarantees the noresidue that exhausts.Every hole adds dimethyl sulfoxide 150 μ l, and 5min vibrates on agitator.By microplate reader, at 490nm place, measure the light absorption value (OD) in every hole, calculate cell-damaging rate (seeing formula 1), and use medicine inhibition concentration software for calculation 1.0.0 version (LOGIT method) to calculate the poisonous concentration TC of half of medicine
50.
2.4 antigen inhibitory action experiments
Experiment component Experimental agents group and cell matched group.HepG2.2.15 cell is made single cell suspension after digestion, regulates cell concentration to 1 * 10
4individual mL
-1, inoculation 96 well culture plates, every hole 180 μ L, 37 ℃, 5%C0
2cultivate 24h, cell is tested after growing up to monolayer.According to Drug toxicity trails result, take to cell is initial concentration without the concentration of overt toxicity, medicine gradient surely become 4 kinds of final concentrations 0.4,0.2,0.1,0.05mgmL
-1.Every concentration of every kind of medicine adds 3 holes, establishes not dosing cell simultaneously and contrasts 3 holes and culture fluid blank 3 holes, after drug effect 72h, collects each porocyte supernatant and is placed in 96 well culture plates, and-20 ℃ freezing standby.Adopt HBsAg, HBeAg content in ELISA method antigen diagnose reagent kit operation detection supernatant, calculate medicine to the inhibition percentage of antigen (the same destructive rate of method), and calculate medium effective concentration IC
50(the same TC of method
50, LOGIT method) and therapeutic index (TI), see formula 2:
TI=TC
50/ IC
50(formula 2)
2.5 date processing and statistical analysis
Mtt assay and ELISA method experimental data all with "
" represent, with Excel 2003 statistical softwares, add up.
2.6 result
2.6.1 the toxic action to HepG2.2.15 cell
With mtt assay, record the TC of Herb Polygoni Chinensis's alcohol extract to HepG2.2.15 cell strain
50for 1.322mgmL
-1the foundation that this result is selected as Experimental agents concentration.Under inverted microscope, observe, visible liquor strength is 1.6,0.8mgmL
-1time cell is had to overt toxicity, the dead cell of the adherent minimizing of visible cell, shrinkage, suspension increases; Medicinal liquid 0.4mgmL
-1following concentration is showed no overt toxicity, and cell growth state is good, and survival rate is higher, similar to cellular control unit existing state.Therefore, adopt 0.4mgmL
-1following concentration is carried out the impact experiment of HepG2.2.15 cell HBsAg, HBeAg secretion.The results are shown in Table 2.
The cytotoxicity of table 2 Herb Polygoni Chinensis ethanol extract
2.6.2 the effect to HepG2.2.15 cell HBsAg, HBeAg secretion
Measurement result shows that Herb Polygoni Chinensis has certain inhibitory action to the secretion of HBsAg, HBeAg after 72h in HepG2.2.15 cell strain is cultivated, and along with the increase of Herb Polygoni Chinensis's concentration, its inhibitory action strengthens, and is certain dose-effect relationship.Generally by TI value, evaluate the potential applicability in clinical practice of medicine, TI > 2 is effective low toxicity, and 1 < TI < 2 is for effectively poisonous, and TI < 1 is toxic action.Medicine is at the IC of HBsAg, HBeAg
50and TI value, in Table 3, table 4.
Table 3 Herb Polygoni Chinensis ethanol extract is in HepG2.2.15 cell culture
The inhibitory action of secretion HBsAg and HBeAg (
n=3)
*: compare P < 0.05, * * with cell matched group: compare P < 0.01 with cell matched group.
The evaluating drug effect result of the anti-HBV of table 4 Herb Polygoni Chinensis ethanol extract
2.7 conclusion
Herb Polygoni Chinensis is external has certain inhibitory action to HepG2.2.15 emiocytosis HBsAg, HBeAg.
Claims (1)
1. Chinese medicine preparation has the purposes in the medicine of function for protecting liver and reducing enzyme activity in preparation, it is characterized in that: by weight, Herb Polygoni Chinensis's extract shared ratio in Chinese medicine preparation is 0.5%～99.5%, and surplus is medicament molding adjuvant; The preparation method of Chinese medicine preparation is as follows: 1. do Herb Polygoni Chinensis's coarse powder, the ethanol water reflux, extract, that is 65%～95% by concentration; 2. alcohol extract flows through D
301macroporous resin column, to remove pigment and chlorophyll, collects effluent; 3. effluent reclaims solvent to rare cream; 4. in rare cream, add white sand Icing Sugar, dissolve, mix, make oral liquid; Or rare cream is further made to dry cream, and dry cream breaks into after fine powder and figuration auxiliary materials and mixing, makes tablet or granule.
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|---|---|---|---|---|
| US20050042314A1 (en) * | 2003-08-22 | 2005-02-24 | National Yang-Ming University | Extracts of Polygonum multiflorum Thunb., and preparation process and uses of the same |
| CN1672719A (en) * | 2004-03-24 | 2005-09-28 | 吕圭源 | Application of fleece flower root in preparing medicine for treating fatty liver |
| CN1823941A (en) * | 2005-12-29 | 2006-08-30 | 唐素筠 | Xianxiang medicine for treating hepatitis B and its production method |
| CN101041029A (en) * | 2007-04-24 | 2007-09-26 | 罗顺卿 | Chinese traditional medicine for curing hepatitis B |
| CN101810607A (en) * | 2009-02-25 | 2010-08-25 | 财团法人工业技术研究院 | Hepatitis C resistant plant extracts composition |
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| JP2001072583A (en) * | 1999-09-07 | 2001-03-21 | Sunstar Inc | Prophylactic or therapeutic composition for hyperlipemia |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050042314A1 (en) * | 2003-08-22 | 2005-02-24 | National Yang-Ming University | Extracts of Polygonum multiflorum Thunb., and preparation process and uses of the same |
| CN1672719A (en) * | 2004-03-24 | 2005-09-28 | 吕圭源 | Application of fleece flower root in preparing medicine for treating fatty liver |
| CN1823941A (en) * | 2005-12-29 | 2006-08-30 | 唐素筠 | Xianxiang medicine for treating hepatitis B and its production method |
| CN101041029A (en) * | 2007-04-24 | 2007-09-26 | 罗顺卿 | Chinese traditional medicine for curing hepatitis B |
| CN101810607A (en) * | 2009-02-25 | 2010-08-25 | 财团法人工业技术研究院 | Hepatitis C resistant plant extracts composition |
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| Title |
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| JP特开200172583A 2001.03.21 |
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