CN102342984A - Novel use of coptis total alkaloid extract - Google Patents
Novel use of coptis total alkaloid extract Download PDFInfo
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- CN102342984A CN102342984A CN2010102463673A CN201010246367A CN102342984A CN 102342984 A CN102342984 A CN 102342984A CN 2010102463673 A CN2010102463673 A CN 2010102463673A CN 201010246367 A CN201010246367 A CN 201010246367A CN 102342984 A CN102342984 A CN 102342984A
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Abstract
The invention discloses a novel use of coptis total alkaloid extract in preparation of drugs for preventing and treating hepatic fibrosis and liver cancer generation and metastasis. The invention further discloses a preparation method of the coptis total alkaloid extract, and a pharmaceutical composition containing the coptis total alkaloid extract.
Description
Technical field
The present invention relates to medical technical field purposes, be specifically related to the application of Rhizoma Coptidis total alkaloids extract in the medicine of preparation prevention and treatment hepatic disease, particularly hepatic fibrosis and/or hepatocarcinoma generation and transfer.
Background technology
Along with the raising of living standards of the people, living habit irregular, tired, tobacco and wine are excessive, cause the sickness rate of hepatopathy constantly to rise.Research for hepatopathy in recent years discloses, and the common pathologic basis of multiple chronic hepatopathy is hepatic fibrosis.If without diagnosis in time, reasonably treatment; Because of the impaired hepatocyte of inflammation is difficult for repairing even increasing the weight of damage, less and less until normally functioning hepatocyte, cause hepatic insufficiency at last; Develop into liver cirrhosis even hepatocarcinoma, serious threat people's life quality and life security.So, should strengthen the input of fund and technology, adopt too many levels, multi-level, many target spots means to intervene, open up a kind of new method of treating hepatic fibrosis and hepatocarcinoma safely and effectively.
" Chinese pharmacopoeia (2005 editions) is recorded the dry tuber of these article for ranunculaceae plant Rhizoma Coptidis Coptis chinensisFranch., Coptis deltoidea C.Y.Cheng et Hsiao Coptis deltoidea C.Y.Cheng et Hsiao or Coptis Teeta Wall Coptis teeta Wall., practises and claims to be respectively " flavor connects ", " refined company " and " Coptis Teeta Wall ".Mainly contain various alkaloids in the Rhizoma Coptidis, wherein the highest with berberine (berberine) content, be about 5.56%～7.25%.Include in " the total alkaloids composition of three kind Rhizoma Coptidis of Chinese pharmacopoeia is comparatively similar.Rhizoma Coptidis has antimicrobial, protozoacidal effect; Berberine can excited heart in the Rhizoma Coptidis, increases coronary flow, and when heavy dose, shows as inhibitory action; Berberine has protective effect to myocardial ischemia and myocardial infarction simultaneously, also can be to arrhythmia; The effect that berberine has maincenter to suppress causes dyspnea; Berberine can also be to anti-experimental character diarrhoea and gastric mucosa injury and ulcer, and effects such as antiinflammatory are arranged.
Pharmacological research shows that the inventor has obtained satisfied effect with the prevention of Rhizoma Coptidis total alkaloids extract and treatment hepatic fibrosis and hepatocarcinoma, has found the new purposes of Rhizoma Coptidis and the total bio-extract of Rhizoma Coptidis.
Summary of the invention
An object of the present invention is to provide the application of Rhizoma Coptidis total alkaloids extract in the medicine of preparation prevention and treatment hepatic disease.
The present invention preferably provides the application of Rhizoma Coptidis total alkaloids extract in the medicine of preparation prevention and treatment hepatic fibrosis.
The present invention also preferably provides the application of Rhizoma Coptidis total alkaloids extract in the medicine of preparation prevention and treatment hepatocarcinoma.
Rhizoma Coptidis total alkaloids extract of the present invention is to extract according to the conventional method that this area is used to extract Rhizoma Coptidis total alkaloids.Rhizoma Coptidis total alkaloids extract of the present invention is with Rhizoma Coptidis; Like Coptis deltoidea C.Y.Cheng et Hsiao Coptisdeltoidea C.Y.Cheng et Hsiao or Coptis Teeta Wall Coptis teeta Wall. is raw material, extracts according to following method to obtain: get a certain amount of medicinal raw material and add a certain amount of water; Under heating, extract one or many; Be concentrated into driedly after extracting solution merges, promptly get the Rhizoma Coptidis total alkaloids extract.
Preferably; Rhizoma Coptidis total alkaloids extract of the present invention is with Rhizoma Coptidis; Like Coptis deltoidea C.Y.Cheng et Hsiao Coptisdeltoidea C.Y.Cheng et Hsiao or Coptis Teeta Wall Coptis teeta Wall. is raw material; Obtain according to following method extraction: get the water that a certain amount of medicinal raw material adds 5-20 times of weight; Under decocting under the boiling water state, extract more than 2 times, each more than 0.5 hour; Be concentrated into driedly after extracting solution merges, promptly get the Rhizoma Coptidis total alkaloids extract.
More preferably; Rhizoma Coptidis total alkaloids extract of the present invention is with Rhizoma Coptidis; Like Coptis deltoidea C.Y.Cheng et Hsiao Coptisdeltoidea C.Y.Cheng et Hsiao or Coptis Teeta Wall Coptis teeta Wall. is raw material; Obtain according to following method extraction: get the water that a certain amount of medicinal raw material adds 8-15 times of weight; Extract 2-4 time each 0.5-2 hour down at 100 ℃; Be concentrated into driedly after extracting solution merges, promptly get the Rhizoma Coptidis total alkaloids extract.
Most preferably; Rhizoma Coptidis total alkaloids extract of the present invention is with Rhizoma Coptidis; Like Coptis deltoidea C.Y.Cheng et Hsiao Coptisdeltoidea C.Y.Cheng et Hsiao or Coptis Teeta Wall Coptis teeta Wall. is raw material; Obtain according to following method extraction: get the water that a certain amount of medicinal raw material adds 10 times of weight; Extract 2 times each 1 hour down at 100 ℃; Be concentrated into driedly after extracting solution merges, promptly get the Rhizoma Coptidis total alkaloids extract.
Preferably, the tuber with above-mentioned Rhizoma Coptidis is a raw material.
Preferably, the ratio of weight and number of said medical material and water is 1: 10; Extraction time is 1 hour.
Contain berberine Alkaloid chemical compounds such as magnoflorine, Columbamine, jateorhizine, coptisine, palmatine, epiberberine and berberine in the Rhizoma Coptidis total alkaloids extract of the present invention.
Pharmacological experiment study shows that above-mentioned total alkaloids extract can effectively be improved hepatic fibrosis and the hepatic injury that carbon tetrachloride causes; And suppress the growth and the migration of hepatoma carcinoma cell, can be used for prevention and treatment hepatic fibrosis and hepatocarcinoma.
Rhizoma Coptidis total alkaloids extract of the present invention can use separately, perhaps is equipped with other drug and processes compound preparation.
The present invention further provides the application of the pharmaceutical composition that comprises above-mentioned Rhizoma Coptidis total alkaloids extract in the medicine of preparation treatment or prevention hepatic fibrosis and/or hepatocarcinoma.Said pharmaceutical composition can further contain acceptable accessories.Said preparation of pharmaceutical compositions can be become various dosage forms as required, for example: the dosage form of oral administration such as tablet, capsule (comprising hard capsule, soft capsule and microcapsule), powder, pill, granule and syrup; The dosage form of non-oral administration such as injection, suppository, gel and patch.Except that these regular dosage forms; Oral fast release solid formulation (for example tablet, granule etc.) and the slow releasing preparation (tablet, granule, fine granular, pill, capsule, syrup, Emulsion, suspension, solution) that is used for oral or non-oral administration the present invention can also be used for, also these preparations can be prepared through conventional method.Preparation among the present invention can be the coating or the form of coating not, depends on the needs.The present invention particularly preferably is the dosage form that this pharmaceutical composition is used for oral administration.As acceptable accessories; Comprise the various excipient that are used for solid preparation, lubricant, binding agent, disintegrating agent, stabilizing agent, foaming agent, coating materials etc.; Or be used for solvent, solubilizing agent, suspending agent, isotonic agent, buffer agent, emollient, emulsifying agent of semi-solid preparation, liquid preparation etc.; In addition, also can use other medical additive such as antiseptic, antioxidant, coloring agent, sweeting agent and flavoring agent etc. as required.
The dosage of Rhizoma Coptidis total alkaloids of the present invention; Because of the difference of patient's state, body weight, administering mode etc. there are differences; For example under non-oral administration, can be 0.05-200mg/kg/ days, preferred 0.1-100mg/kg/ days like intramuscular, intravenous, enteral administration; During oral administration, be 0.1-600mg/kg/ days, preferred 1-400mg/kg/ days.
Description of drawings
Fig. 1 Rhizoma Coptidis total alkaloids extract is to CCl
4Cause the preventive and therapeutic effect serum biochemistry index of rat liver fibrosis; (A) serum AST index; (B) Serum ALT index; (C) SOD in serum index;
Fig. 2 Rhizoma Coptidis total alkaloids extract causes the pathological examination of the preventive and therapeutic effect of rat liver fibrosis to CCl4; (A) normal rats; (B) model group rat; (C) Rhizoma Coptidis total alkaloids extract low dose group rat; (D) dose groups rat in the Rhizoma Coptidis total alkaloids extract; (E) Rhizoma Coptidis total alkaloids extract high dose group rat;
Fig. 3 Rhizoma Coptidis total alkaloids extract is to the inhibitory action of hepatoma cell strain MHCC-97L growth.Wherein the application concentration of Rhizoma Coptidis total alkaloids extract is the molar concentration of calculating with the amount of berberine.
Fig. 4 Rhizoma Coptidis total alkaloids extract is to the inhibitory action of the hepatoma carcinoma cell MHCC-97L migration of height migration; (A) the Rhizoma Coptidis total alkaloids extract can show and suppress the migration in the plane of MHCC-97L cell; (B) the Rhizoma Coptidis total alkaloids extract can show and suppress the migration that the MHCC-97L cell passes extracellular matrix.Wherein the application concentration of Rhizoma Coptidis total alkaloids extract is the molar concentration of calculating with the amount of berberine.
The specific embodiment
Below further specify the present invention through embodiment, but be not used in the present invention is imposed any restrictions.
The preparation of embodiment 1 Rhizoma Coptidis total alkaloids extract
The Rhizoma Coptidis medical material picks up from Chinese Sichuan Province, and " Chinese pharmacopoeia (2005 editions) [Rhizoma Coptidis] item is contained medical material down through being accredited as.The 0.5kg medical material is added in the 5kg water in 1: 10 ratio, be heated to 100 ℃ of boiling water and decocted 1 hour, filter, filtering residue repeats to decoct once by above-mentioned condition.Merging filtrate is evaporated to driedly on Rotary Evaporators, promptly gets Rhizoma Coptidis total alkaloids extract 40g.With high performance liquid chromatography-mass spectrometry method the Rhizoma Coptidis total alkaloids extract is carried out component analysis; And adopting the standard substance comparison, the result shows the extract obtained berberine Alkaloid chemical compounds such as magnoflorine, Columbamine, jateorhizine, coptisine, palmatine, epiberberine and berberine that contain; Adopting the magnoflorine in the high effective liquid chromatography for measuring Rhizoma Coptidis total alkaloids extract is 1.44%; Columbamine is 1.79%, and jateorhizine is 5.13%, coptisine is 9.84%, and palmatine is 15.79%; Epiberberine is 8.99%, and berberine is 53.73%.
Embodiment 2 Rhizoma Coptidis total alkaloids extracts are to CCl
4
The effect of inductive hepatic fibrosis rats
1, detection method
32 180～220g male SD rats are divided into the Rhizoma Coptidis total alkaloids extract group of extracting among negative control group and model control group, positive control silymarin group, the embodiment 1,8 every group at random by body weight.The model group rat carries out pathology detection in respectively getting 2 rats in the 4th week, the 5th week, the prediction degree of hepatic fibrosis.The Rhizoma Coptidis total alkaloids extract group dosage that extracts among positive control drug and the embodiment 1 is respectively 150mg/kg body weight, 120mg/kg body weight.Negative control group is 2 subcutaneous injection 3mg/kg olive oil weekly, and all the other each groups are 2 subcutaneous injection 50%CCL weekly
4Olive oil 3mg/kg body weight, negative control group and model group are pressed 10ml/kg every day and are irritated the stomach normal saline, and all the other each groups are irritated the stomach relative medicine every day by each group dosage.In 6 weeks of successive administration, after 1 hour, under the etherization situation, the eye socket venous plexus is taken a blood sample in the last administration, and centrifuging and taking serum detects ALT, AST in full automatic biochemical apparatus.Dissect and take out hepatic tissue, half puts-80 ℃ of preservations, is used for detecting hepatic tissue hydroxyproline and SOD, MDA, GSH, GSH-PX content.Other gets half puts 10% formaldehyde fixed, does pathology detection.
2, hepatic tissue biochemical analysis
Superoxide dismutase (SOD): relatively significance reduction (P＜0.05) of the activity of SOD and negative control group in the model group liver tissues of rats; The activity of SOD and model control group compare in the silymarin group liver tissues of rats, and significance raises; The activity of SOD and model control group compare in the Rhizoma Coptidis total alkaloids extract group liver tissues of rats, significance rising (P＜0.05).
Malonaldehyde (MDA): relatively significance rising (P＜0.05) of the content of MDA and negative control group in the model group liver tissues of rats.The content of MDA and model group relatively there are no significant difference (P＞0.05) in silymarin group, the Rhizoma Coptidis total alkaloids extract group liver tissues of rats.
Glutathione (GSH): relatively significance reduction (P＜0.05) of the concentration of GSH and negative control group in the model group liver tissues of rats; Relatively significance rising (P＜0.05) of the concentration of GSH and model control group in silymarin group, the Rhizoma Coptidis total alkaloids extract group liver tissues of rats.
Glutathione peroxidase (GSH-PX): relatively significance reduction (P＜0.05) of the activity of GSH-PX and negative control group in the model group liver tissues of rats.Relatively significance rising (P＜0.05) of the activity of GSH-PX and model control group in silymarin group, the Rhizoma Coptidis total alkaloids extract group liver tissues of rats.
Hydroxyproline (Hyp): relatively significance rising (P＜0.01) of the content of Hyp and negative control group in the model group liver tissues of rats.The content of Hyp and model control group compare there was no significant difference (P＞0.05) in the silymarin group liver tissues of rats, relatively significance reduction (P＜0.05) of the content of Hyp and model control group in the Rhizoma Coptidis total alkaloids extract group liver tissues of rats.
Table 1, each group of drugs on liver fibrosis in rat liver tissue CCl4 biochemical test results of
Annotate: compare with negative control group,
△P＜0.05;
△ △P＜0.01; Compare with model control group,
*P＜0.05;
*P＜0.01.(down together)
3, serum biochemistry detects
Relatively significance rising (P＜0.05) of the level of ALT, AST and negative control group in the model control group rat blood serum.The level of ALT, AST and model control group compare in silymarin group, the Rhizoma Coptidis total alkaloids extract group rat blood serum, and there are no significant reduces (P＜0.05).
Table 2 in each group of drugs on Hepatic Fibrosis blood biochemical test results
4, pathological examination
1) test item: film-making, HE dyeing, histopathologic examination
2) detect foundation: nonstandard method " histopathologic examination " (GDMLAC/OG900-A/1).
3) detection method
Film-making: the pathological tissue specimen after drawing materials, fix, repair piece, flowing water flushing, dehydration, transparent, waxdip, paraffin embedding, paraffin section, HE dyeing, mounting.
Read sheet: whether observation hepatic tissue lobules of liver structure is disorderly, hepatocellular degeneration, necrosis, pathological changes such as portal area interstitial edema, proliferation of fibrous tissue and cell infiltration.The relevant document of reference [Fan Mingxia, Zhou Kangrong, Shen Jizhang etc. rat CCl
4The Liver Fibrosis Model Mn-DPDP MR research of bringing out [J]. clinical roentgenology magazine, 2004,23 (8): 722-726; Ceng Minde, Wang Tailing, Wang Baoen. liver fibrosis diagnosis and curative effect assessment common recognition [J]. liver, 2002,7 (2): 3-4], hepatic fibrosis is carried out by stages: So: no hepatic fibrosis; S1 (1 grade of hepatic fibrosis): no portal area enlarges fibrosis; S2 (2 grades of hepatic fibrosis): fibrosis or fibrous septum form around the portal area, and leaflet structure keeps; S3 (3 grades of hepatic fibrosis): fibrosis companion leaflet structure is disorderly, no liver cirrhosis; S4 (4 grades of hepatic fibrosis): possible or form with liver cirrhosis certainly.
Statistical analysis: every part of specimen choose successively under the low power field (10 * 10) upper left, upper right a, left side down, bottom right, middle 5 visuals field (common 1.5mm
2) observe, measure and calculate the gross area of fibrous tissue in the visual field.
4) liver outward appearance
The negative control group liver surface is dark red, and the edge is sharp keen, exquisite quality; The model control group liver is lark, and is full coarse, and there is the tuberosity that differs in size on the surface, and matter is hard, and toughness is big; Each medication group color and quality fall between.
5) om observation (result as shown in Figure 2)
Negative control group: detect liver organization 8 examples.Lobules of liver clear in structure under the mirror, the interior hepatic cords of lobule is arranged more neat, and the hepatocyte size is even, no degeneration, necrosis, the portal area does not enlarge, and does not see proliferation of fibrous tissue in the liver.With reference to the pathology classification, the hepatic fibrosis check result is So (8 example).At the average 1.5mm of low power field (10 * 10)
2The fibrous tissue gross area 0.010 ± 0.006mm2 in the scope.
Model control group: detect liver organization 8 examples.The hepatic cords arrangement disorder of hepatic tissue under the mirror, steatosis widely appears in hepatocyte, visible massive inflammatory cells infiltrated around portal area and the central vein, proliferation of fibrous tissue is obvious.With reference to the pathology classification, hepatic fibrosis result by stages is S2 (3 example), S3 (4 example), S4 (1 example), at the average 1.5mm of low power field (10 * 10)
2The fibrous tissue gross area 0.090 ± 0.021mm in the scope
2
Silymarin group: detect liver organization 8 examples.Hepatic tissue destructiveness, hepatic cell fattydegeneration and downright bad degree, inflammation active level and proliferation of fibrous tissue degree weight do not wait under the mirror, and the visible pathological manifestations of 2 examples obviously alleviates than model control group.With reference to the pathology classification, the hepatic fibrosis check result is S1 (2 example), S2 (3 example), S3 (2 example), S4 (1 example).At the average 1.5mm of low power field (10 * 10)
2The fibrous tissue gross area 0.067 ± 0.046mm in the scope
2
Rhizoma Coptidis total alkaloids extract group: detect liver organization 8 examples.Hepatic tissue destructiveness, hepatic cell fattydegeneration and downright bad degree, inflammation active level and proliferation of fibrous tissue degree totally alleviate than model group under the mirror, and the visible pathological manifestations of 3 examples obviously alleviates.With reference to the pathology classification, the hepatic fibrosis check result is S1 (3 example), S2 (2 example), S3 (3 example).At the average 1.5mm of low power field (10 * 10)
2The fibrous tissue gross area 0.041 ± 0.020mm in the scope
2
6) experimental result statistics
Adopt the SPSS16.0 statistical software, relatively adopt One-Way ANOVA to analyze between group, comparative result is seen table 3.
Table 3 liver fibrosis in each group comparison
Annotate: compare with negative control group,
△ △P＜0.01; Compare with model group,
*P＜0.05 draw
*P＜0.01.
Visible by table 3, model control group rat liver fibrosis area and negative control group relatively have significant difference (P＜0.01), results suggest CCl
4Bringing out the rat liver fibrosis modeling is successful on the whole.Silymarin group rat liver fibrosis degree alleviates than model control group to some extent, but there was no significant difference (P＞0.05).Rhizoma Coptidis total alkaloids extract group rat liver fibrosis area and model control group relatively have significance to dwindle (P＜0.05), and the result shows that the Rhizoma Coptidis total alkaloids extract can effectively dwindle the area of rat liver fibrosis.
5, conclusion
Comprehensive above-mentioned research shows that under this experimental condition, the Rhizoma Coptidis total alkaloids extract has preventive and therapeutic effect to the rat liver fibrosis model of tetrachloro-methane induction.
The Rhizoma Coptidis total alkaloids extract of embodiment 3 various dose is to CCl
4
The effect of inductive hepatic fibrosis rats
50 of male SD rats; Body weight 200～220g; Be divided into five groups at random; Be respectively normal group A; Model group B; The Rhizoma Coptidis total alkaloids extract low dose group C that extracts among the embodiment 1, dose groups D in the Rhizoma Coptidis total alkaloids extract that extracts among the embodiment 1, the Rhizoma Coptidis total alkaloids extract high dose group E that extracts among the embodiment 1.Wherein, B, C, D and E organize rat lumbar injection 0.15ml CCl weekly
4, continued for 8 weeks; A group rat is lumbar injection equivalent vegetable oil weekly, continues for 8 weeks; The Rhizoma Coptidis total alkaloids extract 400,600 that extracts among C, D and E group rat difference oral administration gavage embodiment every day 1,800mg/kg continued for 8 weeks; A and B group rat are irritated stomach equivalent distilled water respectively.
The pathology index of monitoring in the present embodiment comprises: a. blood parameters: serum AST, ALT, SOD enzyme activity; B. pathological examination: paraffin section, the situation of hepatic fibrosis and hepatic necrosis is observed and is estimated in HE dyeing.Each treated animal pathology quantitatively evaluating is as shown in table 4.
Each treated animal pathology quantitatively evaluating of table 4
The result shows that the Rhizoma Coptidis total alkaloids extract can show reduction CCl
4The serum alanine transaminase (ALT) that causes, the rising of aspartate transaminase (AST); The vigor of ultra qi peroxidase (SOD) in the rising serum shows that the Rhizoma Coptidis total alkaloids extract can effectively improve CCl
4Hepatic fibrosis that causes and hepatic injury, and the effectiveness of Rhizoma Coptidis total alkaloids extract strengthens (result is as shown in Figure 1) with the rising of dosage.
Embodiment 4 Rhizoma Coptidis total alkaloids extracts are to the effect of the inductive hepatic fibrosis rats of bile duct ligation
1, detection method
47 200～250g male SD rats are divided into 9 of the Rhizoma Coptidis total alkaloids extract groups extracted among 11 of 8 of negative control group, 8 of sham operated rats, 11 of model control group, silymarin group (comprise later stage do for supplement 3), the embodiment 1 at random by body weight.The model group rat is pressed the anesthesia of 10mg/kg body weight lumbar injection 3% pentobarbital sodium, the skin of abdomen sterilization, and the sterile working cuts off the abdominal cavity along the abdominal part median line, isolates bile duct, at bile duct near-end and far-end 2 places ligation bile duct.To cause the bile duct obstruction rat model; Sham operated rats is cut off abdominal part under the anesthesia situation, do not carry out ligation; Negative control does not deal with.The Rhizoma Coptidis total alkaloids extract group dosage that extracts among silymarin group, the embodiment 1 is respectively 150mg/kg body weight, 120mg/kg body weight.Negative control group weekly 1 subcutaneous injection normal saline 50 μ g/ only, 1 subcutaneous injection vitamin K1 50 μ g/ is only weekly for silymarin group, drug group of the present invention.Negative control group and model control group are pressed the 10ml/kg body weight every day and are irritated the stomach normal saline, and all the other each groups are irritated the stomach relative medicine every day by each group dosage.Successive administration 28 days, every day, observed and recorded was 1 time.After 1 hour, under the etherization situation, the eye socket venous plexus is taken a blood sample in the last administration, and centrifuging and taking serum detects ALT, AST, ALP and TBIL in full automatic biochemical apparatus.Dissect and take out hepatic tissue, half puts-80 ℃ of preservations, is used for detecting hepatic tissue SOD, MDA, GSH, MPO content.Other gets half puts 10% formaldehyde fixed, does pathology detection.
2, general clinical observation
Experimental session, the active state of negative control group and sham operated rats SD rat is good, and hair color is smooth, and reaction is flexibly.The urine jaundice appears in rat behind model control group and the drug group modeling 2～3d, and jaundice performances such as afterbody xanthochromia, ear Huang, hair xanthochromia appear in postoperative 7d.Silymarin group, Rhizoma Coptidis total alkaloids extract group symptom of rats all make moderate progress than model group.In the experimentation, dead 3 of model group, dead 8 of silymarin group (comprise later stage do for supplement 3, dead in the week), dead 1 of Rhizoma Coptidis total alkaloids extract group.Dead animal is bile ascites and causes death.
3, hepatic tissue biochemical analysis
Superoxide dismutase (SOD): relatively significance reduction (P＜0.01) of the activity of SOD and negative control group in the model control group liver tissues of rats; Relatively significance reduction (P＜0.01) of the activity of SOD and sham operated rats in the model control group liver tissues of rats; The activity of SOD reduces in the inductive liver fiber of the results suggest bile duct ligation rat liver.Relatively significance rising (P＜0.01) of the activity of SOD and model control group in the Rhizoma Coptidis total alkaloids extract group liver tissues of rats.Results suggest Rhizoma Coptidis total alkaloids extract can improve liver fiber degree through the activity that improves SOD in the hepatic fibrosis rats hepatic tissue.
Malonaldehyde (MDA): relatively significance rising (P＜0.05) of the content of MDA and negative control group in the model control group liver tissues of rats, relatively significance reduction (P＜0.01) of the activity of MDA and sham operated rats in the model control group liver tissues of rats; The content of MDA raises in the inductive hepatic fibrosis rats liver of results suggest bile duct ligation.The content of MDA and model group relatively there are no significant difference (P＞0.05) in silymarin group, the Rhizoma Coptidis total alkaloids extract group liver tissues of rats.
Glutathione (GSH): relatively significance rising (P＜0.01) of the concentration of GSH and negative control group in the model control group liver tissues of rats, relatively significance rising (P＜0.01) of the concentration of GSH and sham operated rats in the model control group liver tissues of rats.The concentration of GSH and model control group compare there was no significant difference (P＞0.05) in silymarin group, the Rhizoma Coptidis total alkaloids extract group liver tissues of rats.GSH is a kind of oxidant and free radical scavenger, can reduce poisonous substance peroxidating ability through redox reaction, is the key factor of weighing the antioxidant ability of organism size.Vitamin K1 is participated in oxidation-reduction process, guarantees that phosphoric acid shifts and high-energy phosphate compound matter homergy in the body.The abnormal change of GSH in the model group liver tissues of rats, whether relevant with the injection vitamin K1, remain further to be studied.
Myeloperoxidase (MPO) (MPO): the concentration of MPO and negative control group compare in the model control group liver tissues of rats, there was no significant difference (P＞0.05), and the concentration of MPO and sham operated rats compare there was no significant difference (P＞0.05) in the model control group liver tissues of rats.Concentration and the model control group of MPO relatively decrease in silymarin group, the Rhizoma Coptidis total alkaloids extract group liver tissues of rats, but there are no significant difference (P＞0.05).MPO is a kind of enzyme that mainly is present in the neutrophilic granulocyte azurophilic granule; But its catalysis superoxide anion and hydroperoxidation generate hydroxy radical; Pair cell causes major injury; Its active reaction the infiltration number and the activity of neutrophilic granulocyte, be acknowledged as an index of the assess inflammation order of severity.Because 1 subcutaneous injection vitamin K1 50 μ g/ is only weekly in this laboratory animal modeling process; Show according to literature research; Vitamin K1 can be used for treating acute, chronic hepatitis; Enhance hepatocyte vigor and absorbability; Elimination virus; Reduce the serum bilirubin of icterohepatitis, alleviate spasm, transaminase lowering.Whether the concentration of MPO and negative control group compare there was no significant difference in the model control group liver tissues of rats, relevant with the injection vitamin K1, remain further to be studied.
Table 5 in each group of drugs on liver fibrosis in bile duct ligation rat liver biochemical test results of
4, serum biochemistry detects
Relatively significance rising (P＜0.01) of the content of ALT, AST, ALP and TBIL and negative control group, sham operated rats in the model control group rat blood serum, the inductive hepatic fibrosis rats modeling success of results suggest bile duct ligation; The level of AST and TBIL and model control group compare in the Rhizoma Coptidis total alkaloids extract group rat blood serum, and all significance reduces (P＜0.05).Results suggest, the Rhizoma Coptidis total alkaloids extract possibly improve degree of hepatic fibrosis through the content that reduces AST, TBIL in the hepatic fibrosis rats serum.
Table 6 in each group of drugs on liver fibrosis in rats by bile duct ligation serum biochemical test results
Annotate: compare △ P＜0.05 and △ △ P＜0.01 with negative control group; Compare with model control group,
*P＜0.05 draw
*P＜0.01.
5, pathological examination
1) test item: film-making, HE dyeing, histopathologic examination
2) detect foundation: nonstandard method " histopathologic examination " (GDMLAC/OG900-A/1).
3) detection method: identical with embodiment 2.
4) liver outward appearance
The negative control group liver surface is dark red, clear-cut margin, and matter is soft, and common bile duct does not have expansion; The enlargement of model control group liver is yellow green, and quality is medium hard, the above obviously expansion in common bile duct self-ligating place; Each medication group color and quality fall between.
5) om observation
Negative control group: detect surviving animals liver organization 8 examples, lobules of liver clear in structure under the mirror, hepatic cords is arranged more neat in the lobule; The hepatocyte size is even, no degeneration, necrosis, and the portal area does not enlarge; Do not see proliferation of fibrous tissue and little bile duct proliferation, the pathology integration is 0 fen (8 example).
Sham operated rats: detect surviving animals liver organization 8 examples; Mirror is the same negative control group of performance down; The lobules of liver clear in structure; Hepatic cords is arranged more neat in the lobule; The hepatocyte size is even, no degeneration, necrosis, and the portal area does not enlarge; Do not see proliferation of fibrous tissue and little bile duct proliferation, the pathology integration is 0 fen (8 example).
Model control group: detect surviving animals liver organization 8 examples; The obvious hypertrophy of little bile duct companion's fibrous tissue in the most animals liver, pathology integration: 1 minute (2 example), 2 minutes (1 example), 3 minutes (5 example), many places hepatocyte piecemeal necrosis; Massive inflammatory cells infiltrated is at low power field (10 * 10) 1.5mm
2The little bile duct companion proliferation of fibrous tissue gross area 0.88 ± 0.46mm in the scope
2Detect dead animal (experiment 1-2 is dead after week) liver organization 3 examples, see portal area little bile duct companion proliferation of fibrous tissue under the mirror, the pathology integration: 1 minute (2 example), 2 minutes (1 example), at low power field (10 * 10) 1.5mm
2The little bile duct companion proliferation of fibrous tissue gross area 0.32 ± 0.29mm in the scope
2
The silymarin group: detect surviving animals liver organization 3 examples, performance is basically with the model matched group down for mirror, and little bile duct is accompanied proliferation of fibrous tissue pathology integration: 1 minute (1 example), 2 minutes (1 example), 3 minutes (1 example), and at low power field (10 * 10) 1.5mm
2Fibrosis and the little bile duct proliferation gross area 0.85 ± 0.48mm in the scope
2Detect dead animal (experiment 1-2 is dead after week) liver organization 8 examples, see portal area little bile duct companion proliferation of fibrous tissue under the mirror, the pathology integration: 1 minute (7 example), 2 minutes (1 example), at low power field (10 * 10) 1.5mm
2The little bile duct companion proliferation of fibrous tissue gross area 0.27 ± 0.23mm in the scope
2
Rhizoma Coptidis total alkaloids extract group: detect surviving animals liver organization 8 examples; Hepatic tissue destructiveness, hepatic necrosis degree, inflammation active level and bile duct fibrosis degree totally alleviate than model control group; Little bile duct companion proliferation of fibrous tissue pathology integration: 1 minute (4 example), 2 minutes (3 example), 3 minutes (1 example), at low power field (10 * 10) 1.5mm
2Fibrosis and the little bile duct proliferation gross area 0.49 ± 0.42mm in the scope
2
6, experimental result statistics
Adopt the SPSS16.0 statistical software, relatively adopt One-Way ANOVA relatively between group, comparative result is seen table 7 and table 8.
Table 7 animals in each group died of liver bile duct fibrosis compare
Annotate: compare with negative control group,
△ △P＜0.01.
Table 8 surviving animals in each group of liver bile duct fibrosis compare
Annotate: compare with negative control group,
△ △P＜0.01; Compare with model control group,
*P＜0.05.
7-8 is visible by table; Model control group rats'liver bile duct fibrosis degree integration is divided into the master with 3; Significantly increase (P＜0.01) with negative control group, sham operated rats comparison bile duct fibrosis degree, the ligation of results suggest bile duct causes the modeling of rats'liver bile duct fibrosis and is successful on the whole; Silymarin group rat is because of the surviving animals negligible amounts, and hepatic duct fibrosis degree and model control group are not seen remarkable reduction (P＞0.05); Rhizoma Coptidis total alkaloids extract group rat (except that individual animal) hepatic duct fibrosis degree and model control group significantly reduce (P＜0.05); The result shows that the Rhizoma Coptidis total alkaloids extract has therapeutic effect to rats'liver bile duct fibrosis.
6, conclusion
Comprehensive above-mentioned result of study; Under this experimental condition; The Rhizoma Coptidis total alkaloids extract can be through improving SOD activities of liver, reducing AST activity and TBIL content; Reduce hepatic fibrosis hypertrophy area and improve degree of hepatic fibrosis, show that the Rhizoma Coptidis total alkaloids extract has preventive and therapeutic effect to the inductive rat liver fibrosis model of bile duct ligation.
The effect that embodiment 5 suppresses liver cancer cell growth and migration
1, the Rhizoma Coptidis total alkaloids extract suppresses liver cancer cell growth
Hepatoma cell strain MHCC-97L cell inoculation in 96 porocyte culture plates, treat adherent fully after, add the Rhizoma Coptidis total alkaloids extract that extracts among the embodiment 1 of variable concentrations and (count 512 with content of berberine in the Rhizoma Coptidis total alkaloids; 256,128,64; 32,16,8; 4,2 μ M) handle cell 24,48 and 72 hours; Adding Thiazolyl blue in each time point end hatched 4 hours; Supernatant is removed in suction, after the adding dimethyl sulfoxide fully dissolves, on microplate reader, measures cell survival rate with 595nm.The result shows that the Rhizoma Coptidis total alkaloids extract can effectively suppress the growth of MHCC-97L cell.The cell median surviving dose of 24 hours Rhizoma Coptidis total alkaloids extract-treated is about 300 μ M; The cell median surviving dose of 48 hours Rhizoma Coptidis total alkaloids extract-treated is about 150 μ M (result as shown in Figure 3).
2, the Rhizoma Coptidis total alkaloids extract suppresses the hepatoma carcinoma cell migration
Hepatoma cell strain MHCC-97L cell inoculation with high animal migration is in 6 well culture plates; After treating that cell is paved with whole culture hole; Central authorities scrape the space that one width is about 1mm in the hole with the minicell scraper plate; The culture fluid (counting 100 or 200 μ M with content of berberine in the Rhizoma Coptidis total alkaloids) that adds the Rhizoma Coptidis total alkaloids extract that extracts among the embodiment 1 that contains variable concentrations was handled cell 24 hours, and cellular control unit adds the normal cultured base and cultivates.The result shows that the transfer ability of MHCC-97L cell obviously receives the inhibition of Rhizoma Coptidis total alkaloids extract.The analog cells in the extracellular matrix of the migration, resuspended in serum-free medium containing drugs MHCC-97L cells were seeded on the surface of the extracellular matrix material covered with a polycarbonate membrane Transwell? 96-well plates, Transwell lower solution containing induction of serum-free medium for 24 hours, 24 hours post-treatment removed the top cell, adding
GR for migrating to lower fuel cells were stained, fluorescence microplate reader measured.The result shows that the Rhizoma Coptidis total alkaloids extract can effectively suppress the MHCC-97L cell and move to (result as shown in Figure 4) in lower floor's culture medium through extracellular matrix.
In sum; Can find out through detection of serum index and cytologic experiment; From Rhizoma Coptidis, extracting the Rhizoma Coptidis total alkaloids extract obtain has hepatocyte fibrosis symptom and obviously alleviates effect; Growth and transfer to hepatoma carcinoma cell all have the obvious suppression effect, can be used to treat hepatic fibrosis and/or hepatocarcinoma.
Claims (8)
1. the application of Rhizoma Coptidis total alkaloids extract in the medicine of preparation treatment or prevention hepatic fibrosis and/or hepatocarcinoma.
2. application as claimed in claim 1 is characterized in that, said Rhizoma Coptidis total alkaloids extract extracts according to following method and obtains: get a certain amount of medicinal raw material and add a certain amount of water; Under heating, extract one or many; Be concentrated into driedly after extracting solution merges, promptly get the Rhizoma Coptidis total alkaloids extract.
3. application as claimed in claim 1 or 2; It is characterized in that; Said Rhizoma Coptidis total alkaloids extract extracts according to following method and obtains: gets the water that a certain amount of medicinal raw material adds 5-20 times of weight, under decocting under the boiling water state, extracts more than 2 times, and each more than 0.5 hour; Be concentrated into driedly after extracting solution merges, promptly get the Rhizoma Coptidis total alkaloids extract.
4. application as claimed in claim 1 or 2 is characterized in that, said Rhizoma Coptidis total alkaloids extract extracts according to following method and obtains: get the water that a certain amount of medicinal raw material adds 8-15 times of weight, extract 2-4 time each 0.5-2 hour at 100 ℃ down; Be concentrated into driedly after extracting solution merges, promptly get the Rhizoma Coptidis total alkaloids extract.
5. application as claimed in claim 1 or 2 is characterized in that, said Rhizoma Coptidis total alkaloids extract extracts according to following method and obtains: get the water that a certain amount of medicinal raw material adds 10 times of weight, extract each 1 hour 2 times down at 100 ℃; Be concentrated into driedly after extracting solution merges, promptly get the Rhizoma Coptidis total alkaloids extract.
6. application as claimed in claim 1 is characterized in that, comprises magnoflorine, Columbamine, jateorhizine, coptisine, palmatine, epiberberine and berberine in the said Rhizoma Coptidis total alkaloids extract.
7. comprise the application of pharmaceutical composition in the medicine of preparation treatment or prevention hepatic fibrosis and/or hepatocarcinoma like the arbitrary described extract of claim 1-5.
8. application as claimed in claim 7, wherein said pharmaceutical composition further contains acceptable accessories.
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| CN107583003A (en) * | 2016-07-08 | 2018-01-16 | 香港大学 | Prevention or compound, the preparation method and use for the treatment of diabetic eye diseases |
| CN109893651A (en) * | 2017-12-11 | 2019-06-18 | 中国科学院大连化学物理研究所 | β2Adrenoceptor antagonists and application |
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| CN109893651A (en) * | 2017-12-11 | 2019-06-18 | 中国科学院大连化学物理研究所 | β2Adrenoceptor antagonists and application |
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