CN102274262B - Application of Scorzonera total extract in preparation of medicaments for treating hepatitis or protecting liver - Google Patents
Application of Scorzonera total extract in preparation of medicaments for treating hepatitis or protecting liver Download PDFInfo
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Abstract
The invention discloses a scorzonera total extract and application thereof to the preparation of medicaments for treating hepatitis or protecting liver. A preparation method for the scorzonera total extract comprises the following steps of: 1) crushing scorzonera whole herbs into powder, extracting by using 30 to 95 percent ethanol, and concentrating to obtain ethanol extractum; 2) loading the ethanol extractum onto a macroporous resin column to perform chromatography; and 3) eluting by using distilled water, eluting the resin column by using 30 to 95 percent of alcohol water solution to obtain eluent, collecting, concentrating and drying the eluent to form powder, namely the scorzonera total extract. The invention also relates to new application of the scorzonera total extract to the preparation of the medicaments for treating the hepatitis or protecting the liver and provides possibility for the preparation and marketing of the two medicaments.
Description
Technical field
The present invention relates to drug world, particularly a kind of Radix Scorzonerae austriacae total extract and the purposes in preparation treatment hepatitis or hepatic thereof.
Background technology
Liver is the most complicated important metabolism organ of human body maximum, function, and it all plays an important role in the substance metabolismes such as sugar, lipid, protein, vitamin, hormone, bile; Liver also has the function of aspects such as secretion, drainage, biotransformation simultaneously; Therefore liver also is the important shielding organ of body, and its function of detoxification has important protective effect to body, can cause dysbolismus when liver dysfunction, and influence other organ functions, threat to life then in the time of seriously.
Hepatitis is one of disease of serious harm human health, it mainly be by based on the infringement liver one group of pathogen due to.At present, based on viral hepatitis, clear and definite viral hepatitis has five types at least, and wherein hepatitis B is the keypoint control infectious disease in the global viral hepatitis, especially in developing country, also comprise China, not only its sickness rate and prevalence rate height, and cause influence especially severe, patient's poor prognosis, most patient transfers to chronic, may develop into liver cirrhosis, hepatocarcinoma etc.Wherein hepatitis B has infectiousness widely, is global infectious disease.
Hepatitis is hepatitis B particularly, is difficult to radical cure at present, does not also have specific drug in the treatment.In the prior art for the treatment of hepatitis is general adopt in, the method for doctor trained in Western medicine medicine treatment carries out, the reasonable way of doctor trained in Western medicine is to use Drug therapys such as interferon, but its cost height, general patient holds and can't stand that there is obvious toxic and side effects in medicine in addition; China's tradition Chinese medicine also has reasonable medicine at present to the treatment of hepatitis, and its advantage is a less expensive, and the patient is acceptant, and the toxic and side effects of this class medicine is smaller simultaneously, but its treatment cycle is long, and effect is slow.
Except that hepatitis, patient's attack rate of suffering from other hepatic disease in China is equally very high, shows according to the national statistics data, and chronic hepatopathy and liver cirrhosis are to be the 6th of the big cause of death, and annual death toll is more than 3,000 people, and this numerical value is still in continuation is soaring.Therefore develop effective the liver protecting and can be applicable to prevent or treat the medicine of liver related disease, real is the task of top priority.
Radix Scorzonerae austriacae is the dry herb of feverfew Scorzonera austriaca Wild.May～six excavate, dry.10～40 centimetres of this product total lengths.Root is sturdy, inverted cone-shaped, about 3 centimetres of diameter.The stem majority clusters in the collar top, branch not, and smooth no hair, stem foot is by the fibrous sheath shape relict of tearing of dense brown.Basal leaf is linear, and base portion sheath shape enlarges, and by thick cotton wool, the edge is flat in the sheath, and base edge has cotton wool; The stem leaf minority, 1～3 piece, flakey.The head inflorescence list is given birth to the stem top.Its function on Chinese medicine is scattered inflammatory swelling, detoxifcation synthetism, diuresis with curing mainly; Cure mainly diseases such as carbuncle skin ulcer, gonorrhea, women's leukorrhagia, mastitis.Radix Scorzonerae austriacae is as traditional Tibetan medicine (hide name: draw the Abbado), all herbal medicine, and bibliographical information Radix Scorzonerae austriacae herb contains sesquiterpenoids, triterpenes, Coumarins, chemical compounds such as flavonoid.People such as Li Quanmiao, Wang Guangshu has studied extraction separation and structure evaluation (Li Quanmiao, the Wang Guangshu of flavonoid glycoside compound in the Radix Scorzonerae austriacae; The extraction separation of flavonoid glycoside composition and structure are identified [J] in the Radix Scorzonerae austriacae; Jilin University's journal (medicine); But further elaboration is not made in the pharmacological action of its effective site total extract 2010 01 phases).In other pertinent literatures, do not see in addition the report and the explanation of Radix Scorzonerae austriacae total extract in treatment hepatitis and hepatoprotective effect yet.
Summary of the invention
The objective of the invention is for a kind of Radix Scorzonerae austriacae total extract that is used to prepare treatment hepatitis and the liver protecting medicine is provided.
In order to solve the problems of the technologies described above, technical scheme provided by the present invention is:
A kind of Radix Scorzonerae austriacae total extract, total flavones quality percentage composition is 50%～80%, preparation method is:
Step 1) is broken into powder with the Radix Scorzonerae austriacae herb, with 30%～95% ethanol extraction, concentrates and obtains ethanol extract;
Step 2) macroporous resin column on the described ethanol extract is carried out chromatography;
After step 3) was used the distilled water eluting, the alcohol-water solution eluting resin column of reuse 30%～95% obtained eluent, and collecting also, the concentrate drying eluent obtains the Radix Scorzonerae austriacae total extract to Powdered.
Adopt macroporous adsorbent resin that the Radix Scorzonerae austriacae herb is carried out separation and purification in an embodiment of the present invention, obtain the Radix Scorzonerae austriacae total extract.
Macroporous adsorbent resin (macroporou absorption resin) is a class organic polymer adsorbent that is prepared from through polyreaction by additives such as polymerization single polymerization monomer and cross-linking agent, porogen, dispersants.From absorption property, it is stable that it has physicochemical property, is insoluble to acid, alkali and organic solvent, and selectivity is better, is not subjected to inorganic matter to influence numerous advantages.With regard to self-characteristic, it has, and specific surface area is big, exchange velocity is very fast, mechanical strength is high, extract is polluted little, thermally-stabilised characteristics such as good.Compare with other isolation technics, it has advantages such as the relative amount, the product that improve effective ingredient are nonhygroscopic, with short production cycle, resin regeneration is convenient, reusable, thereby is widely used in the separation and purification of natural product in recent years.
This macroporous adsorbent resin preprocess method is: get a certain amount of macroporous resin, and with 95% soak with ethanol 24h～28h, wet method dress post, with 95% pure eluting, to eluent mix with distilled water show muddiness till, with distilled water flush away ethanol, macroporous resin is poured out, and room temperature is dried.
The present invention utilizes 30%～95% alcohol-water solution eluting resin column, collects and concentrates pure washing liquid and obtain the Radix Scorzonerae austriacae total extract.
Utilize 30%～95% alcohol-water solution to extract and two processes of eluting resin column, the content basically identical of Radix Scorzonerae austriacae total extract in the resulting material, its difference is that the high more needed preparation time of concentration is short more, so also basically identical of its drug effect.
As preferably, the present invention adopts 70% ethanol aqueous wash deresination post, collects and concentrated ethanol washing liquid obtains the Radix Scorzonerae austriacae total extract.
The present invention through vacuum drying, according to a conventional method makes solid or liquid medicine dosage form with receivable carrier medically with resulting Radix Scorzonerae austriacae total extract.
In an embodiment of the present invention, the inventor has carried out acute toxicity test in mice to the Radix Scorzonerae austriacae total extract of preparation.In pilot study, the heavy dose of Radix Scorzonerae austriacae total extract of mouse stomach does not also cause death, and can not measure median lethal dose(LD 50).In order to understand the toxic reaction situation of mice to the Radix Scorzonerae austriacae total extract, provide the safe medication reference data for further protecting the liver test, adopt mtd test.The inventor has carried out the acute maximal dose resistance test of mice, observes the Radix Scorzonerae austriacae total extract and gives the toxic reaction and the death condition that are produced behind the mice, for safe medication provides certain reference material.Experimental result shows, its mice is movable normal in experimental period, and chroma of hair is ingested and drained normally, respectively organizes mice in seven days and does not all have death, cuts open mice extremely after seven days, perusal mice internal organs, no abnormality seen phenomenon.
In one embodiment of the invention, the inventor has set up carbon tetrachloride acute liver damage model, has investigated carbon tetrachloride and has caused the biochemical indicator that chmice acute hepatic injury and carbon tetrachloride cause the rat acute hepatic injury.Experiment divides six groups: normal group, carbon tetrachloride model group, positive drug group (bifendate), low dose group, middle dosage group, high dose group, to T check between experimental data employing group, the result shows that the Radix Scorzonerae austriacae total extract has tangible liver-protecting activity.Model group and normal group have utmost point significant difference, and the modeling success is described.And middle dosage group, high dose group compare with model group, and utmost point significant difference is all arranged.Middle dosage group, high dose group and positive drug group relatively find that the liver-protecting activity of Radix Scorzonerae austriacae total extract obviously is better than bifendate positive drug group.Therefore, Radix Scorzonerae austriacae has significant liver-protecting activity, has great exploitation and is worth.
In one embodiment of the invention, the inventor has detected the Radix Scorzonerae austriacae total extract to the DPPH measured by esr technique.
DPPH is 1, the abbreviation of the bitter diazanyl free radical of 1-diphenyl-2-.It is a kind of stable free radical in organic solvent, and its alcoholic solution is darkviolet, has single electronics, so can accept an electronics or hydrion, near wavelength is the 517nm place strong absworption peak is arranged.When having free radical scavenger to exist, the single electron of DPPH is captured and its color is shoaled, absorbance at the 517nm place descends, and the decline degree is linear, therefore can borrow the ultraviolet-visible spectrophotometer to detect the situation that the DPPH free radical is captured, thus the oxidation resistance of evaluation test sample.
Experimental result shows that the Radix Scorzonerae austriacae total extract is to DPPH measured by esr technique IC
50Be 30ug/ml, illustrate that the radical scavenging activity of Radix Scorzonerae austriacae extract is strong, proved the liver-protecting activity of Radix Scorzonerae austriacae total extract.
In one embodiment of the invention, the inventor has detected the synthetic inhibitory action of HBV DNA in the Radix Scorzonerae austriacae total extract pair cell.The Radix Scorzonerae austriacae total extract was cultivated 8 days in the 2.2.15 cell of hepatitis B element (HBV) dna clone infection human liver cancer cell.Experimental result shows that its half-inhibition concentration to HBV DNA is IC
50Be 3.04ugmL
-1 Positive drug 1 μ molL
-1Lamivudine (3TC) be 70.2 ± 4.5% to the suppression ratio of HBV DNA.Proved that the Radix Scorzonerae austriacae total extract have good inhibitory effect to hepatitis B.
Description of drawings
Fig. 1 is the standard curve of reference substance Radix Scorzonerae austriacae flavone glycosides B.
The specific embodiment
The invention discloses a kind of Radix Scorzonerae austriacae total extract and the purposes in preparation treatment hepatitis or hepatic thereof, those skilled in the art can use for reference this paper content, suitably improve technological parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Method of the present invention and application are described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
Technical scheme provided by the invention is:
A kind of Radix Scorzonerae austriacae total extract, total flavones quality percentage composition is 50%～80%, preparation method is:
Step 1) is broken into powder with the Radix Scorzonerae austriacae herb, with 30%～95% ethanol extraction, concentrates and obtains ethanol extract;
Step 2) macroporous resin column on the described ethanol extract is carried out chromatography;
After step 3) was used the distilled water eluting, the alcohol-water solution eluting resin column of reuse 30%～95% obtained eluent, and collecting also, the concentrate drying eluent obtains the Radix Scorzonerae austriacae total extract to Powdered.
In order to make those skilled in the art understand technical scheme of the present invention better, the present invention is described in further detail below in conjunction with specific embodiment.
Embodiment 1: the preparation of Radix Scorzonerae austriacae total extract
Get a certain amount of macroporous resin, with 95% soak with ethanol 24h, wet method dress post, with 95% pure eluting, mix apparent muddiness with distilled water (1: 5) to eluent till.With distilled water flush away ethanol, macroporous resin to be poured out, room temperature is dried.
The Radix Scorzonerae austriacae herb is ground into powder, and the ethanol extraction with 50% promptly gets the Radix Scorzonerae austriacae ethanol extract.With macroporous resin column pretreated on the Radix Scorzonerae austriacae ethanol extract, earlier with water-solubility impurities such as distilled water flushing protein, polysaccharide, the ethanol water eluting of reuse 70%, collecting also, the concentrate drying eluent obtains total extract to Powdered.
Embodiment 2: the preparation of Radix Scorzonerae austriacae total extract
Get a certain amount of macroporous resin, with 95% soak with ethanol 24h, wet method dress post, with 95% pure eluting, mix apparent muddiness with distilled water (1: 5) to eluent till.With distilled water flush away ethanol, macroporous resin to be poured out, room temperature is dried.
The Radix Scorzonerae austriacae herb is ground into powder, and the ethanol extraction with 70% promptly gets the Radix Scorzonerae austriacae ethanol extract.Extractum is scattered in the water, use extracted with diethyl ether, water layer is flung to behind the ether macroporous resin column pretreated on the Radix Scorzonerae austriacae ethanol extract, earlier with water-solubility impurities such as distilled water flushing protein, polysaccharide, the ethanol water eluting of reuse 30%, collect eluent and concentrate drying to Powdered, get total extract.
Embodiment 3: the preparation of Radix Scorzonerae austriacae total extract
Get a certain amount of macroporous resin, with 95% soak with ethanol 24h, wet method dress post, with 95% pure eluting, mix apparent muddiness with distilled water (1: 5) to eluent till.With distilled water flush away ethanol, macroporous resin to be poured out, room temperature is dried.
The Radix Scorzonerae austriacae herb is ground into powder, and the ethanol extraction with 95% promptly gets the Radix Scorzonerae austriacae ethanol extract.Extractum is scattered in the water, use petroleum ether extraction, water layer is flung to behind the ether macroporous resin column pretreated on the Radix Scorzonerae austriacae ethanol extract, earlier with water-solubility impurities such as distilled water flushing protein, polysaccharide, the ethanol water eluting of reuse 95%, collect eluent and concentrate drying to Powdered, get total extract.
Embodiment 4: the preparation of Radix Scorzonerae austriacae total extract
Get a certain amount of macroporous resin, with 95% soak with ethanol 24h, wet method dress post, with 95% pure eluting, mix apparent muddiness with distilled water (1: 5) to eluent till.With distilled water flush away ethanol, macroporous resin to be poured out, room temperature is dried.
The Radix Scorzonerae austriacae herb is ground into powder, and the ethanol extraction with 30% promptly gets the Radix Scorzonerae austriacae ethanol extract.Extractum is scattered in the water, use petroleum ether extraction, water layer is flung to behind the ether macroporous resin column pretreated on the Radix Scorzonerae austriacae ethanol extract, earlier with water-solubility impurities such as distilled water flushing protein, polysaccharide, the methanol aqueous solution eluting of reuse 50%, collect eluent and concentrate drying to Powdered, get total extract.
Embodiment 5: the preparation of Radix Scorzonerae austriacae total extract
Get a certain amount of macroporous resin, with 95% soak with ethanol 24h, wet method dress post, with 95% pure eluting, mix apparent muddiness with distilled water (1: 5) to eluent till.With distilled water flush away ethanol, macroporous resin to be poured out, room temperature is dried.
The Radix Scorzonerae austriacae herb is ground into powder, and the ethanol extraction with 60% promptly gets the Radix Scorzonerae austriacae ethanol extract.Extractum is scattered in the water, use petroleum ether extraction, water layer is flung to behind the ether macroporous resin column pretreated on the Radix Scorzonerae austriacae ethanol extract, earlier with water-solubility impurities such as distilled water flushing protein, polysaccharide, the methanol aqueous solution eluting of reuse 60%, collect eluent and concentrate drying to Powdered, get total extract.
Embodiment 6: the assay of flavone in the Radix Scorzonerae austriacae total extract
1 Radix Scorzonerae austriacae effective site determination of total flavonoids method
1.1 instrument, reagent and material
1.1.1 instrument
AR1530 electronic balance (U.S.)
101-L-S electric heating constant temperature air dry oven (Shanghai make a leapleap forward medical apparatus and instruments factory)
TP150 supersound extraction device (day roc electronics new technique (Shanghai) Co., Ltd.)
8302 type thermostat water baths: the Ying Yu of Gongyi City gives magnificent instrument plant
Agilent8453 ultraviolet-uisible spectrophotometer (U.S.)
Uv-spectrophotometric instrument UVmini-1240 (day island proper Tianjin)
RE-52A Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant)
SHB-IIIA circulation ability of swimming is used vacuum pump (Shanghai Yu Kang science and education instrument and equipment company limited) more
1.1.2 reagent and material
This experiment medical material is adopted in area, Tonghua.Be accredited as Radix Scorzonerae austriacae through professor Zhang Jingmin of crude drug teaching and research room of pharmaceutical college of Jilin University.
Reference substance: Radix Scorzonerae austriacae flavone glycosides B (self-control), shown in I,
Formula I
Name: 5,7,3 ', 4 '-tetrahydroxy-8-C-β-D-glucose flavone c-glycoside
Chemical reagent: Beijing chemical industry group company analytical pure
D101 type macroporous resin: resin branch company of Tianjin pesticide limited company
AB-8 type macroporous resin: Anhui Samsung resin Science and Technology Ltd.
1.2 the foundation of Radix Scorzonerae austriacae effective site determination of total flavonoids method
1.2.1 the preparation of reference substance solution
Precision takes by weighing 0.0030g Radix Scorzonerae austriacae flavone glycosides B in the 25mL volumetric flask, adds dissolve with methanol, and standardize solution is made 120 μ gmL
-1Solution.
1.2.2 the preparation of need testing solution
Precision takes by weighing Radix Scorzonerae austriacae total extract 0.05g that embodiment 1-5 makes in the 25mL volumetric flask, adds dissolve with methanol, and standardize solution is made 120 μ gmL
-1Solution.
1.2.3 determining of coloration method
Get the reference substance solution 1ml of above-mentioned Experiment Preparation, be settled in the 25ml volumetric flask with 60% ethanol.Other gets reference substance solution 1ml, adds 5% aluminum trichloride solution 5mL colour developing, is settled in the 25ml volumetric flask with 60% ethanol.Above-mentioned two kinds of solution are carried out UV scanning.The result shows and not use the reference substance of developer at 270nm place two absworption peaks to be arranged, and the use developer has a regular chromatographic peak at the 273nm place.Therefore this experiment selects for use 5% aluminum trichloride solution to develop the color.
1.2.4 determining of maximum absorption wavelength
Get the reference substance solution 1ml of above-mentioned Experiment Preparation, add 5% aluminum trichloride solution 5mL colour developing, be settled in the 25ml volumetric flask with 60% ethanol; Other gets the need testing solution 1ml of embodiment 1-5 preparation, adds 5% aluminum trichloride solution 5mL colour developing, is settled in the 25ml volumetric flask with 60% ethanol.Above-mentioned two kinds of solution are carried out UV scanning,, determine that maximum absorption wavelength is 273nm through the UV scanning collection of illustrative plates of comparative sample and reference substance.
1.2.5 the drafting of standard curve
Accurately the reference substance solution 0.5,1,2,3,4 of the above-mentioned Experiment Preparation of absorption, 5mL develop the color 60% ethanol standardize solution as stated above in the 25mL volumetric flask.Retinue reagent is blank, and the 273nm place measures absorbance.Experimental result sees Table 1 and Fig. 1.
Table 1 standard curve table
With reference substance concentration is abscissa, and absorbance is the vertical coordinate mapping, gets regression equation y=0.0391x+0.0248, r=0.9999.Experimental result shows that reference substance is in 2.4～24 μ g/mL scopes, and linear relationship is good.
1.2.6 stability test
The reference substance 5mL that gets above-mentioned Experiment Preparation develops the color in the 25mL volumetric flask as stated above, and retinue reagent be blank, respectively at 0,10,20,30,40,50,60, during 120min, at 273nm place mensuration absorbance, the results are shown in Table 2.
Table 2 stability test
Experimental result shows, at room temperature color stability in the 120min.
1.2.7 precision test
The reference substance 5mL that gets 6 parts of above-mentioned Experiment Preparation respectively develops the color by above-mentioned coloration method in the 25mL volumetric flask, and retinue reagent is blank, and the 273nm place measures absorbance.Experimental result sees Table 3.
The experiment of table 3 precision
Experimental result shows that instrument precision is good.
1.2.8 repeatability test
Get need testing solution 1mL that embodiment 1-5 makes in the 25mL volumetric flask, by above-mentioned coloration method colour developing, retinue reagent be blank, 273nm place mensuration absorbance.Parallel test 5 times, experimental result sees Table 4.
The experiment of table 4 repeatability
Experimental result shows that this method repeatability is good.
1.2.9 average recovery test
Precision takes by weighing 5 parts of dry Radix Scorzonerae austriacae, every part of about 0.05g, and the accurate reference substance 1mL that adds, pressing maneuver is handled, colour developing as stated above, retinue reagent is blank, the 273nm place measures absorbance.Experimental result sees Table 5.
The experiment of table 5 response rate
Experimental result shows that RSD is 2.30%, and average recovery rate is 97.55%, meets the requirements.
The mensuration of 2 samples
It is an amount of to get the sample that embodiment 1-5 makes, and 60% dissolve with methanol is settled in the 100mL volumetric flask.
Experimental result sees Table 6, and the result shows that general flavone content is 50%～80% in the Radix Scorzonerae austriacae, and stable content, can satisfy the demand of follow-up test.
General flavone content in table 6 Radix Scorzonerae austriacae
Following examples material therefor and instrument are:
Animal: wistar rat, male and female half and half, body weight 200 ± 20g, secondary; Kunming mouse (SPF level), body weight 20 ± 2g, male and female half and half are by the Bethune of Jilin University medical board experimental animal central supply.
Medicine and reagent: glutamate pyruvate transaminase (GPT/ALT) lot number: 2009028 (Changchun Sai Hui power technology Co., Ltds); Glutamic oxaloacetic transaminase, GOT (GOT/AST) lot number: 2008044 (Changchun remittance power technology Co., Ltds); Bifendate lot number: 08100101 (Beijing consonance pharmaceutical factory); Galactose ammonia model: TCI (Tokyo HuaCheng Industry Co., Ltd); Acetaminophen: dragon source, Queshan pharmaceutcal corporation, Ltd; CCl
4(Tianjin recovery fine chemistry industry institute); Formaldehyde (the federal chemical reagent work in Shenyang City.
Instrument: 8302 type thermostat water baths: the Ying Yu of Gongyi City gives magnificent instrument plant; Anke TGL-16C type centrifuge; Uv-spectrophotometric instrument UVmini-1240 (day island proper Tianjin)
Embodiment 7: the acute toxicity test of Radix Scorzonerae austriacae total extract
Get 20 of body weight 20 ± 2g mices, male and female half and half, the administration first day, fasting 12h can't help water, with the Radix Scorzonerae austriacae total extract of embodiment 1 preparation, presses 100mg/10g, irritates stomach.Raise one week of mice, observe mice physiological performance and death condition.
The observation experiment phenomenon finds that its mice is movable normal in experimental period, and chroma of hair is ingested and drained normally, respectively organizes mice in seven days and does not all have death, cuts open mice extremely after seven days, perusal mice internal organs, no abnormality seen phenomenon.
Experimental result shows, presses maximum concentration 100mg/10g mouse stomach and calculates, and amounts to people's pharmaceutical dosage (the about 60 kilograms of calculating of the general body weight of people) and is equivalent to the day for human beings and uses dosage 0.66mg/kg.No death in seven days of 20 mices and toxic reaction.So according to the statistics rule, existing assurance more than 99.99% proves that the Radix Scorzonerae austriacae total extract must be greater than 100mg/10g to the LD50 of mouse stomach administration.
According to the method described above, the result of the Radix Scorzonerae austriacae total extract gained of embodiment 2-5 preparation is close with the result of embodiment 1, and has statistical significance.
Embodiment 8: the Radix Scorzonerae austriacae total extract causes the pharmacological testing of chmice acute hepatic injury to carbon tetrachloride
The grouping of 1 laboratory animal
Behind the normal feedstuff feed 3 days, be divided into 6 groups, 12 every group, be respectively: normal group, carbon tetrachloride (CCl by the body weight random packet
4) model group, positive drug group (bifendate), low dose group, middle dosage group, high dose group.
Normal group is got distilled water and is irritated stomach, dosage: 20ml/kg.
Carbon tetrachloride model group (CCl
4), irritate stomach, with edible vegetable oil preparation 0.1%CCl
4, dosage: 10ml/kg.
Positive drug bifendate group is irritated stomach, dosage: 100mg/kg.
The Radix Scorzonerae austriacae total extract of embodiment 1 preparation is divided into low, in, high dose group is irritated stomach, is respectively 80mg/kg according to trial test, 160mg/kg, 320mg/kg.
2 carbon tetrachloride acute liver damage modellings
The mice of each grouping, by given dose ratio, gastric infusion, once a day, continuous 7 days, the last administration was after 2 hours, and except that normal group, all the other each treated animals are pressed the CCl of the disposable filling stomach 0.1% of dosage of 10mL/kg body weight
4The Oleum Glycines diluent duplicates the acute liver damage model.
3 estimate the biochemical indicator of use in medicament-induced hepatotoxicity
Alanine aminotransferase in the serum (ALT) activity: alanine aminotransferase (ALT) claims glutamate pyruvate transaminase (GPT) again, mainly is present in the histiocyte, has only minute quantity to be released in the blood under the normal condition, so this enzymatic activity is very low in the serum.When pathological changes took place tissue, ALT was released in the blood in a large number in the cell, made that this enzymatic activity increases in the serum.Serum alt is mainly derived from liver, and when various hepatic injury took place, this enzymatic activity significantly increased, thus the alanine aminotransferase activity be estimate the hepatic injury degree must survey index.
Serum Mid-Heaven Gate radon propylhomoserin aminotransferase (AST) activity: Tianmen radon propylhomoserin aminotransferase (AST) claims glutamic oxaloacetic transaminase, GOT (GOT) again, mainly is present in hepatocyte, cardiac muscle, muscle, is present in the erythrocyte on a small quantity.When liver generation pathological changes, AST is released in the blood in a large number in the cell, makes that this enzymatic activity increases in the blood.
In our test, mainly select for use biochemical indicator in two serum of ALT, AST to reflect the degree of the damage of the liver poison of medicine and liver.
4 blood samplings and serum separate
After the modeling, water is can't help in fasting, plucks eyeball behind the 16h and gets blood, and room temperature is placed 2h, and centrifugal 15 minutes of 5000rpm/min collects supernatant.
The mensuration of AST and ALT in 5 blood plasma
(1) mensuration of GPT/ALT
Principle: adopt reitman-frankel method, ALT acts on substrate L-alanine and α-Tong Wuersuan and generates acetone acid and glutamic acid under certain condition in the sample, acetone acid and 2,4 dinitrophenylhydrazines develop the color under alkali condition, measure absorbance at the 510nm wavelength, calculate its activity.
Reagent constituent: L-alanine, α-Tong Wuersuan, 2,4 dinitrophenylhydrazines, sodium hydroxide
Method for measuring and step:
Wavelength: 510nm
Temperature: 37 ℃
Reagent is prepared: before mensuration, be 0.4mol/L with distilled water by dilution in 1: 9 with sodium hydroxide, with the substrate solution hygral equilibrium to room temperature.Carry out application of sample by table 7:
Table 7AST determination of activity application of sample table
Each pipe adds 0.4mol/L sodium hydroxide 2.5ml, and mixing under the 510nm wavelength, with the reagent blank zeroing, was measured and respectively managed the absorbance A value after 3 minutes.
Calculate: ALT=A sample/A standard * 100 (U/L)
(2) mensuration of AST
Principle: adopt reitman-frankel method, GOT/AST acts on substrate L-aspartic acid and α-Tong Wuersuan generates oxaloacetic acid and glutamic acid, oxaloacetic acid changes into acetone acid and 2,4 dinitrophenylhydrazines generate red phenylhydrazone in alkali condition, under 510nm absorption maximum is arranged.
Reagent constituent: L-aspartic acid, α-Tong Wuersuan, 2,4 dinitrophenylhydrazines, sodium hydroxide
The active mensuration of AST in the blood serum sample:
With reference to the method for ALT determination of activity, change substrate solution into AST substrate liquid, measure the AST unit of activity.
6 statistical dispositions
Group difference adopts the t check.In the T with matched group checks, the P＞no significant difference of 0.05 explanation, P＜there were significant differences in 0.05 explanation.P＞0.01 explanation does not have utmost point significant difference, and P＜0.01 explanation has utmost point significant difference.
7 experimental datas (seeing Table 8)
Table 8 Radix Scorzonerae austriacae total extract to the influence of mice serum liver function (x ± s, n=10)
Annotate: P＜0.01 (normal group and model group); P＜0.01 (model group and positive drug group); P＞0.05 (model group and low dose group); P＜0.01 (model group and middle dosage group); P＜0.01 (model group and high dose group)
8 experimental results
The effects carbon tetrachloride cause the chmice acute liver injury model.Experiment divides six groups: normal group, carbon tetrachloride model group, positive drug group (bifendate), low dose group, middle dosage group, high dose group, to T check between experimental data employing group, the result shows that the Radix Scorzonerae austriacae total extract has tangible liver-protecting activity.Model group and normal group have utmost point significant difference, and the modeling success is described.And middle dosage group, high dose group compare with model group, and utmost point significant difference is all arranged.Middle dosage group, high dose group and positive drug group relatively find that the liver-protecting activity of Radix Scorzonerae austriacae total extract obviously is better than bifendate positive drug group.Therefore, the Radix Scorzonerae austriacae total extract has significant liver-protecting activity, has great exploitation and is worth.
According to the method described above, the result of the Radix Scorzonerae austriacae total extract gained of embodiment 2-5 preparation is close with the result of embodiment 1, and has statistical significance.
Embodiment 9: the Radix Scorzonerae austriacae total extract causes the pharmacological testing of rat acute hepatic injury to carbon tetrachloride
The grouping of 1 laboratory animal
Get 42 of Wistar rats, be divided into 6 groups, 7 every group, be respectively: normal group, carbon tetrachloride (CCl by the body weight random packet
4) model group, positive drug group (bifendate), low dose group, middle dosage group, high dose group.
Normal group is got distilled water and is irritated stomach, dosage: 15ml/kg
Positive drug bifendate group is irritated stomach, dosage: 70mg/kg
The Radix Scorzonerae austriacae total extract of embodiment 1 preparation is divided into low, in, high dose group is irritated stomach, is respectively 50mg/kg according to trial test, 100mg/kg, 200mg/kg
2 carbon tetrachloride acute liver damage modellings
The mice of each grouping, by given dose ratio, gastric infusion, once a day, continuous 7 days.
Other respectively organized rat sc 25% carbon tetrachloride 0.5ml/100g except that the blank group in the 3rd day, the 7th day after the administration
Duplicate the acute liver damage model.
3 blood samplings and serum separate
After the modeling, water is can't help in fasting, puts to death rat heart behind the last administration 12h and gets blood, and room temperature is placed 2h, and centrifugal 15 minutes of 5000rpm/min collects supernatant.
Get 42 of Wistar rats, be divided into 6 groups at random, ig administration, every day 1 time, totally 7 days by table 2.
The mensuration of ALT in 4 blood plasma
Operate by method described in the embodiment 6, measure ALT content.
5 statistical dispositions
Group difference adopts the t check.In the T with matched group checks, the P＞no significant difference of 0.05 explanation, P＜there were significant differences in 0.05 explanation.P＞0.01 explanation does not have utmost point significant difference, and P＜0.01 explanation has utmost point significant difference.
6 experimental datas (seeing Table 9)
Table 9 Radix Scorzonerae austriacae total extract causes the influence (X ± SD) of acute poisoning liver damage rat blood serum ALT to carbon tetrachloride
Annotate: P＜0.01 (normal group and model group); P＜0.01 (model group and positive drug group); P＞0.05 (model group and low dose group); P＜0.01 (model group and middle dosage group); P＜0.01 (model group and high dose group)
7 experimental results
The effects carbon tetrachloride cause the rat acute liver injury model.Experiment divides six groups: normal group, carbon tetrachloride model group, positive drug group (bifendate), low dose group, middle dosage group, high dose group, to T check between experimental data employing group, the result shows that the Radix Scorzonerae austriacae total extract has tangible liver-protecting activity.Model group and normal group have utmost point significant difference, and the modeling success is described.And middle dosage group, high dose group compare with model group, and utmost point significant difference is all arranged.Middle dosage group, high dose group and positive drug group relatively find that the liver-protecting activity of Radix Scorzonerae austriacae total extract obviously is better than bifendate positive drug group.Therefore, Radix Scorzonerae austriacae has significant liver-protecting activity, has great exploitation and is worth.
According to the method described above, the result of the Radix Scorzonerae austriacae total extract gained of embodiment 2-5 preparation is close with the result of embodiment 1, and has statistical significance.
Embodiment 10: the Radix Scorzonerae austriacae total extract is to the DPPH measured by esr technique
The preparation of 1DPPH stock solution
Accurately take by weighing DPPH reagent 0.0131g,, and quantitatively change in the 500mL measuring bottle, use the dehydrated alcohol standardize solution, shake up, get concentration and be about 6.5 * 10 with dehydrated alcohol (analytical pure) dissolving
-5MolL
-1The DPPH stock solution, it is standby to place refrigerator and cooled to hide.
The preparation of 2 sample solutions
It is 0.2,5,10,20,40,50,60,80 that embodiment 1 prepared Radix Scorzonerae austriacae total extract is mixed with concentration respectively with dehydrated alcohol, 100ugmL
-1Or concentration is 0.1,0.2,0.4,0.6,0.8, and 1mg/mL series solution is standby.
3 Radix Scorzonerae austriacae total extracts are removed the mensuration of DPPH free radical ability
The method of reference literature, getting 2.5mL concentration is 6.5 * 10
-5MolL
-1DPPH solution, add 0.5mL solvent (dehydrated alcohol), mixing leaves standstill 30min, is reference liquid with the alcoholic solution with ultraviolet-visible spectrophotometer, at the mensuration absorbance A0 of 517nm place; Other gets 2.5mL concentration is 6.5 * 10
-5MolL
-1DPPH solution, add the 0.5mL sample solution, mixing leaves standstill 30min, at the mensuration absorbance Ai of 517nm place; Get the 2.5mL solvent then, add the 0.5mL sample solution, record absorbance Aj with quadrat method, parallel testing 3 times.
Calculate clearance rate by following formula:
S(％)＝[1-(Ai-Aj)/A0]×100％
The absorbance of A0:2.5mL DPPH solution+0.5mL solvent;
The absorbance of Ai:2.5mL DPPH solution+0.5mL sample solution;
The absorbance of Aj:2.5mL solvent+0.5mL sample solution.
And draw the DPPH free radical scavenging activity to the sample solution concentration curve, read out the concentration IC that the DPPH free radical scavenging activity is 50% o'clock sample solution by curve
50, this moment IC
50The physical significance of value is: make the free radical number reduce the concentration of 50% o'clock required sample, at this sample concentration of instigating the absorption value decline 50% under wavelength 517nm of DPPH solution, concentration is with mgmL
-1Or ugmL
-1Expression.Antioxidant for clearing DPPH free radical ability adopts IC
50Value representation, IC
50More little, the radical scavenging activity of antioxidant is strong more.
4 experimental results (seeing Table 10)
Table 10 Radix Scorzonerae austriacae total extract is to the DPPH measured by esr technique
Experimental result shows, the IC of Radix Scorzonerae austriacae total extract
50Be 30ug/ml, illustrate that the radical scavenging activity of Radix Scorzonerae austriacae total extract is strong, proved the liver-protecting activity of Radix Scorzonerae austriacae total extract.
According to the method described above, the result of the Radix Scorzonerae austriacae total extract gained of embodiment 2-5 preparation is close with the result of embodiment 1, and has statistical significance.
Embodiment 11: the synthetic inhibitory action of HBV DNA in the Radix Scorzonerae austriacae total extract pair cell
1 medicine and reagent
Medicine: the Radix Scorzonerae austriacae total extract according to embodiment 1 preparation, is pale brown toner end.Be made into 4 ℃ of preservations of 10mg/ml mother solution with distilled water, be made into desired concn with the HepG2.2.15 cell culture fluid during use.
Cell strain: the 2.2.15 cell strain (HepG2.2.15 cell strain) that hepatitis B element (HBV) dna clone infects human liver cancer cell is provided by Beijing Ditan Hospital.Passage is cultivated: HepG 2.2.15 cell is cultivated with the DMEM culture medium (containing penicillin 100U/mL, streptomycin 100U/mL, G418380 μ g/mL) that contains 10% hyclone.37 ℃ of cell culture conditions, 5%CO
2Cultivate in the incubator, an about week goes down to posterity once.
Reagent: hyclone (Promague company, the U.S.); DMEM (Dulbeccosmodified Eagle ' s medium) culture medium (Promague company, the U.S.); G418 (Sigma company, the U.S.); Hepatitis B virus e antigen diagnostic kit (ELISA method Huamei Bio-Engrg Co.);
2 key instruments
Superclean bench (SW-CT-IFD, Suzhou); Water isolation type electro-heating standing-temperature cultivator (Shanghai); Desk centrifuge (TGL-16G, Beijing);-80 ℃ of cryogenic refrigerators (SANYO, Japan); Carbon dioxide incubator (SANYO MCO-17AIC, Japan); Inverted microscope (OLYMPUS, Japan); Water bath with thermostatic control agitator (HZS-H, Harbin); Electronic balance (prunus mume (sieb.) sieb.et zucc. Teller); Microplate reader (BIO-RA03550 type); γ-calculating instrument (U.S. DPC company);
3 experimental techniques
The collection of the cultivation of cell, the intervention of medicine, cell and supernatant: the HepG 2.2.15 cell of the trophophase of taking the logarithm, concentration is 5 * 10
4ML
-1, every hole adds the 1mL single cell suspension in 24 well culture plates.Cell grows to when converging behind the 24h, and experimental group adds the Radix Scorzonerae austriacae total extract (10ugmL that contains variable concentrations
-1, 5ugmL
-1, 2.5ugmL
-1, 1.25ugmL
-1, 0.625ugmL
-1) culture fluid 1mL; Not dosing negative control group adds the DMEM culture fluid 1mL that contains 10% hyclone; The positive drug matched group adds 1 μ molL
-1Lamivudine (3TC), continue to cultivate.Collected supernatant and cell on the 8th day after dosing ,-20 ℃ of frozen being equipped with, examined.
The synthetic inhibitory action of HBV DNA in the Radix Scorzonerae austriacae total extract pair cell: extract DNA in the cell, every hole adds 400 μ L lysis buffer (10mmolL
-1Tris HCl, pH8.0/0.1molL
-1EDTA/0.5%SDS) suspension cell is hatched 1h for 37 ℃; The 500mgL that adds 100 μ L
-1E.C. 3.4.21.64 in 50 ℃ of following digestion 2h; Add the saturated phenol 500 μ L of equal-volume TE, jog 1min, 12000rmin
-1Centrifugal 3min moves to upper water in another centrifuge tube mutually; Add equal-volume phenol-chloroform-isoamyl alcohol mixed liquor (V: V: V=25: 24: 1), the same step extracting once moves to upper water in another centrifuge tube after centrifugal mutually; (V: V=24: 1) the same step extracting once to add equal-volume chloroform-isoamyl alcohol; Add 2 times of volume dehydrated alcohol, fully mixing places-70 ℃ of 15min; 12000rmin
-1Centrifugal 5min abandons supernatant, and drying at room temperature 10min adds 1 μ L HindIII, 37 ℃ of enzyme action 30min to water white transparency.Quantitatively, A260/A280 ratio is at 1.8-2.0 down for the DNA spectrophotometer.Get sample on the DNA 10 μ g, 0.8% agarose gel electrophoresis.Adopt the capillary siphonage to change film then, use on-radiation hepatitis B source of disease gene diagnosis kit (Shanghai Medical Univ's preventive medicine institute) to carry out Southern Blotting hybridization, detailed step is seen description.Use Image J 1.36Analysis Software Southern hybridization figure is carried out the expression intensity semi-quantitative analysis.
HBV DNA suppresses percentage rate (%)=(cell contrast A value-administration group A value)/cell contrast A value * 100.
4 experimental datas (seeing Table 11)
Half-inhibition concentration is IC
50Be 3.04ugmL
-1
Table 11 Radix Scorzonerae austriacae total extract is the 8th day inhibitory action to HBV DNA in the 2.2.15 cell
Experimental result shows that the Radix Scorzonerae austriacae total extract was cultivated 8 days in the 2.2.15 cell of hepatitis B element (HBV) dna clone infection human liver cancer cell.Half-inhibition concentration to HBV DNA is IC
50Be 3.04ugmL
-1 Positive drug 1 μ molL
-1Lamivudine (3TC) be 70.2 ± 4.5% to the suppression ratio of HBV DNA.Proof Radix Scorzonerae austriacae effective site total flavones have good inhibitory effect to hepatitis B.
According to the method described above, the result of the Radix Scorzonerae austriacae total extract gained of embodiment 2-5 preparation is close with the result of embodiment 1, and has statistical significance.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (5)
1. a Radix Scorzonerae austriacae total extract is preparing the purposes for the treatment of in the hepatitis medicament, and total flavones quality percentage composition is 50%～80% in the described Radix Scorzonerae austriacae total extract, and preparation method is:
Step 1) is broken into powder with the Radix Scorzonerae austriacae herb, with 30%～95% ethanol extraction, concentrates and obtains ethanol extract;
Step 2) macroporous resin column on the described ethanol extract is carried out chromatography;
After step 3) was used the distilled water eluting, the alcohol-water solution eluting resin column of reuse 30%～95% obtained eluent, and collecting also, the concentrate drying eluent obtains the Radix Scorzonerae austriacae total extract to Powdered.
2. purposes according to claim 1 is characterized in that, the described ethanol of step 1) is 50% ethanol.
3. purposes according to claim 1 is characterized in that step 2) described macroporous resin column is for through pretreated macroporous resin column.
4. purposes according to claim 3 is characterized in that, described pretreatment comprises gets macroporous resin, with 95% soak with ethanol 24h～28h, wet method dress post is with 95% pure eluting, to eluent mix with distilled water show muddiness till, with distillation washing macroporous resin, room temperature is dried.
5. purposes according to claim 1 is characterized in that, the described alcohol-water solution of step 3) is 70% ethanol water.
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| Title |
|---|
| 徐善荣.鸦葱总黄酮制备工艺、质量标准以及活性研究.《中国优秀硕士学位论文全文数据库 医药卫生科技辑》.2011,(第2011年第2期),E057-40,9－20，37. |
| 徐善荣.鸦葱总黄酮制备工艺、质量标准以及活性研究.《中国优秀硕士学位论文全文数据库 医药卫生科技辑》.2011,(第2011年第2期),E057-40,9-20,37. * |
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