CN102028726A - Extract of Chinese herb marsdenia tenacissima as well as preparation method and application thereof - Google Patents
Extract of Chinese herb marsdenia tenacissima as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an extract of Chinese herb marsdenia tenacissima as well as a preparation method and application thereof, belonging to the technical fields of extracts of Chinese herbs and preparation methods thereof. The extract containing total phenolic acid and total saponin which are effective parts of marsdenia tenacissima can be extracted by reflux with ethanol or can be prepared by separately extracting the extracts of the total phenolic acid and the extracts of the total saponin and mixing the extracts together. The extract of Chinese herb marsdenia tenacissima can be used for preparing medicines for resisting against hepatitis B virus, reducing transaminase level and regulating immunity. The extract of Chinese herb marsdenia tenacissima has the effect of protecting liver and reducing enzyme and certain protection effect on animal liver injury caused by CC14, and meanwhile, can improve non-specific immunity function in a proper dosage range.
Description
Technical field
The invention belongs to Chinese medicine extract and preparation method thereof technical field, be specifically related to Chinese medicine Caulis Marsdeniae Tenacissimae extract and its production and application.
Background technology
It is 3.5 hundred million that the hepatitis B virus number is carried in the whole world, and the number of the infected of hepatitis C has also reached 1.5 hundred million.In China, hepatitis B infected rate is about 10%, and the people of infection hepatitis C has 4,000 ten thousand approximately.Above data as seen, it is also very huge that hepatitis C infects colony, can not be ignored.
In the treatment of hepatitis B, the achievement that China obtains is very big, the crowd infection rate of hepatitis B virus descends also very fast, but from absolute numeral, also still huge, even according to 7.18% present calculation, we also have more than 9,000 ten thousand hepatitis B surface antigen carriers nearly in the whole nation, some has been the chronic viral hepatitis B patient in the middle of these crowds, and the somebody may proceed to liver cirrhosis, even has a minority may also have primary hepatocarcinoma.Show according to external and the geographic data of China Hong Kong and Taiwan, certain also data in some continent, chronic viral hepatitis B the infected, the patient of surface antigen positive just, finally probably have four/for a moment to develop into liver cirrhosis, this is very big from absolute number, so the social danger of hepatitis B virus also can not be ignored.
And hepatitis C has its feature, in case about 85% can become chronicity after infecting, rate has only 5% if the adult infects the hepatitis B chronicity, and the chronicity rate that infects hepatitis C is 85%, this ratio is very high, so not treating great majority, hepatitis C all can not become chronicity, so just more important to the timely diagnosis and the treatment of hepatitis C.Hepatitis C probably has 10% to 15% can develop into liver cirrhosis every year, has 1% to 7% can develop into hepatocarcinoma, and this ratio is very high, and exactly this point has been ignored by us.
The antiviral drugs of generally acknowledging both at home and abroad now mainly is two classes, and a class is an interferon, and a class is oral nucleoside medicine.Interferons divides two kinds again, a kind of common interferon, and a kind of is long lasting interferon, nucleoside medicine probably has four or five kinds, two big classes, seven or the eight kinds of medicines of adding up altogether.
This two classes medicine is each has something to recommend him, pluses and minuses are respectively arranged, curative effect is also different, such as interferons, antivirus action is not only arranged, immunoregulation effect is also arranged simultaneously, its advantage applies is having than higher e antigen serology conversion, be exactly that the great three positive said of common people changes into small three positive, and effectively the back curative effect can compare lastingly
General guide both domestic and external is thought, if chronic viral hepatitis B patient transaminase level is than higher, dna level is not very high, and patient is relatively younger again, so at first can consider interferon therapy, might obtain reasonable curative effect, pay the utmost attention to long-acting interferon as far as possible, wish by the limited course of treatment, such as 1 year, a year and a half or 2 years, can realize e antigen serology conversion, patient can be more stable after the drug withdrawal, can avoid taking medicine for a long time.Therefore interferon is still one of choice drug of chronic viral hepatitis treatment, but chronic hepatitis B patient leukopenia occurs in using the interferon anti-reflecting virus therapeutic process.Therefore being badly in need of a kind of medicine substitutes present interferon hepatopathy is treated.
Summary of the invention
Purpose of the present invention provides a kind of Chinese medicine Caulis Marsdeniae Tenacissimae extract for the treatment of hepatitis.
Another object of the present invention has provided the preparation method of this Chinese medicine Caulis Marsdeniae Tenacissimae extract.
The 3rd purpose of the present invention has been to provide the application of this Chinese medicine Caulis Marsdeniae Tenacissimae extract in preparation anti-hepatitis B virus, transaminase lowering level and adjusting immune drug.
Technical scheme of the present invention is as follows:
A kind of Chinese medicine Caulis Marsdeniae Tenacissimae extract is characterized in that, described Chinese medicine extract comprises the total phenolic acid of effective site and the total saponins of Caulis Marsdeniae Tenacissimae, by following (i) and (ii) two kinds of different paths obtain:
(i), the crude drug Caulis Marsdeniae Tenacissimae pulverized after, add 4～20 times of amounts, concentration and be 60%～90% ethanol, reflux, extract, 1～3 time, extract the Caulis Marsdeniae Tenacissimae extract that acquisition in 1～3 hour contains total phenolic acid and total saponins at every turn;
Or (ii), total phenolic acid and total saponins in the crude drug Caulis Marsdeniae Tenacissimae are extracted respectively, then total phenolic acid and total saponins are mixed, obtain to contain the Caulis Marsdeniae Tenacissimae extract of total phenolic acid and total saponins; Wherein total phenolic acid is by extracting with 10%～95% alcohol heating reflux or the supersound extraction acquisition; Total saponins is to obtain by a kind of extraction in solvent method, the sedimentation method, the cholesterol sedimentation method or the two-phase acid-hydrolysis method.
Chinese medicine Caulis Marsdeniae Tenacissimae extract described in the technique scheme, wherein, the extract in the described approach (i) is by after the crude drug Caulis Marsdeniae Tenacissimae is pulverized, and adds 6～20 times of amounts, concentration and be 60%～80% ethanol, reflux, extract, 3 times, extracts acquisition in 2 hours at every turn;
Described approach (ii) in total phenolic acid carry out behind 10%～95% ethanol ultrasonic extraction that macroporous resin is refining to be obtained.
Chinese medicine Caulis Marsdeniae Tenacissimae extract described in the technique scheme, wherein, the extract in the described approach (i) is by after the crude drug Caulis Marsdeniae Tenacissimae is pulverized, and adds 20 times of amounts, concentration and be 60% ethanol, reflux, extract, 3 times, extracts acquisition in 2 hours at every turn.
The application of Chinese medicine Caulis Marsdeniae Tenacissimae extract described in the technique scheme in preparation anti-hepatitis B virus, transaminase lowering level and adjusting immune drug.
The preparation method of the Chinese medicine Caulis Marsdeniae Tenacissimae extract described in the technique scheme, by following (i) and (ii) two kinds of different paths acquisitions, wherein:
(i), the crude drug Caulis Marsdeniae Tenacissimae pulverized after, add 10～20 times of amounts, concentration and be 60%～90% ethanol, reflux, extract, 1～3 time, extract the Caulis Marsdeniae Tenacissimae extract that acquisition in 1～3 hour contains total phenolic acid and total saponins at every turn;
Or (ii), total phenolic acid and total saponins in the crude drug Caulis Marsdeniae Tenacissimae are extracted respectively, then total phenolic acid and total saponins are mixed, obtain to contain the Caulis Marsdeniae Tenacissimae extract of total phenolic acid and total saponins; Wherein total phenolic acid is by extracting with 10%～95% alcohol heating reflux or the supersound extraction acquisition; Total saponins is to obtain by a kind of extraction in solvent method, the sedimentation method, the cholesterol sedimentation method or the two-phase acid-hydrolysis method.
Preparation method described in the technique scheme, wherein, the extract in the described approach (i) is by after the crude drug Caulis Marsdeniae Tenacissimae is pulverized, and adds 15～20 times of amounts, concentration and be 60%～80% ethanol, reflux, extract, 3 times, extracts acquisition in 2 hours at every turn;
Described approach (ii) in total phenolic acid carry out behind 10%～95% ethanol ultrasonic extraction that macroporous resin is refining to be obtained.
Preparation method described in the technique scheme, wherein, the extract in the described approach (i) is by after the crude drug Caulis Marsdeniae Tenacissimae is pulverized, and adds 20 times of amounts, concentration and be 60% ethanol, reflux, extract, 3 times, extracts acquisition in 2 hours at every turn.
Chinese medicine Caulis Marsdeniae Tenacissimae extract of the present invention can also add pharmaceutically acceptable carrier, technical method according to this area is prepared into various dosage forms such as mixture, granule, tablet, capsule, drop pill, pill, patch, unguentum, tincture, lozenge, spirit, suppository, liniment, injection, is applied to anti-hepatitis B virus, transaminase lowering level, regulates immunity, metabolism syndrome etc.
Effective ingredient in Chinese, be meant that the class in Chinese medicine simply or the herbal mixture extract or the content of a few class chemical constituents reach more than 50% of total extract, and this class or a few class known chemical composition be considered to effective ingredient, and the mixture of this class or several constituents promptly is considered to effective site.
The present invention has following beneficial effect:
The prepared Caulis Marsdeniae Tenacissimae extract that contains total phenolic acid of effective site and total saponins can effectively prevent leukopenia and have to alleviate myelosuppressive effect, and no obvious toxic-side effects is economical, convenient.Its main component steroidal saponin constituents and liposoluble ingredient, these compositions have effects such as antimutagenic, antibiotic, antiviral, and phenolic acid makes the effect to the normal cell reverse of human liver cancer cell, people's adenocarcinoma of stomach cell and human lung adenocarcinoma cell in addition except that the function with kill cancer cell; And have antioxidation, an immunoregulation effect preferably.
The specific embodiment
For making technical scheme of the present invention be convenient to understand, the present invention is further illustrated below in conjunction with the specific embodiment.
Embodiment 1:The preparation of Caulis Marsdeniae Tenacissimae extract
The weighting raw materials Caulis Marsdeniae Tenacissimae, crushing screening adds 4 times of amounts, concentration and is 90% ethanol, reflux, extract, 1 time, extracts 1 hour at every turn, obtains to contain the Caulis Marsdeniae Tenacissimae extract of total phenolic acid and total saponins.
Embodiment 2:The preparation of Caulis Marsdeniae Tenacissimae extract
The weighting raw materials Caulis Marsdeniae Tenacissimae, crushing screening adds 20 times of amounts, concentration and is 60% ethanol, reflux, extract, 3 times, extracts the Caulis Marsdeniae Tenacissimae extract that obtained to contain total phenolic acid and total saponins in 2 hours at every turn.
Embodiment 3:The preparation of Caulis Marsdeniae Tenacissimae extract
The weighting raw materials Caulis Marsdeniae Tenacissimae, crushing screening adds 6 times of amounts, concentration and is 75% ethanol, reflux, extract, 2 times, extracts the Caulis Marsdeniae Tenacissimae extract that obtained to contain total phenolic acid and total saponins in 2 hours at every turn.
Embodiment 4:The preparation of Caulis Marsdeniae Tenacissimae extract
The weighting raw materials Caulis Marsdeniae Tenacissimae, crushing screening extracts or supersound extraction obtains to contain the extract of the total phenolic acid of effective composition with 10% alcohol heating reflux;
Medicinal residues after the reflux, extract, are extracted by solvent method, be specially: use methanol or Diluted Alcohol slightly to carry as solvent, extracting solution is after reclaiming solvent, dilute with water, again through n-butanol extraction or purification with macroreticular resin, after getting thick saponin, the reuse silica gel column chromatography separates the extract that obtains to contain effective composition total saponins;
The extract that contains total phenolic acid of effective composition and total saponins that obtains is mixed, obtain containing the Caulis Marsdeniae Tenacissimae extract of total phenolic acid of effective composition and total saponins.
Embodiment 5:The preparation of Caulis Marsdeniae Tenacissimae extract
The weighting raw materials Caulis Marsdeniae Tenacissimae, crushing screening is used 95% ethanol ultrasonic extraction, obtains to contain the extract of the total phenolic acid of effective composition;
Adopt the sedimentation method to extract to the thick saponin that obtains among the embodiment 4, be specially: thick saponin is dissolved in small amount of methanol or the ethanol, dropwise add ether, acetone or ether-acetone (1: 1) mixture, the precipitant of separating out after even is the extract that contains effective composition total saponins, continues to add ether and can obtain the higher extract that contains effective composition total saponins of purity;
The extract that contains total phenolic acid of effective composition and total saponins that obtains is mixed, obtain containing the Caulis Marsdeniae Tenacissimae extract of total phenolic acid of effective composition and total saponins.
Embodiment 6:The preparation of Caulis Marsdeniae Tenacissimae extract
The weighting raw materials Caulis Marsdeniae Tenacissimae, crushing screening behind 60% ethanol ultrasonic extraction, adopts macroporous resin refining, obtains to contain the extract of the total phenolic acid of effective composition;
Adopt the cholesterol sedimentation method to extract to the thick saponin that obtains among the embodiment 4, be specially thick saponin is dissolved in the small amount of ethanol, add the saturated alcoholic solution of cholesterol, utilization utilizes steroidal saponin to generate the molecular complex of slightly solubility with cholesterol and obtains precipitation, must precipitate after the filtration successively and wash to remove saccharide, pigment, oils and fats and free cholesterol through water, alcohol, ether, go out cholesterol with ether extraction after super-dry, the residue that obtains is the purer extract that contains effective composition total saponins.
The extract that contains total phenolic acid of effective composition and total saponins that obtains is mixed, obtain containing the Caulis Marsdeniae Tenacissimae extract of total phenolic acid of effective composition and total saponins.
Embodiment 7:The preparation of Caulis Marsdeniae Tenacissimae extract
The weighting raw materials Caulis Marsdeniae Tenacissimae, crushing screening behind 60% ethanol ultrasonic extraction, adopts macroporous resin refining, obtains to contain the extract of the total phenolic acid of effective composition;
Adopt the two-phase acid-hydrolysis method to extract to the thick saponin that obtains among the embodiment 4, the aglycon of generation can be entered in the organic facies solvent and reduce time of contact of aglycon and acid, avoided the destruction of aglycon.The extract that contains effective composition total saponins.
The extract that contains total phenolic acid of effective composition and total saponins that obtains is mixed, obtain containing the Caulis Marsdeniae Tenacissimae extract of total phenolic acid of effective composition and total saponins.
Below the beneficial effect of further setting forth the present invention and had by the pharmacodynamics test of Caulis Marsdeniae Tenacissimae extract of the present invention.
Test example one:Caulis Marsdeniae Tenacissimae extract is to the effect of hepatic injury
Respectively with 10.54,5.27, high, medium and low three dosage of 2.63g crude drug/kg body weight irritate stomach and give mice.
1, experiment material
1.1 be subjected to the reagent thing
Caulis Marsdeniae Tenacissimae extract of the present invention, 1 gram extractum is equivalent to the 2.70g crude drug approximately.
1.2 dosage design
The clinical people's consumption per day of Caulis Marsdeniae Tenacissimae extract is 36～45g crude drug, and getting intermediate value is 40.5g, and test is pressed the conversion of animals and human beings kg body weight with dosage, and (simple method: rat is pressed 200g, coefficient 0.018; Mice is pressed 20g, coefficient 0.0026), middle dosage is equivalent to people's clinical equivalent dosage approximately, respectively establishes a dosage group by 1/2 and 2 times of clinical dosage again.Rat oral gavage dosage is 7.30,3.65,1.82g crude drug/kg, and mouse stomach dosage is 10.54,5.27,2.63g crude drug/kg.4,2,1,0.5,0.25g/L 2 dosage levels are the highest administration concentration below the cytotoxicity to occur to give 2.2.15 cell drug dosage, and 5 dilution factors are respectively:.
1.3 medicine and reagent
Liver-protecting tablet, 5 constant virtues, Heilongjiang Province sunflower pharmaceutcal corporation, Ltd, lot number: 20080631; Animals administer dosage is pressed the conversion of animals and human beings kg body weight, and rat clinical equivalent dosage is 0.4g/kg (ig), and mice is 0.6g/kg (ig).Levamisole hydrochloride (levamisole), Shandong Kernel and Hall Pharmaceutical Industry Co., Ltd., lot number 080702; Cyclophosphamide for injection: lot number: 20070102, SHANXI POWERDONE PHARMACEUTICAL.,LTD.Mice is 40mg/kg (ig).Bifendate drop pill, Beijing XieHe medicine Factory, lot number: 08030202; Olive oil, Chemical Reagent Co., Ltd., Sinopharm Group, lot number: F20081006; The ginsenoside; India ink is Beijing Solarbio company product; ANIT (Alfa Aesar, lot 10137323); ALT (ALT), aspartic acid aminotransferase (AST), total bilirubin (TBIL) are Beijing northization fine chemicals company limited reagent for clinical diagnosis subsidiary factory product; Na
+-K
+-ATP enzyme reagent kit, Coomassie brilliant blue protein determination kit, MDA measure test kit, SOD measures test kit and (is Nanjing and builds up Science and Technology Ltd.'s product, product batch number: 20090807); Interleukin-6 (IL-6) is put inspection-free test agent box, provides lot number by Beijing pul great achievement bio tech ltd: 080225; All the other reagent are all analytical pure.
1.4 instrument
BS120 full automatic biochemical apparatus (Mairui Biological Medical Electronic Co., Ltd., Shenzhen); Agilent-8453 ultraviolet spectrophotometer (hewlette-packard); The SN-695B type is put and is exempted from γ measuring instrument (Shanghai day ring instrument one factory); TGL-16C centrifuge (Anting Scientific Instrument Factory, Shanghai).
1.5 laboratory animal
SPF level Kunming mouse, 18～22g.Provide credit number by Test Animal Centre, Academy of Military Medical Sciences, P.L.A: [SCXK-(army), 2007-004].
1.6 detect index and method
Serum ALT, AST activity, IL-6 content and hepatic tissue SOD activity and MDA content are all operated by the test kit description.Liver histopathology is observed fixedly hepatic tissue of 40g/L formalin, specimens paraffin embedding slices, and conventional haematoxylin Yihong (HE) dyeing is observed, is taken pictures under the light microscopic.
1.7 statistical procedures software
The SPSS for Windows of SPSS Inc. 17.0.Adopt single sample variance analytic statistics methods, adopt method of least square to compare in twos between each group.
2 methods and result
2.1 Caulis Marsdeniae Tenacissimae extract causes the influence of mouse liver injury to carbon tetrachloride
Get mice, be divided into six groups at random, normal control group, model control group, liver-protecting tablet group, high, medium and low three the dosage groups of Caulis Marsdeniae Tenacissimae extract.Every group 10, gastric infusion, each 0.5ml/, every day 1 time, the normal control group is given distilled water, a continuous week with method.Except that the normal control group, 1h after the last administration, every group of ip 0.1% CCl
4Soybean oil solution, behind the fasting 16h, mouse orbit is got blood, and separation of serum is measured ALT and AST.Get the accurate title of fresh lobus sinister hepatic tissue and decide 0.2g, add normal saline homogenate in ice bath of 9 times of amounts, homogenate is got supernatant with the centrifugal 10min of 4000r/min, presses the test kit description and measures the activity of SOD and the content of MDA.Other cuts the liver sub-fraction, and to put into 10% formalin solution fixing, makes pathological section.
Table 1 Caulis Marsdeniae Tenacissimae extract causes the influence of mouse liver injury to carbon tetrachloride
| Group | Number of animals (only) | ALT(u/L) | AST(u/L) | SOD(nmol/mgprot) | MDA(u/mgprot) |
| The normal control group | 10 | 34.3±3.6 | 126.1±16.2 | 23.55±2.89 | 4.18±1.90 |
| Model group | 10 | 625.6±226.9 ## | 543.5±218.9 ## | 30.51±6.96 ## | 7.00±2.29 ## |
| The liver-protecting tablet group | 10 | 480.4±157.4 ## | 380.4±95.6 ##，* | 46.09±6.04 ##，** | 7.40±1.93 # |
| The Caulis Marsdeniae Tenacissimae extract height | 10 | 459.7±210.9 ##，* | 352.9±124.1 ##，** | 44.26±7.62 ##，** | 5.43±2.03 |
| In the Caulis Marsdeniae Tenacissimae extract | 10 | 472.9±127.5 ##，* | 418.4±152.9 ## | 42.93±4.23 ##，** | 6.08±2.32 # |
| Caulis Marsdeniae Tenacissimae extract is low | 10 | 494.4±155.6 ## | 514.1±153.8 ## | 37.04±3.82 ** | 6.79±1.59 ## |
Compare with the normal control group
#P＜0.05
##P＜0.01; Compare with model group
*P＜0.05
*P＜0.01.
The result shows: Caulis Marsdeniae Tenacissimae extract height, middle dosage group are to CCl
4Cause that ALT, AST and MDA rising has tangible reduction effect.The Caulis Marsdeniae Tenacissimae extract low dose group has the trend that comparatively significantly reduces ALT in the mice serum, AST and MDA content.But high, medium and low three the dosage group significances of Caulis Marsdeniae Tenacissimae extract improve CCl
4SOD content in the mouse liver.
2.2 the immunization of extract of the present invention
(the mice carbon granule is cleaned up examination 2.2.1 extract of the present invention is to the influence of mouse monokaryon cytophagy
Test):
Select for use Kunming mouse 20-24g/ only, complete male 60,12 every group, press table 2 grouping administration, irritate stomach every day 1 time, continuous 7 days, 30 minutes tail vein injection 10% india ink 0.1ml/10g behind the last get blood 20 μ l in 30 seconds, 5 minutes eye socket posterior veins, join and fill 2ml0.1%Na
2Co
3In the test tube of solution, shake up, measure optical density, claim liver, spleen weight, calculate phagocytic index K and engulf coefficient a in wavelength 675nm.Result such as table 2.
Table 2 extract of the present invention is to the influence of mouse monokaryon cytophagy
Brief summary: dosage and ginsenoside can obviously improve the mouse monokaryon cytophagy in the Caulis Marsdeniae Tenacissimae extract.But large and small dosage is produce effects not.
2.2.2 extract of the present invention is to the influence (hemolysin test) of mouse humoral immune function:
Select for use Kunming mouse 20-24g/ only, half and half 60 of male and female, every group 10, press table 20 grouping administration, irritate stomach every day 1 time, continuous 14 days, respectively organized lumbar injection 5% chicken erythrocyte suspension 0.2ml immunity on the 8th day, all the other respectively organize subcutaneous injection cyclophosphamide 20mg/kg except that immune matched group, the next day 1 time totally 4 times; Posterior orbit was got blood 20 μ l in the 15th day, joined to fill in the 1ml normal saline to shake up, and then added 5% chicken erythrocyte suspension 0.5ml; Temperature was bathed 30 minutes in 37 ℃ of water-baths, cessation reaction in ice bath, and centrifuging and taking supernatant 1ml joins in the test tube that fills 3ml Dou Shi liquid, leaves standstill 540nm wavelength colorimetric 10 minutes.Produce intensity with optical density (OD) size as hemolysin (antibody).The t inspection statistics significance of difference, result such as table 3.
Table 3 Caulis Marsdeniae Tenacissimae is to the influence of mouse humoral immune function
Brief summary: each dosage group of Caulis Marsdeniae Tenacissimae extract has the trend that improves the serum hemolysin growing amount, but no statistical significance.
Experimental result shows that Caulis Marsdeniae Tenacissimae extract has:
1, function for protecting liver and reducing enzyme activity: mice serum glutamate pyruvate transaminase (ALT), glutamic oxaloacetic transaminase, GOT (AST) rising that carbon tetrachloride (CCl4) is caused have certain inhibitory action.Histological examination, to the animal liver damage that is caused by CCl4, Caulis Marsdeniae Tenacissimae extract has the certain protection effect.
2, the result of the test to immunization shows: (1) shows mice hemolysin test result: each dosage group of Caulis Marsdeniae Tenacissimae extract of the present invention has the trend that improves the serum hemolysin growing amount, but no statistical significance; (2) mice carbon granule phagocytosis test result is shown: dosage can improve the mononuclear cell phagocytic activity in the Caulis Marsdeniae Tenacissimae extract of the present invention, points out Caulis Marsdeniae Tenacissimae extract of the present invention suitably can improving non-specific immunity in the dosage range.
Test example two:Caulis Marsdeniae Tenacissimae extract is anti-HBV effect experiment in HepG 2.2.15 cell culture
1 materials and methods
1. laboratory sample: extract of the present invention, be liquid, dilute with culture medium during experiment.
2. experimental technique: experiment is in the human hepatoma cell line HepG2 .2.15 of hepatitis B virogene transfection cell, and after the dosing 7 days: the research medicine was to the inhibitory action of hepatitis B virus.To the toxicity of HepG2.2.15 cell culture, measure the pathological changes that medicine causes cell with inverted microscope observation of cell pathological changes (CPE) method, be index with Reed-Muench method calculating poisonous concentration of half (TC50) and maximal non-toxic concentration (TC0).In cell culture supernatant HBsAg and the excretory inhibitory action of HBeAg: measure the antigen titre of cell culture supernatant with euzymelinked immunosorbent assay (ELISA) (ELISA), send out calculation of half inhibitory concentration (IC50) and selection index (SI) is an index with Reed-Muench.In pair cell is cultivated HBV-DNA inhibitory action: measure the intracellular HBV-DNA of cell culture with real-time fluorescence quantitative PCR method (qPCR), and, calculate suppression ratio (%) and IC50 and SI under the different pharmaceutical concentration respectively with after the house-keeping gene GAPDH correction in the cell.
3. result and conclusion: the 1st batch of the poisonous concentration of toxic half (TC50) of sample copy invention extract pair cell in cell culture is 0.05%, the 2nd batch is 0.18%, the 1st batch of non-toxic concn (TCO) is 0.0137%, the 2nd batch is 0.041%, the HBV-DNA suppression ratio is all less than 50% in the pair cell under Caulis Marsdeniae Tenacissimae extract maximal non-toxic concentration in 2 batches of experiments, and IC50 is respectively greater than 0.0137% and 0.041%; To supernatant HBsAg and the equal unrestraint effect of HBeAg.Positive drug 3TC when 1 μ g/ml in the pair cell HBV-DNA be about 800%.
Experimental result shows that Caulis Marsdeniae Tenacissimae extract has the hepatitis virus resisting effect, suppresses 2.2.15 cell HBsAg, and HBeAg duplicating and expressing, and Caulis Marsdeniae Tenacissimae has certain inhibitory action to hepatitis B virus.
The above, it only is preferred embodiment of the present invention, be not that the present invention is done any formal and substantial restriction, all those skilled in the art, in not breaking away from the technical solution of the present invention scope, when can utilizing the above technology contents that discloses, and a little change of making, modify the equivalent variations with differentiation, be equivalent embodiment of the present invention; Simultaneously, all foundations essence technology of the present invention all still belongs in the scope of technical scheme of the present invention change, modification and the differentiation of any equivalent variations that above embodiment did.
Claims (7)
1. the Chinese medicine Caulis Marsdeniae Tenacissimae extract is characterized in that, described Chinese medicine extract comprises the total phenolic acid of effective site and the total saponins of Caulis Marsdeniae Tenacissimae, by following (i) and (ii) two kinds of different paths obtain:
(i), the crude drug Caulis Marsdeniae Tenacissimae pulverized after, add 4～20 times of amounts, concentration and be 60%～90% ethanol, reflux, extract, 1～3 time, extract the Caulis Marsdeniae Tenacissimae extract that acquisition in 1～3 hour contains total phenolic acid and total saponins at every turn;
Or (ii), total phenolic acid and total saponins in the crude drug Caulis Marsdeniae Tenacissimae are extracted respectively, then total phenolic acid and total saponins are mixed, obtain to contain the Caulis Marsdeniae Tenacissimae extract of total phenolic acid and total saponins; Wherein total phenolic acid is by extracting with 10%～95% alcohol heating reflux or the supersound extraction acquisition; Total saponins is to obtain by a kind of extraction in solvent method, the sedimentation method, the cholesterol sedimentation method or the two-phase acid-hydrolysis method.
2. Chinese medicine Caulis Marsdeniae Tenacissimae extract according to claim 1, it is characterized in that: the extract in the described approach (i) is by after the crude drug Caulis Marsdeniae Tenacissimae is pulverized, add 6～20 times of amounts, concentration and be 60%～80% ethanol, reflux, extract, 3 times, extract acquisition in 2 hours at every turn;
Described approach (ii) in total phenolic acid carry out behind 10%～95% ethanol ultrasonic extraction that macroporous resin is refining to be obtained.
3. Chinese medicine Caulis Marsdeniae Tenacissimae extract according to claim 1 and 2, it is characterized in that: the extract in the described approach (i) is by after the crude drug Caulis Marsdeniae Tenacissimae is pulverized, add 20 times of amounts, concentration and be 60% ethanol, reflux, extract, 3 times, extract acquisition in 2 hours at every turn.
4. the application of the described Chinese medicine Caulis Marsdeniae Tenacissimae extract of arbitrary claim in preparation anti-hepatitis B virus, transaminase lowering level and adjusting immune drug in the claim 1～3.
5. the preparation method of the Caulis Marsdeniae Tenacissimae extract of Chinese medicine described in the claim 1, by following (i) and (ii) two kinds of different paths obtain, wherein:
(i), the crude drug Caulis Marsdeniae Tenacissimae pulverized after, add 10～20 times of amounts, concentration and be 60%～90% ethanol, reflux, extract, 1～3 time, extract the Caulis Marsdeniae Tenacissimae extract that acquisition in 1～3 hour contains total phenolic acid and total saponins at every turn;
Or (ii), total phenolic acid and total saponins in the crude drug Caulis Marsdeniae Tenacissimae are extracted respectively, then total phenolic acid and total saponins are mixed, obtain to contain the Caulis Marsdeniae Tenacissimae extract of total phenolic acid and total saponins; Wherein total phenolic acid is by extracting with 10%～95% alcohol heating reflux or the supersound extraction acquisition; Total saponins is to obtain by a kind of extraction in solvent method, the sedimentation method, the cholesterol sedimentation method or the two-phase acid-hydrolysis method.
6. preparation method according to claim 5, it is characterized in that: the extract in the described approach (i) is by after the crude drug Caulis Marsdeniae Tenacissimae is pulverized, add 15～20 times of amounts, concentration and be 60%～80% ethanol, reflux, extract, 3 times, extract acquisition in 2 hours at every turn;
Described approach (ii) in total phenolic acid carry out behind 10%～95% ethanol ultrasonic extraction that macroporous resin is refining to be obtained.
7. according to claim 5 or 6 described preparation methoies, it is characterized in that: the extract in the described approach (i) is by after the crude drug Caulis Marsdeniae Tenacissimae is pulverized, and adds 20 times of amounts, concentration and be 60% ethanol, reflux, extract, 3 times, extracts acquisition in 2 hours at every turn.
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| CN111690034A (en) * | 2020-07-07 | 2020-09-22 | 南京宸翔医药研究有限责任公司 | Preparation method and pharmaceutical composition of digitized green intelligent high-purity marsdenia tenacissima component group and marsdenia tenacissima glycoside H |
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