CN1427723A - Composition for treatment of B-type hepatitis and liver cirrhosis - Google Patents
Composition for treatment of B-type hepatitis and liver cirrhosis Download PDFInfo
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- CN1427723A CN1427723A CN01808972A CN01808972A CN1427723A CN 1427723 A CN1427723 A CN 1427723A CN 01808972 A CN01808972 A CN 01808972A CN 01808972 A CN01808972 A CN 01808972A CN 1427723 A CN1427723 A CN 1427723A
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Abstract
The invention provides a mixed extract of <i>Phellodendri</i> cortex and <i>Patrinia</i> that has a therapeutic effect on hepatitis B and liver cirrhosis.
Description
Technical field
The present invention relates to a kind of hardened compositions of hepatitis B regulating liver-QI that is used for the treatment of.More particularly, the present invention relates to comprise the pharmaceutical composition of the mixed extract of Cortex Phellodendri (Phellodendri cortex) as active component and Herba Patriniae (Patriniascabiosaefolia FISCH), said composition anti-hepatitis B virus activity and liver cirrhosis are effective, and has an antivirus action, active and the effectiveness in the hepatocellular regenerative process of fibrosis of immunological adjuvant also relates to the method for preparing this based composition.
Background technology
Although hepatitis B virus (HBV) is a DNA viruses, it is easy to sudden change.The HBV genome is made up of the double-stranded cyclic DNA of about 3.2kb, and has extremely compact texture.Open reading frame in the gene (ORF) is arranged in extremely folding mode, makes that an ORF and another ORF are partly or entirely overlapping, to support the different fragments of hereditary information.Do not resemble other DNA viruses, in infected cell, HBV experiences transcriptive process,reversed during its genome duplication.That is, produce full-length RNA at first, it is called as protogene group RNA (pgRNA) to represent its role in the genome duplication process.
In reproduction process, by the host RNA polymerase II DNA chain is transcribed into the RNA chain, then as template,, be different from the duplicating of common DNA viruses of the DNA chain that directly duplicates them by viral reverse transcription synthetic gene group DNA.As a result, compare with common RNA viruses, HBV demonstrates high-frequency sudden change.Although the sudden change in the HBV genome takes place at random, the speed and the frequency of propagation are constant.
Because the host produces body fluid and cell immune response to the deutero-albumen of HBV, in this class reaction, constantly undergo mutation in the virus of survival.Carry out although the hbv replication cascade is independent of host chromosome, the part of viral DNA can be incorporated in the host genome.When HBV DNA had been incorporated in the host genome, it is unstable that viral genome becomes, and the X protein of expressing therefrom starts transcribing of host cell, thereby cause the inhibition to the p53 gene function.If these conditions exist, hepatocarcinoma will take place.
Most of primary hepatoma originates from viral hepatitis.Hepatitis virus has been divided into A-E5 type.Recently, a kind of new Hepatitis virus-G type is cloned.Thereby the known hepatitis virus that has 6 types.Wherein, on epidemiology, hepatitis B virus (HBV) is relevant with the carcinogenesis in the liver with hepatitis C virus (HCV).The outer capsid of HBV directly contacts with host's immune system, because that part of being exposed from the teeth outwards of virus.
Thereby HBV keeps the change of its antigenic surface structure so that escape host's immune system, thereby survives.This class change in the antigenic structure demonstrates spectral signature in the evolution of disease or persistent infection, and becomes important factor.Because this class factor makes the host produce invalid immunoreation to virus, the T cell comprises the endocrine function of steroid, the afunction of medicine etc., and acute hepatitis develops into chronic form.
When the symptom of hepatitis continues at least 6 months, and in biochemical and serological research, find improvement, this hepatitis is classified as chronic hepatitis.Chronic hepatitis is divided into chronic persistent hepatitis and chronic active hepatitis again, and the latter develops into cirrhosis, liver failure, and because the progressive development even the death of tissue injury in the liver.The chronic hepatitis of being brought out by HBV or HCV demonstrates the height tendentiousness that develops into cirrhosis, and if further develop, can develop into hepatocarcinoma.Liver cirrhosis can comprise viral infection by a variety of causes, parasite, and posteriority syphilis, ethanol, medicine or chemical toxicity, gallbladder block (biliaryobstruction), hyperemia, hemachromatosis, hepatolenticular degenerations etc. cause.
In liver cirrhosis, observe the disappearance of hepatic parenchymal cells, follow the withering of reticulin structure of dimension canal curvature, cellulosic generates, the propagation of remaining hepatocyte and general connective tissue, and little by little atrophy of liver.Owing to following necrosis and regenerating in essence, the propagation of extracellular matrix causes the distortion of normal hepatocytes structure, and because connective tissue is bred, is changed by division or merging lobule.This change strangler venous blood flows, and causes a hypertension, and it causes ascites again, and side Zhi Xunhuan (since esophageal varicosis or umbilical vein expansion, the formation of so-called Jellyfish head (medusa head)), splenomegaly etc.The brief summary propagation form of finding in the male cirrhosis of HBV comprises inner cell (intimatecell), and the pro-cancer stage is observed the existence of the colony that is similar to the clone.Yet, observe and the initial relevant genetic abnormality of cancer in this stage end.
Based on their mechanism of action, the therapeutic agent that has been used for the treatment of chronic hepatitis B is divided into antiviral agent and immunological adjuvant.For example, antiviral agent comprises Ara-A, Ara-Amp, acycloguanosine, and suramin.As a kind of therapy of new treatment chronic hepatitis B, lamivudine, 3TC is in clinical experimental stage.Lamivudine is the nucleic acid derivative as oral antiviral agent, and because it suppresses the reverse transcriptase activity of HIV specifically, it has been used as a kind of AIDS therapy in western countries since nineteen ninety-five.Lamivudine is tested at chronic hepatitis B patient at first on one's body by Dienstas etc.During administration, the patient turns to HBV DNA feminine gender, and 19% patient shows and to return to normal ALT scope that this is illustrated in the HBV DNA feminine gender that prolongs in the serum.Yet Lamivudine has a shortcoming, promptly since toxicity it can not long term administration, and if stop administration, the propagation resilience of virus also returns to the initial period.Recently, find administration 6-12 month extended period, in the patient who shows the administration resistance, HBV mutation tolerance Lamivudine occurred.
The interferon that only has very limited effectiveness, a kind of immunological adjuvant also has been used to treat hepatitis B.Yet the standard administration continues 3-6 month.Although obtain better result by prolongation administration expectation, be accompanied by a large amount of side effect, and if stop administration, virus is recovered the propagation quilt and is returned to initial conditions.In treatment chronic hepatitis B process, follow the HBe antigen negative, the improvement of ALT level can be used as therapeutic index.Yet although some patients show the HBe antigen negative, they still keep active hepatitis state.
After all, so far also without any the method for practicality of treatment liver cirrhosis.
Disclosure of an invention
Therefore, an object of the present invention is to provide a kind of pharmaceutical composition that is used for the treatment of chronic hepatitis B.
Another object of the present invention provides a kind of pharmaceutical composition that is used for the treatment of liver cirrhosis.
Another object of the present invention provides a kind of pharmaceutical composition that is used for antibody activity and T cell proliferation.
Another object of the present invention provides a kind of method for preparing the pharmaceutical composition for the treatment of hepatitis B.
Pharmaceutical composition by the treatment hepatitis B can reach purpose of the present invention, and this pharmaceutical composition comprises the Cortex Phellodendri for the treatment of effective dose and the mixed extract of Herba Patriniae.In addition, the compositions that comprises Cortex Phellodendri and Patrinia mixed extract according to the present invention also is used for liver cirrhosis treatment and antibody activity and T cell proliferation.
Patrinia comprises Patrinia scabiosaefolia FISCH., Patrinia villosa JUSS., and Patrinia sibirica JUSS., waiting is a kind ofly to comprise the perennial wild plant that grows in the hills of Korea S and the mountain region everywhere in the temperate zone.It uses with Herb (whole plant) form, and in the Orient in the medical science, in oculopathy, streptococcus pyogenes, edema is used for antiinflammatory action in the leucorrhea etc.
The heat extraction substrate of having reported Patrinia has antitumor action (as anti-uterus carcinoma, the esophageal carcinoma, intestinal cancer, and pulmonary carcinoma), and suppresses staphylococcus aureus, the growth of streptococcus etc. consumingly external.Extract is also known to have regeneration of damaged impairing the liver cell, prevents that their from degenerating, and improves the ability of portal venous circulation, causes cell cultured supernatant regeneration.It also acts on the central nervous system with calm neural, demonstrates intensive analgesic activity and hypotensive effect (hypotensive effect) and Urina Hominis (processed) effect.
The root of Patrinia comprises quintessence oil, various saponin, the alkaloid of saccharide and trace such as coumarin.In root corium, can find acetic acid, formic acid, valeric acid and alkaloid comprise chatinine and valerianine.Wherein, known valeric acid has intensive analgesic activity.Exsiccant seed contains the albumen of 19.4-19.9% and the fat of 30-34.4%.Yet, also almost the active component of Patrinia or the details of pharmacology's performance are not studied.
Phellodendron bark (cortex phellodendron), another kind of plant of the present invention is the skin of phellodendronamurense RUPRECHT, phellodendron amurense RUPRECHT is in the East Asia grows wild, and is foremost during Cortex Phellodendri belongs to kind.The mutation of other Cortex Phellodendri comprises P.latifoliolatumNakai, KAWAMOTO, P.japonicum OHWI, P.insulare NAKAI, P.molleNAKAI, and P.sargent.Cortex Phellodendri comprises several alkaloids of yellow or yellowish-brown pigment and 1.5-4.5%.Alkaloidal main component is a berberine.Also comprise palmatine (palmatine), magnoflorine, guanidine, jateorhizine, phellodendrine, Kan Naxin, Dauricine and, obacunone, obakulactone, β-sitoesterol etc. as bitter substance.These materials have intensive antibacterial activity, antihypertensive activity (hypotensiveactivity), and to central nervous system's inhibitory action, acetylcholine effect and antiinflammatory action, and known osteopathia and the jaundice of being used for traditionally.Cortex Phellodendri also is used for the treatment of typhoid fever, cholera and be used to nourish harmonization of the stomach normalization intestinal flora.Therefore, Cortex Phellodendri has been used for the treatment of gastritis, stomachache, and jaundice etc., as the bitter tonic agent, microbial ecological agent (probiotics) or antiinflammatory astringent.
Plant extract of the present invention has an important feature, and promptly except the propagation that suppresses HBV, it demonstrates the therapeutical effect to liver cirrhosis.Therefore, use the pharmaceutical composition of the present invention of the mixed extract of Patrinia and Cortex Phellodendri to demonstrate, and have the effect of curing inflammation and strengthening helper T cell in the host immune system simultaneously direct antivirus action by the hepatitis b virus infected hepatitis that causes.By the hepatocyte in the regenerated fiber hepatic tissue, recover the central vein in the liver, it also demonstrates the effect of treatment liver cirrhosis.
Comprise the Cortex Phellodendri for the treatment of effective dose and the mixed extract of Patrinia according to compositions of the present invention.Can be by the mixture of extraction Cortex Phellodendri and Patrinia or by obtaining mixed extract in conjunction with Cortex Phellodendri extract and Patrinia extract.
In the present invention, be in the extract of two kind of plant of admixture because cooperative effect demonstrates the good therapeutical effect of level.Therefore, the ratio of preferred two kind of plant is 2: 5-5: in 2 weight ranges.Think when the extract of this two kind of plant mixes in above-mentioned weight ratio, antivirus action of the present invention, cirrhosis therapeutical effect and immunoadjuvant function are best.
Compositions of the present invention can be regarded as a kind of of optimized therapeutic agent, because except having direct anti-HBV effect, it has realized in treatment hepatitis B process effectiveness greatly by strengthening the immune system relevant with the T cell, and demonstrates and have the very low hepatocellular effect of toxic regenerated fiberization.
According to another aspect of the present invention, a kind of method of pharmaceutical compositions is provided, this method comprises with aqueous solvent to be extracted the mixture of Cortex Phellodendri and Patrinia and filters extract; The precipitation of saturated filtrate and the coagulated protein that obtains by centrifugal removal under the pressure that improves; May be dissolved in material in the organic solvent with adding organic solvent in filtrate with removal, separate the also step in dry water-bearing layer then.
In a preferred embodiment of the invention, by mixing and grind the Patrinia and the Cortex Phellodendri of 1: 1 weight ratio, use tab water (tab water) or distilled water, heat is extracted mixture under the pressure that improves, remove precipitation by centrifugal extract, under the pressure that improves, boil once more extract with solidify subsequently by filter the residual protein of removing obtain Patrinia and Cortex Phellodendri in conjunction with extract., centrifugal with the saturated once more filtrate of steam with the removal precipitation to solidify residual protein, and then filter.Adding solvent such as chloroform or hexane in filtrate can be by this solvent-extracted any extraction composition to remove, and comprises resin or is dissolved in fibrous material in the filtrate.Use Talcum or analog purification water-bearing layer and lyophilizing so that blended extract powder to be provided thereafter.
Mixed extract of the present invention as above-mentioned preparation can be mixed with suitable pharmaceutical composition separately or with pharmaceutical carrier, is used as the hardened therapeutic agent of treatment hepatitis B regulating liver-QI by oral administration or injection then.
Can change the treatment effective dose of mixed extract of the present invention according to desired effects.Usually, for the adult patients oral administration, every day, the effective dose of every kg body weight can be in the 5-50mg scope, preferred 10-40mg.If suitable, mixed extract of the present invention also can with other medicines as being suitable for the therapeutic agent of hepatitis, antiinflammatory and antiviral agent are used in combination.Can comprise diphenyl dimethyl dicarboxylic ester with the medicine that compositions of the present invention is used in combination, Oleum Bulbus Allii, Radix Glycyrrhizae glucin, and silymarin.
The accompanying drawing summary
Fig. 1 a is the chart (HBe antigen and antibody) of the therapeutical effect of expression clinical implementation example 1.
Fig. 1 b is the chart (HBs antigen and antibody) of the therapeutical effect of expression clinical implementation example 1.
Fig. 2 a is the chart (HBe antigen and antibody) of the therapeutical effect of expression clinical implementation example 2.
Fig. 2 b is the chart (HBs antigen and antibody) of the therapeutical effect of expression clinical implementation example 2.
Fig. 3 a is the chart (HBe antigen and antibody) of the therapeutical effect of expression clinical implementation example 3.
Fig. 3 b is the chart (HBs antigen and antibody) of the therapeutical effect of expression clinical implementation example 3.
Fig. 4 a-4d is that expression is to accepting contrast respectively, DMN, the picture of the therapeutical effect of the liver cirrhosis of each group of the DMN+150mg/kg present composition and the DMN+500mg/kg present composition.
Fig. 5 represents the ultraviolet spectra available from the Patrinia extract.
Fig. 6 represents the ultraviolet spectrogram available from Cortex Phellodendri extract.
Fig. 7 represents available from the ultraviolet spectra according to the mixed extract of Patrinia of the present invention and Cortex Phellodendri.
Fig. 8 represents the HPLC chromatograph available from the Patrinia extract.
Fig. 9 represents available from the HPLC chromatograph according to the mixed extract of Patrinia of the present invention and Cortex Phellodendri.
Figure 10 represents the HPLC chromatograph available from Cortex Phellodendri extract.
Figure 11 represents available from the HPLC chromatograph according to the mixed extract of Patrinia of the present invention and Cortex Phellodendri.
Implement best mode of the present invention
Based on preparation and clinical implementation example, now the present invention will be described in more detail.Yet, be appreciated that these embodiment only are described for explanation, can not be interpreted as limiting the protection domain of request in this application, protection domain limits in accompanying Claim.Embodiment 1: the preparation of mixed extract
Get the sample aliquot of 100g powder, powder is by mixing and grinding exsiccant Cortex Phellodendri of equivalent and Patrinia acquisition, to wherein adding 3, the 000ml distilled water.Under the pressure that improves, because the saturated extraction of carrying out 50 minutes.The extract that collection obtains, and remove residue.Centrifugal subsequently chloroform extract precipitates to remove, and filters aqueous extract.It is 1500ml that the filtrate that obtains is concentrated into total amount, and then centrifugal.Thereafter, (121 ℃, 15 Pascals) saturated filtrate is 15 minutes under the pressure that improves, and by the concretion that centrifugal removal obtains, especially protein-contg precipitation is then filtered.Filtrate is moved to separatory funnel, and add the 400ml chloroform to extract resin and fibrous material etc.Then, separation and removal chloroform layer.Re-extract twice, and add the 200ml normal hexane extracting residual protein to the water-bearing layer, resin, fibrous material and other are dissolved in the material of normal hexane.Collect the water-bearing layer then, be heated to 60-80 ℃, stir the Talcum that adds 500g then.Filter the removal Talcum by suction, and filtrate is slowly filtered again, be lyophilized into powder then.Provide productive rate (based on dry weight) to be about about 15g extract of 15% like this from Cortex Phellodendri and Patrinia mixture.Embodiment 2: the preparation of alcohol extract
In the powder of 100g, add 1 by mixing and grinding exsiccant Cortex Phellodendri of equivalent and Patrinia acquisition, the methanol of 500ml 70%, and follow and intermittently stir placement 60 hours.Then mixture being carried out suction filters so that alcohol extract to be provided.Subsequently, after removing alcohol, be retained in the alcohol extract in the reflux condenser and sneak into 1, the 000ml distilled water, and with mixture heated to 100 ℃ maintenance 5 minutes, so that water-soluble substance dissolves.By adding the extract of an amount of Talcum preliminary purification muddiness, then stir and the suction filtration.The filtrate of purification is moved to separatory funnel, and add the 300ml chloroform to extract resin and fibrous material etc.Separate then and remove chloroform layer.In the water-bearing layer, add the 200ml normal hexane with the extraction residual protein, resin, fibrous material and other are dissolved in the material of normal hexane.Collect the water-bearing layer then, be heated to 60-80 ℃, stir an amount of Talcum of adding then.Filter the removal Talcum by suction, and filtrate is slowly filtered again, be lyophilized into powder then.Provide productive rate (based on dry weight) to be about about 10g extract of 10% like this from Cortex Phellodendri and Patrinia mixture.Example of formulations 1: tablet
The mixed extract of the freeze-dried powder form that 250mg is prepared in the foregoing description 1 in the U-shaped blender with the 260mg lactose, a kind of excipient, 35mg Avicell (microcrystalline Cellulose), 15mg starch gluconic acid sodium salt, a kind of disintegrating agent, with the low hydroxypropyl cellulose of 80mg, a kind of binding agent mixed 20 minutes.After mixing is finished, add the 10mg magnesium stearate, a kind of lubricant, then additional mixing is 3 minutes.After quantitative test and the moisture resistance test mixture is pressed into tablet and peplos, produces every tablet of tablet that contains the 250mg mixed extract.Example of formulations 2: syrup
Suitable amount of sucrose is dissolved in the water of scheduled volume, to wherein add 80mg to hydroxymethyl-benzoic acid salt (para-oxymethyl benzoate) and 16mg to propoxy benzoic acid salt as antiseptic.The mixed extract that adds the freeze-dried powder form that 4.5g prepares subsequently in the foregoing description 1, and remain on dissolving fully under 60 ℃ in temperature, after the mixture cooling, make 150ml by adding distilled water, a kind of syrup dosage form is provided.Example of formulations 3: capsule
The mixed extract of the freeze-dried powder form that 500mg is prepared in the foregoing description 1 is filled in the hard gelatin capsule to produce a kind of capsule formulation.Example of formulations 4: beverage
The mixed extract of the freeze-dried powder form that 500mg is prepared in the foregoing description 1 is dissolved in an amount of water, adds additive such as vitamin C, fumet such as citric acid, and sodium citrate, high fructose syrup, and add certain amount of sodium benzoate as antiseptic.Mixture is made cumulative volume is 100ml by adding entry then, to produce a kind of beverage formulations.Example of formulations 5: injection
The mixed extract of the freeze-dried powder form that 200mg is prepared in the foregoing description 1 contains in the normal saline of 1% polyoxyethylene hydrogenated Oleum Ricini at 200mg by heating for dissolving, contains the injection dosage form of 0.1% mixed extract with preparation.Clinical implementation example 1: to suffering from patient's administration present composition of chronic hepatitis B
Male's chronic hepatitis B patient oral administration of 38 years old is contained in the foregoing description 1 capsule formulation of the present composition of preparation, with the dosed administration of 2 capsules every day twice (2000mg/ days).The results are shown in Fig. 1.Fig. 1 illustrates with RIA (radioisotope analysis) method and checks the result of anti-HBV antibody, and points out the therapeutical effect of the present composition to hepatitis B.(Fig. 1 a:HBe antigen, Fig. 1 b:HBs antigen).Clinical implementation example 2: to suffering from the chronic hepatitis B patient administration present composition
36 years old male patient's oral administration that diagnosis is suffered from chronic hepatitis B contains the capsule formulation 11 months of the present composition, with the dosed administration of 2 capsules every day twice (2000mg every day).The result is the antibody that has produced anti-hepatitis B virus, thereby has almost removed HBe antigen and HBs antigen, makes the related antigen titre be reduced to about 0.(Fig. 2 a and Fig. 2 b) in the present embodiment because hepatitis is chronic, detects high-caliber antigen before the treatment beginning.The administration present composition causes producing the antigenic antibody of anti-HBe, and the result has reduced antigen, and is same owing to similar reason has also reduced HBs antigen.Therefore, administration finds that antigen valence is reduced to about 0 after initial 6 months.Clinical implementation example 3: to suffering from the chronic hepatitis B patient administration present composition
34 years old male patient's oral administration suffering from the chronic hepatitis relevant with HBV contained the capsule formulation 8 months of the present composition, with the dosed administration of 2 capsules every day twice (2000mg every day).The results are shown among Fig. 3 a and Fig. 3 b.As can be seen from the figure, the antigen titre of HBV is reduced to about 0.
The bright present composition of above-mentioned clinical implementation illustration has fabulous curative effect to the treatment hepatitis B.Experimental example 1: to the therapeutical effect of liver cirrhosis
Be the effect of the check present composition to liver cirrhosis, (40mg/kg i.p.) induces liver cirrhosis by injecting N-Nitrosodimethylamine (DMN) separately for rat.DMN causes tissue change, its in people's liver cirrhosis, find those are extremely similar, therefore, usually be used for the liver cirrhosis animal experiment.
24 Mus are divided into 4 groups every group 6, first group with comparing, only accept 40mg/kgDMN for second group, the 3rd winding is subjected to 40mg/kgDMN, follow 2 weeks of the dosed administration present composition with 150mg/kg, the 4th winding is subjected to 40mg/kgDMN, follows 2 weeks of the dosed administration present composition with 500mg/kg.To the 3rd group and the 4th group of administration present composition time, to first group and second group of administration normal saline.
After 2 weeks, fixing all animals of execution in 4% formalin for blood-sample withdrawal and excision hepatic tissue.Determine these indexes such as ALT, AST, bilirubin, albumin etc. from blood sample.For observing tectology,, and under optical microscope, check with the AZAN dyeing hepatic tissue of dyeing pathological product such as hyaline degeneration and fibrin according to pathological development.
By tissue being dipped into mordant (10% moisture potassium dichromate (k
2Cr
2O
7) the moisture trichloroacetic acid (CCl of 50ml+10%
3COOH) 5ml), under the temperature that improves, be dipped into then and carry out AZAN (azocazmine) dyeing in the azocazmine solution.After the dyeing, in aniline-alcohol, break up continuously, dyeed satisfactorily by other people and the remainder organized is decoloured until karyon, and with the fixing stain of acetic acid-alcohol.Then, use aniline blue-orange G solution-treated tissue, and break up with 100% ethanol, discharge opeing and sealing are to provide the microscope slide of making.
Fig. 4 is illustrated in the result of microscopically check.In Fig. 4 a, normal hepatic tissue shows that central vein is high-visible.In Fig. 4 b, blue region can be accredited as the fibrosis tissue in the inductive liver cirrhosis of DNA.In Fig. 4 c, when the administration 150mg/kg present composition, cirrhosis is enhanced, and this from the minimizing in blue dyeing zone as can be seen.In Fig. 4 d, when the present composition of administration 500mg/kg dosage, the fibrosis zone reverts to almost normal level as can be seen.Central vein also demonstrates the pattern that expression progressively recovers.
The The above results proof has the ability of regenerated fiber liver according to compositions of the present invention, and liver cirrhosis is had therapeutical effect.Experimental example 2: the immunoadjuvant function (strengthening the effect of the antibody generation that relies on the T cell in vivo) between the extract separately that compares the present composition and Cortex Phellodendri and Patrinia
25 Balb/c Mus (body weight 17-20g) are divided into 5 groups, 5 every group.(SRBC) migrates to intraperitoneal with sheep red blood cell (SRBC).Then, to the extract separately of every kind of Cortex Phellodendri of Mus oral administration and Patrinia, it is the composition of the present composition and is dissolved in the present composition in the water, and dosage is the 3-100mg/kg body weight, from transplanting day beginning administration 3 days (0 day, 1 day, 2 days).
For matched group, by same route of administration administration normal saline.After 4 days, put to death animal and their spleen of aseptic separation.Shred tissue, use the mesh separating Morr. cell, and in the culture dish that contains 3ml HBSS (Hank ' the equilibrated saline solution of s) with the plunger separating Morr. cell of syringe.
The cell suspension that obtains is moved in the 15ml conical tube, and left standstill 5 minutes.Centrifugal 10 minutes of the supernatant of removal 2ml and 1200rpm.Remove supernatant, cell is resuspended among the 2ml EBSS, dilution is 30 times in EBSS, is used for the plaque forming cells with 100 (μ l) aliquot and analyzes.With EBSS (25 μ l, target cell) washing SRBC, will among EBSS (25 μ l), dilute 2 times GPC and agarose (in EBSS, containing 0.85%, 350 μ l) mixing, and be poured in the culture dish to solidify, allow CO then at 37 ℃
2Placed about 1 hour in the incubator.Jerne plaque assay according to improvement is determined antibody forming cell's number.With antibody forming cell's number/10
6Form numeration and the record plaque and the cell of cell.
As a result, with regard to the present composition, antibody forming cell's number obviously greater than administration antibody forming cell's number of component extract separately, shows to have obtained more intensive immunological adjuvant effect in the present invention.This is confirmed in following table 1.Table 1. present composition is to the influence of the antibody producing of dependence T cell
Experimental example 3: the present composition is to the influence of t cell activation
| Dosage | Produce the cell number (per 10 of antibody 6Cell) | ||
| Cortex Phellodendri extract | The Patrinia extract | Mixed extract of the present invention | |
| Contrast | 2672±135 | ?2672±135 | ?2672±135 |
| ?3mg/kg | ?2845±245 | ?3212±454 | ?3690±98 |
| ?10mg/kg | ?2996±273 | ?4036±46 | ?6027±444 |
| ?30mg/kg | ?3391±484 | ?3920±105 | ?6610±374 |
| ?100mg/kg | ?4208±548 | ?4408±72 | ?8215±533 |
Determine the influence of the present composition with mixed immunity cell effect method to t cell activation.When hybrid separation when having the immunocyte of the antigenic Mus of different MHC classifications for two kinds, discern the antigenic T cell proliferation on other kind Mus cell.
From the Mus of two strains, B6C3F1 (H-2k) and BDF1 (H-2d), it has different MHC classification antigen mutually, and aseptic separation spleen also discharges splenocyte.The isolating immunocyte that obtains is adjusted to 2.5 * 10
6Cell/ml, and divide to 96 well culture plates with the amount in 100 μ l/ holes, and make 200 μ l.
To wherein adding the concentration of the present composition, culture plate is placed 37 ℃ of CO to 0.01-100 μ g/ml
2Cultivated 3 days in the incubator.Cultivate to stop preceding 18 hours, add 1 μ Ci/ hole [
3H]-thymidine.Use automated cell catcher harvesting, and pass through to measure [
3H]-thymidine picked-up determines the degree of immune cell propagation.
The result who enumerates in following table 2 points out that the mode with the content that relies on the present composition has strengthened the propagation of T cell.Table 2. present composition is to the influence of T cell proliferation
Experimental example 4: acute toxicity test
| Experimental condition | Propagation (DPM/250,000 cell) |
| Contrast | 3809±419 |
| This compositions 0.01 (μ g/kg) | 4329±522 |
| This compositions 0.03 | 5245±611 |
| This compositions 0.1 | 6769±916 |
| This compositions 0.3 | 7146±410 |
| This compositions 1 | 9008±1046 |
| This compositions 3 | 10243±1034 |
| This | 10792±611 |
| This compositions 30 | 11178±1332 |
| This | 11045±1364 |
(body weight 19 ± 1g) is divided into 5 groups, 6 every group with 30 normal female ICR Mus.Determine the oral median lethal dose(LD 50) (LD of mixed extract of the present invention in the Mus of oral administration mixed extract with the Behrens-Karber method
50P.o.), dosage is an A group 3g/kg body weight, and B organizes 6g/kg, and C organizes 9g/kg, and D organizes 12g/kg, and E organizes 15g/kg.Median lethal dose(LD 50) is the virtual value of expression medicine acute toxicity index.The safety of its indication present composition.
The result who enumerates in following table 3 represents not have Mus death, even in the group of administration 15g/kg body weight mixed extract, thereby can not determine the median lethal dose(LD 50) value.Therefore, the oral median lethal dose(LD 50) of mixed extract of the present invention is higher than the 15g/kg body weight, and this confirms that mixed extract of the present invention is very safe for human body.In other words, clearly, mixed extract of the present invention can be by administration safely and is not had remarkable toxicity.
Following obduction and the histopathological analysis of carrying out.In the ending of test, with all surviving animals of etherization and pass through sacrificed by exsanguination.Separate organ and with the naked eye check deformity.For carrying out histopathological analysis, organ is fixed at least 10 days in 10% neutral formalin solution, dehydration, be embedded into paraffin embedding center (Fisher then, Histomatic information processes machine 166A), and cuts into 5 μ l slices with rotary microtome (AO rotary microtome), its with hematoxylin-eosin staining after, be examined subsequently.
Find as follows from the dissection of all animals in every group and the histopathology of microscopy.When to Mus administration mixed extract of the present invention during to the 15g/kg body weight, the anomaly of in hepatic tissue, having no way of and producing in administration.At kidney, myocardial cell, gastrointestinal tract, pancreas, lung, spleen, the adrenal gland, brain, testis, ovary, bone marrow etc. are not observed anomaly yet.Therefore can infer that when with the maximal dose of 15g/kg body weight during to the Mus administration, mixed extract of the present invention does not demonstrate the side effect that the acute toxicity of any organ is produced owing to it.Table 3. is by the fatal dose (LD of oral administration mixed extract
50)
Annotate:
*Z: from the half value of the dead animal number of two successive dosage
*D: the difference experimental example 5 between two successive dosage: characterize mixed extract by the UV absorption spectrum
| Test group | Dosage (g/kg) | Oral (p.o.) | Intraperitoneal (i.p.) | *Z | **d |
| Total number of animals in dead animal number/group | Total number of animals in dead animal number/group | ||||
| ?A | ?3 | ?0/6 | ?0/6 | ?- | ?- |
| ?B | ?6 | ?0/6 | ?0/6 | ?0 | ?3.0 |
| ?C | ?9 | ?0/6 | ?3/6 | ?1.5 | ?3.0 |
| ?D | ?12 | ?0/6 | ?4/6 | ?3.5 | ?3.0 |
| ?E | ?15 | ?0/6 | ?6/6 | ?5.0 | ?3.0 |
Use ultraviolet spectrophotometer (Pharmacia, Ultrospec2000) and HPLC (HP1090) check mixed extract according to Patrinia of the present invention and Cortex Phellodendri.Fig. 5 is the ultra-violet absorption spectrum result of Patrinia extract (0.5mg/ml), is illustrated in the 282nm place and absorbs increase.Fig. 6 is the ultra-violet absorption spectrum result of Cortex Phellodendri extract (0.25mg/ml), and it absorbs increase at 278nm and 325nm place.Fig. 7 is the ultra-violet absorption spectrum result according to the mixed extract of Patrinia of the present invention and Cortex Phellodendri (0.5mg/ml), is illustrated in 281nm and 316nm place and absorbs increase.Experimental example 6: characterize mixed extract by HPLC
For with HPLC segregation, mixed extract of the present invention and Patrinia or the independent extract of Cortex Phellodendri are dissolved in respectively in the water, to concentration be 10mg/ml.From dissolved extract solution, get 10 μ l aliquots, and be used for HPLC (HP1090) analysis.
(analysis condition)
Post: Luna 5u C18; 4.6mm i.d. * 250mmL (Phenomenex)
Mobile phase: the gradient system of 2% acetic acid and methanol
Flow rate: 1ml/ branch
Detect: detect with photodiode array detector at the 254nm place
In repeated trials, observe the peak feature of extract separately.Fig. 8 represents the HPLC scattergram of Patrinia extract.Under the aforesaid operations parameter, 10.245,12.772, characteristic peak appearred in 14.104,17.087,18.769 minutes.Wherein, represent 3 at 10.952 minutes peak, the 4-protocatechuic acid, and expression content is 0.022%.Chlorogenic acid is represented at peak at 17.087 minutes, and its content is 0.238%.Represent caffeic acid (caffeine acid) at 18.769 minutes peak, and its content is 0.107%.When under condition same as described above, analyzing mixed extract of the present invention,, show the above-mentioned three kinds of materials that contain derived from Patrinia as can be seen from Figure 9 by HPLC, content be 0.020% 3,4-protocatechuic acid, 0.270% chlorogenic acid and 0.044% caffeic acid.The additional features peak occurred at 17.299,22.048 and 22.449 minutes.
Be to analyze berberine and palmatine, the abundantest alkaloid in the Cortex Phellodendri extract uses following condition.
(analysis condition)
Post: Luna 5u C18; 4.6mm i.d. * 250mmL (Phenomenex)
Mobile phase: second eyeball: water: KH
2PO
4: SDS=500: 500: 3.4: 1.7
Flow rate: 1ml/ branch
Detect: detect with photodiode array detector at the 254nm place
In the HPLC of Cortex Phellodendri extract analyzes, as shown in figure 10, detected palmatine at 10.629 minutes, content is 0.988%, detects berberine at 11.314 minutes, content is 1.963%.Yet, in mixed extract of the present invention, detecting palmatine at 11.751 minutes, content is 0.229%, detects berberine at 12.650 minutes, content is 0.405%, the tendency of the productive rate that this is equivalent to reduce in the mixed extract (Figure 11).
Industrial usability
Has the hepatocellular effect of fibrillatable in the hepatitis type B virus of inhibition and the Liver Regeneration sclerosis according to composition of the present invention. Especially, because it does not show disadvantageous side effect or induced drug resistance, but the long term administration said composition. Said composition also has the advantage that keeps the composition activity during the administration that prolongs. Therefore, composition of the present invention can be advantageously used in treatment hepatitis B and cirrhosis.
Claims (6)
1. pharmaceutical composition for the treatment of hepatitis B, it comprises the Cortex Phellodendri for the treatment of effective dose and the mixed extract of Herba Patriniae.
2. pharmaceutical composition for the treatment of liver cirrhosis, it comprises the Cortex Phellodendri for the treatment of effective dose and the mixed extract of Patrinia.
3. as the pharmaceutical composition that requires in claim 1 or 2, wherein said mixed extract has 1 by being extracted on the dry basis: 0.2-1: the Cortex Phellodendri of 5 weight ratios and the mixture of Patrinia prepare.
4. as the pharmaceutical composition that requires in claim 1 or 2, wherein said mixed extract is by with 1: 0.2-1: 5 weight ratios mixing Cortex Phellodendri extract and Patrinia extract prepare.
5. as the pharmaceutical composition that requires in claim 1 or 2, wherein said mixed extract is that dosage with the 10-50mg/kg body weight is to adult's administration.
6. as the pharmaceutical composition that requires in claim 1 or 2, it has been formulated into the tablet that adds pharmaceutical excipient, capsule, solution, suspension, syrup, or the form of beverage.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2000-0023564A KR100389131B1 (en) | 2000-05-02 | 2000-05-02 | Composition for B type Hepatitis and Cirrhosis of Liver Comprising Phellodendron amurense Rupr. cortex and Patrinia scabiosaefolia FISCH. Extract |
| KR200023564 | 2000-05-02 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1427723A true CN1427723A (en) | 2003-07-02 |
| CN100341537C CN100341537C (en) | 2007-10-10 |
Family
ID=19667915
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB018089720A Expired - Fee Related CN100341537C (en) | 2000-05-02 | 2001-05-02 | Composition for treatment of B-type hepatitis and liver cirrhosis |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JP4087114B2 (en) |
| KR (1) | KR100389131B1 (en) |
| CN (1) | CN100341537C (en) |
| AU (1) | AU2001256809A1 (en) |
| WO (1) | WO2001082941A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005239699A (en) * | 2004-01-30 | 2005-09-08 | Kobayashi Pharmaceut Co Ltd | Hepatic fibrosis inhibitory agent |
| JP2007297289A (en) * | 2006-04-27 | 2007-11-15 | Kitasato Gakuen | Antiviral agent |
| RU2593007C1 (en) * | 2015-03-12 | 2016-07-27 | Государственное бюджетное учреждение здравоохранения города Москвы Московский клинический научно-практический центр Департамента здравоохранения города Москвы | Method of treatment hepatic cirrhosis of various etiology |
| KR101934673B1 (en) * | 2018-05-23 | 2019-01-02 | 경희대학교 산학협력단 | Compositions for Treatment and Prevention of Inflammatory Disease Comprising Extract of Patrinia scabiosaefolia as Active Ingredient |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1056424A (en) * | 1990-05-11 | 1991-11-27 | 陈江辉 | The syrupy compound method of Fuganning |
| KR100328467B1 (en) * | 1994-12-21 | 2002-06-28 | 김광순 | Composition for improving liver function and treating hepatitis containing extract of valerianaceae |
| JPH107569A (en) * | 1996-06-18 | 1998-01-13 | Kureha Chem Ind Co Ltd | Hsp47 synthetic suppressant containing berberine derivative |
| KR100207293B1 (en) * | 1996-09-16 | 1999-07-15 | 김광순 | Immunostimulating composition containing mixed aqueous extract of phellodendri cortex and patrinia scabiosaefolia |
| KR100193216B1 (en) * | 1996-12-26 | 1999-06-15 | 김광순 | Treatment of acquired immunodeficiency syndrome containing mixed water extracts of Hwangbaekpi and Matari plants |
| KR19990015612A (en) * | 1997-08-07 | 1999-03-05 | 한영복 | Hepatitis C Therapeutic Composition Containing Mixed Extracts of Hwangbaekpi and Matari Plants |
| CN1236623A (en) * | 1998-05-21 | 1999-12-01 | 姜鹏凌 | Medicine for treating hepatitis B |
| CN1238187A (en) * | 1998-08-04 | 1999-12-15 | 姜彬 | Chinese herbal medicine for radically treating hepatitis B |
| KR100316596B1 (en) * | 1999-03-30 | 2001-12-12 | 김철호 | Extract of Phellodendria Cortex having activity of anticancer, antihepatitis and immunological enhancement |
-
2000
- 2000-05-02 KR KR10-2000-0023564A patent/KR100389131B1/en active IP Right Grant
-
2001
- 2001-05-02 JP JP2001579815A patent/JP4087114B2/en not_active Expired - Fee Related
- 2001-05-02 CN CNB018089720A patent/CN100341537C/en not_active Expired - Fee Related
- 2001-05-02 WO PCT/KR2001/000723 patent/WO2001082941A1/en active Application Filing
- 2001-05-02 AU AU2001256809A patent/AU2001256809A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| KR20010104497A (en) | 2001-11-26 |
| CN100341537C (en) | 2007-10-10 |
| JP2003531861A (en) | 2003-10-28 |
| JP4087114B2 (en) | 2008-05-21 |
| WO2001082941A1 (en) | 2001-11-08 |
| KR100389131B1 (en) | 2003-06-25 |
| AU2001256809A1 (en) | 2001-11-12 |
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