CN101116670B

CN101116670B - Application of compound capable of providing active methyl or of taking part in the diversion of methyl in the preparation of medicine for treating virosis

Info

Publication number: CN101116670B
Application number: CN2006100368451A
Authority: CN
Inventors: 邹清雁; 张晓元; 李远友; 李公信; 王尚武; 丁先风
Original assignee: GUANGZHOU HEZHU BIOTECHNOLOGY CO Ltd
Current assignee: SHAOGUAN HUAGONG HIGH-TECH INDUSTRY Research Institute
Priority date: 2006-08-01
Filing date: 2006-08-01
Publication date: 2010-11-10
Anticipated expiration: 2026-08-01
Also published as: WO2008017264A1; CN101116670A

Abstract

The invention relates to the application of a compound capable of providing active methyl or being involved in methyl transfer used in preparing medicine for curing viral diseases, wherein the compound capable of providing active methyl or being involved in methyl transfer is made from the combination of one or more of folic acid, vitamin B12, vitamin B6, vitamin B2, choline, betaine, serine and methionine; antiviral compound is made from the combination of one or more of interferons compounds and nucleoside analogues compounds; the viral diseases includes various types of viral hepatitis, encephalitis B, influenza and poultry influenza. The invention also provides a combination which is used to cure viral disease including at least one type of compound capable of providing active methyl or being involved in methyl transfer as well as at least one type of antiviral compound. The invention can substantially improve comprehensive antiviral effect of body and cell, and particularly the inhibition effect of known antiviral compounds against various viruses.

Description

Active methyl can be provided or participate in of the application of the chemical compound of Methyl transporters as the medicine of preparation treatment viral disease

Technical field

The present invention relates to the novel application of compound that a class can provide active methyl or participate in Methyl transporters, especially relate to of the application of this compounds as the medicine of preparation treatment viral disease.

Background technology

The multiple disease that viral infection causes, serious harm human beings'health and life.So far, the whole world has found to make virus kind more than 1200 of human disease, tens thousand of kinds of virus subtypes and variant.Since the eighties, newfound epidemic infectious diseases has 2nd/3rd from twentieth century, cause by viral infection, wherein, and the sickness rate height, the viral disease that hazardness is big has the acute and chronic hepatitis due to A, B, C, D, E and the G kind hepatitis virus; 8 kinds of retinitis, keratitis, interstitial pneumonia, encephalitis, genital herpes, herpes zoster and lip portion herpess etc. that the herpes virus hominis causes; Bronchitis, pneumonia, measles, parotitis and poliomyelitis etc. that respiratory virus infection causes; Acute gastroenteritis due to the enterovirus, traveller and infantile diarrhea etc.; First, Influenza B virus often causes the seasonal whole world or some areas to be very popular; The mortality rates such as hemorrhagic fever with renal syndrome, lassa fever, ebola hemorrhagic fever and dengue fever that caused by multiple virus are very high.

China is the viral hepatitis district occurred frequently, has 1.2 hundred million people to carry hepatitis B virus approximately.Hepatitis C also is a kind of common viral hepatitis, worldwide is widely current, and is the topmost cause of disease that Europe, the United States, Japan and other countries cause liver cirrhosis and hepatocarcinoma, also be China's post-transfusion hepatitis main diseases because of.The anti-HCV positive rate is about 1%～3% in the population of China, estimates that therefore the positive crowd of anti-HCV of China has 3,000 ten thousand approximately.At the year ends 2002, SARS virus begins to extend.Be badly in need of effectively specific treatment strategy and treatment new drug clinically.

Lamivudine and interferon are to use maximum antiviral drugs at present, are mainly used in the treatment of hepatitis B and hepatitis C.The curative effect of interferon or lamivudine and other drug (comprising Chinese herbal medicine) therapeutic alliance chronic hepatitis B is still needed and is further confirmed.

Meta-analysis shows, the HBeAg positive patient is after common interferon-ALPHA (common IFN α) is treated 4～6 months, treatment group and not treatment group HBV DNA change bright rate (hybrid method) and are respectively 37% and 17%, the HBeAg negative conversion rate is respectively 33% and 12%, and the HBsAg negative conversion rate is respectively 7.8% and 1.8%.

Random contrast clinical trial shows both at home and abroad, oral 100mg lamivudine every day (lamivudine) can obviously suppress the HBV dna level, HBeAg serology conversion ratio prolongs with treatment time and improves, and treats that the HBeAg frequence of seroconversion is respectively 16%, 17%, 23%, 28% and 35% after 1,2,3,4 and 5 year.Adefovir ester is the no ring analogues of 5 '-single phosphoric acid deoxidation vidarabine.The clinical trial of randomized, double-blind placebo shows, HBeAg positive chronic hepatitis B patient, oral adefovir ester can obviously suppress the HBV dna replication dna, (＜1000 copies/ml) be respectively 28%, 45% and 56%, HBeAg serology conversion ratio is respectively 12%, 29% and 43% to HBVDNA negative conversion rate when using 1,2,3 year.The male hepatitis B patient of HBeAg over half is still arranged, existing Drug therapy, HBeAg can not turn out cloudy, and reason is unclear.

It is the main paathogenic factor of chronic hepatopathy that hepatitis C infects.Standard care adds ribavirin for poly-dealing with alcohol alpha-interferon, and approximately only effective in cure to the patient of half, the reason of second half failure is unclear.

Therefore, seeking hepatitis B and the more effective medicine of hepatitis C, is a big difficult point of present hepatopathy research field.For other viral diseases, also there is similar problem, need to seek better medicine.

Summary of the invention

The object of the present invention is to provide a class that the novel application of compound of active methyl or participation Methyl transporters can be provided, be of the application of this compounds as the medicine of preparation treatment viral disease, this compounds is enhancing body and the comprehensive antiviral effect of cell obviously, especially obviously strengthens the inhibition effect of known antiviral compound to each viroid.

The chemical compound that active methyl is provided or participates in Methyl transporters of the present invention is selected from one or more combination of folic acid, vitamin B12, vitamin B6, vitamin B2, choline, betanin, serine and methionine, is preferably betanin or betanin, folic acid, vitamin B12 three share.

Antiviral compound of the present invention is selected from one or more combination of interferons chemical compound, nucleoside analogues compounds.

The nucleoside analogues compounds comprises: Abacavir, acyclovir, Ah Fu Wei, amprenavir, atazanavir, Delavirdine, didanosine, Yi Feiweilun, emtricitabine, En Fuwei ground, famciclovir, ganciclovir, indinavir, lamivudine, viracept see nelfinaivr, how flat, the Oseltamivir of Wella, penciclovir, rimantadine, ritonavir, Saquinavir, stavudine, tenofovir, only cut down Xi Luowei, Zha Namiwei, zidovudine, foscarnet sodium, etc.

Described viral disease comprises various viral hepatitis, encephalitis B, influenza and avian influenza.

The present invention also provides a kind of compositions that is used for the treatment of viral disease.

A kind of compositions that is used for the treatment of viral disease of the present invention comprises: at least a chemical compound that active methyl is provided or participates in Methyl transporters; And at least a antiviral compound.

The described chemical compound that active methyl is provided or participates in Methyl transporters is selected from one or more combination of folic acid, vitamin B12, vitamin B6, vitamin B2, choline, betanin, serine and methionine, is preferably betanin or betanin, folic acid, vitamin B12 three share.

Described antiviral compound is selected from one or more combination of interferons chemical compound, nucleoside analogues compounds

The present invention also provides a kind of pharmaceutical composition that is used for the treatment of viral disease.

A kind of pharmaceutical composition that is used for the treatment of viral disease of the present invention comprises: at least a chemical compound that active methyl is provided or participates in Methyl transporters of dose therapeutically effective; At least a antiviral compound of dose therapeutically effective; And acceptable carrier on the materia medica.

Described antiviral compound is selected from the interferons chemical compound, one or more combinations of nucleoside analogues compounds.

Described pharmaceutical composition is mixed with and is selected from injection, injectable powder, capsule, tablet, oral liquid, one of oral, chewable tablet, inhalant, cutaneous permeable agent dosage form.

Described pharmaceutical composition is mixed with the dosage form that is selected from one of injection, injectable powder, capsule, tablet, oral liquid, chewable tablet.The clinical practice approach is based on oral, but also can make oral, sublingual administration, intranasal or various drug administration by injection or rectum or suction or transdermal means administration with various excipient, and various dosage forms or the mode that can make in the practical application enter human body.

The present invention is by various animal models and experimental results show that, the chemical compound energy enhancing body anti-virus ability of active methyl or participation Methyl transporters can be provided, obviously the comprehensive antiviral effect of enhancing body and cell especially obviously strengthens the inhibition effect of known antiviral compound to various viruses.Because this compounds convenient sources, extensively, the extracting method maturation, therefore the invention provides a series of inexpensive, treat the medicine of viral disease efficiently.

The specific embodiment

Definition: when specific descriptions are of the present invention, be necessary some term that will use is herein defined and explains, in order to the understanding of the present invention.

Active methyl can be provided or participate in the chemical compound of Methyl transporters: be meant the relevant element general name of (Methylation) function that methylates in a class and the cell, they generally comprise: folic acid, vitamin B12, vitamin B6, vitamin B2, choline, betanin, serine and methionine etc., they have all participated in the transfer process of one carbon unit (methyl) in the cell.The major physiological function of active methyl is as methylated source and donor; the process of methylating is exactly under the effect with the relevant in vivo methylase of these methyl micromolecule in the body; connect mutually with respective substance; bringing into play corresponding biological function, playing a role aspect the expression of cell differentiation, gene regulation, inhibition aberrant gene and the cancer formation as dna methylation.Have at least in the body 37 kinds of methylase peace treaties have 100 surplus kind of material can change its biologic activity through methylating, therefore, methylating has crucial physiology biological significance in human body.

Antiviral compound: be meant that a class has the treatment or the chemical compound of prophylactic action to viral disease, this compounds comprises that studying, in the exploitation and commercially available chemical compound.This compounds includes but not limited to: interferons chemical compound, nucleoside analogues compounds etc.This compounds can be that one or more chemical compounds are used in combination in the present invention, also can be successively to use.

Embodiment one, betanin are to CCl ₄The influence of the rat acute hepatic injury of damage

Present embodiment utilizes CCl ₄Make the hepatic injury mouse model, observe the protective effect of betanin the Mouse Liver infringement.

1, materials and methods: 32 of 1.1 healthy male HIH mices, provide by Zhongshan University's medical college Experimental Animal Center, body weight 18～20g, sub-cage rearing, standard diet is freely drunk water, and is used for experiment after one week at laboratory rearing.

1.2 divide 4 groups at random, blank group, carbon tetrachloride (2.0ml/kg) model group and high and low dose betaine group, 8 every group.The betaine group dosage of high and low two dosage is 600mg/kg and 300mg/kg respectively, gastric infusion, continuous 10 days, CCl ₄Behind the contamination 24h, win eyeball, get blood, separation of serum.With the mice sacrificed by decapitation, get liver immediately, wash residual blood with normal saline, be fixed in 10% formalin that pH7.4PBS is made into paraffin embedding, section, hematoxylin-eosin staining then.Observe down in light microscopic.

1.3 alanine aminotransferase in the serum (ALT) and aspartic transaminase (AST) are measured with reitman-frankel method, unit is a U/L serum.

2, result and discussion

2.1 morphological observation is the result show, CCl ₄Group, mouse liver cell bar rope arrangement disorder, karyopyknosis appears in the extensive hydropic degeneration of hepatocyte, necrocytosis, and inflammatory cell infiltration and central vein hyperemia are arranged.And give betaine group, and hepatic injury then obviously alleviates, and the lobules of liver structure is clear, and visible hepatic cords still radially distributes around central vein.The small amount of water sample degeneration is only arranged, a small amount of hepatic necrosis, but do not have obvious inflammatory cell infiltration and central vein hyperemia.Light microscopy checking shows that betanin obviously alleviates CCl ₄Damage to hepatic tissue.

2.2 serum zymetology result shows that betanin can reduce CCl ₄The ALT of the acute liver damage mice of causing, the AST activity also is dosage relevant (table 1).

Table 1 betanin is to CCl ₄Hepatic injury mice serum transaminase's influence (x ± s, n=8)

^*P＜0.05, ^*P＜0.01 (same CCl ₄Group relatively)

2.3 the result shows: betanin has protective effect to acute chemical hepatic injury.

Embodiment two, betanin are to CCl ₄The influence of chronic hepatic injury Mouse Liver animal model

1, purpose: observe betanin to mice CCl ₄The therapeutical effect of chronic injury hepatitis.

2, material and method:

2.1 the female BALB/C mice of materials A, animal: 18-22g, is provided by Zhongshan Medical Univ.'s Experimental Animal Center by 30.B, reagent: 1) CCl ₄Guangzhou chemical reagents corporation product.2) betanin, and Sigma company product (St.Louis, MO, USA).3) defend barbital sodium, Guangzhou chemical reagents corporation product.

2.2 the female BALB/C mice of method mice chronic hepatitis model, random packet.Behind the fasting 12h, press 0.5ml/kg body weight lumbar injection CCl ₄(10% paraffin oil solution), secondary weekly, totally eight times.

Experiment is divided into three groups, and betaine group is irritated stomach by the 600mg/kg body weight every day, and normal control group and damage matched group give corresponding normal saline.Last administration was killed Mus on the 3rd day, get hematometry ALT and serum albumin (A, g/L).Get a fritter hepatic tissue of hepatomegaly leaf same area simultaneously, after 10% formalin fixed, do check pathological section.Total survival rate that animal is also added up in experiment.

3,3.1CCl as a result ₄Inflammatory cell infiltration is obvious around the poisoning group, lobules of liver, visible proliferation of fibrous tissue, and big the hepatic necrosis in lobule center is obvious, and part of hepatocytes fat becomes the apparition of cavity sample; The liver histological of betanin treatment group changes and CCl ₄The poisoning group has obvious difference, and liver proliferation of fibrous tissue and obvious hepatic necrosis are not seen in most visuals field, and inflammatory cell infiltration and fat-like degeneration are lighter.

3.2 serum zymetology and serum albumin check result see Table 2.

Table 2 betanin is to CCl ₄Chronic hepatic injury mice serum transaminase and sero-abluminous influence (x ± s, n=10)

^*P＜0.01 (same CCl ₄Group relatively)

4, the result shows, betanin can improve the degeneration of chronic hepatic injury hepatic tissue cavity sample, liver fatization and Fibrotic morphological change, obviously reduces in the serum albuminous content in the liver transaminase and lifting serum, liver function protecting.

Embodiment three: betanin associating alpha-interferon and lamivudine are to the influence of the hepatitis B virus cell strain of In vitro culture

Principle: the 2.2.1.5 cell strain is the hepatoma cell strain of the external energy active secretion hepatitis B virus of setting up, it derives from the HepG2 hepatoma carcinoma cell, known alpha-interferon can suppress 2.2.1.5 cell strain secretion hepatitis B virus by signal pathway in its cell, lamivudine is a nucleoside analog, can directly suppress viral dna replication.By HBsAg, the HBeAg of detection culture fluid supernatant and the content of HBV DNA, can identify the biologic activity of lamivudine antivirus action and alpha-interferon.Present embodiment be with can providing active methyl or participate in the betanin of one of chemical compound of Methyl transporters, with alpha-interferon and lamivudine synchronization in the 2.2.1.5 cell strain, observe their inhibitory action to hepatitis B virus.

1, material and method

1.1 material: the 2.2.1.5 cell strain, draw from institute of Materia Medica,Chinese Academy of Medical Sciences; RPMI1640 culture medium (Sigma company product), homemade newborn calf serum (newborn calf serum, NCS; Provide by Sijiqing Bioengineering Material Inst., Hangzhou City), betanin (Sigma company product), interferon-alpha (Egen Corp.'s production); Lamivudine (Britain GlaxoSmithKline PLC company provides).MTT (Fluka company product); HBsAg and HBeAg enzyme are exempted from test kit (ABOTT company product).

1.2 cell culture and 2.2.1.5 emiocytosis is antigenic dynamic observes

2.2.1.5 after cell inoculation in 1640 culture medium that contain 10% calf serum, covers with culture bottle, change being inoculated in 96 well culture plates, 1 * 105 cell/ml of concentration, every hole 100 μ l, 37 ℃ of cultivations under 5% gas concentration lwevel.Commutation every day with culture fluid once, it is standby that the supernatant that swaps out is put-20 ℃ of preservations.The 8th day termination test carries out antigen measuring simultaneously with all supernatant.

1.3 administration

Use the pastille culture fluid in inoculation instead after 24 hours, every kind of drug level all adds 8 holes, and experiment is grouped as follows:

The alpha-interferon of A variable concentrations (0,50,100,200,500,1000,2000 and 5000IU/ml) and the betanin (0,0.1 of variable concentrations, 0.2,0.5,1,2,5,10 μ g/ml) and the lamivudine (0,10,50 of variable concentrations, 100,200,500,1000,2000 μ g/ml), join respectively in the culture medium, establish negative control group.

B chooses the betanin (5 μ g/ml) of the alpha-interferon (200IU/ml), lamivudine (200 μ g/ml) and the suitable concentration that are fit to concentration, and compatible combination adds in the culture medium respectively, establishes negative control group.

Commutation every day with culture fluid once, the 8th day termination test.

1.4HBsAg and the HBeAg suppression ratio is measured

The cell conditioned medium liquid of-20 ℃ of preservations put in 37 ℃ of water-baths melt, exempting from test kit with enzyme, to detect that HBsAg and HBeAg tire be contrast, calculates suppression ratio respectively with following formula: suppression ratio=(HBsAg and the HBeAg of control wells tire-experimental port HBsAg and HBeAg tire)/control wells HBsAg and HBeAg tire * and 100%.

(1.5MTT tetramethyl azo azoles salt) method detects cell survival rate, referring to document (Zou Qingyan, Kong Xiangping, etc., piglets brain extract and cerebrolysin are to the neuronic influence of tire Mus cerebral cortex in cultivating, Tianjin medicine, 1994,11 (4): 661-664).The percentage ratio of calculating survivaling cell.Cell survival rate=experimental port OD value/control wells OD value.

2, result

2.1HepG2.2.1.5 the antigenic characteristic test of emiocytosis finds that cell culture 24 hours can measure HBsAg and HBeAg in its growth-promoting media, and increases in time, the antigen secretion is stable to be increased.The results are shown in Table 3.

Antigenic the dynamic observing of table 3:HepG2.2.1.5 emiocytosis

2.2 the influence of secretion of medicine pair cell antigen and cell survival

Three kinds of compositions see Table 4 to the influence of HepG2.2.1.5 emiocytosis antigen and cell survival.The result shows: alpha-interferon 200IU/ml and lamivudine during in the above concentration of 200 μ g/ml the secretion to virus antigen can reach effective inhibitory action, and pair cell does not have obvious toxic and side effects.And betanin has facilitation at 1 μ g/ml with the propagation of interior pair cell, but not obvious to the secretion influence of virus antigen, when the propagation of betanin pair cell when 10 μ g/ml are above just has slight inhibitory action, and not obvious to the secretion influence of virus antigen.Therefore we select for use alpha-interferon (200IU/ml), lamivudine (200 μ g/ml) and betanin (5 μ g/ml) to work in coordination with inhibition test as compatible combination.

Table 4: medicine is to the influence of HepG2.2.1.5 emiocytosis antigen and cell survival

2.3 the coordinate repression of medicine

Select for use alpha-interferon (200IU/ml), lamivudine (200 μ g/ml) and betanin (5 μ g/ml) to work in coordination with inhibition test, get the 8th day supernatant and measure, the results are shown in Table 5 as compatible combination.

Table 5: medicine is to the antigenic coordinate repression of HepG2.2.1.5 emiocytosis

Annotate: "+" shows and adds corresponding medicine; "-" expression does not add corresponding medicine.

The result shows: betanin and interferon, lamivudine are compatible, all show the obvious synergistic effect, to the inhibitory action of virus antigen obviously greater than their simple summation action.And interferon and lamivudine are compatible mutual enhanced effect arranged also.

3, discussion and conclusion:

3.1 betanin when the following concentration of 2 μ g/ml like the secretion of virus is had facilitation, but with compare no significant difference.This may promote that the cell growth is relevant with the betanin of low concentration.We infer, betanin may be by suppressing assembling and the secretion that endocytoplasmic reticulum stress suppress virus protein, because viral assembling is mainly synthesized by the albumen that the inducing cell er stress carries out self.

3.2 alpha-interferon is not obvious in the influence of the propagation of 500IU/ml pair cell, has certain facilitation when low concentration (below the 200IU/ml), but with compare no significant difference.Lamivudine in 500 μ g/ml the time on cell proliferation do not have influence, when greater than 500 μ g/ml, then show the obvious suppression effect.

3.3 betanin can obviously improve the antiviral effect of alpha-interferon, obviously greater than their summation action, its mechanism may be relevant with methylation in the cell for their inhibitory action.Research (Duong FH, et al.S-Adenosylmethionine and betaine correct hepatitis C virus induced inhibition of interferonsignaling in vitro.Hepatology.2006Apr are arranged; 43 (4): 796-806.) show: the proteic expression initiation of HCV STAT1 methylates and suppresses and the signal process of infringement Jak-STAT.Unmethylated STAT1 activity is low, activates Profilin (PIAS1) deactivation because it can be suppressed agent-STAT1.Handle the signal conduction that cell can recover methylating of STAT1 and improve alpha-interferon with SAM (S-adenosylmethionine) and betanin.

3.4 betanin can obviously improve the antiviral effect of lamivudine, the effect that they are united when using is obviously distinguished action effective simple addition really greater than both, its mechanism may stress suppress the intracellular virus assembling and secrete relevant by suppressing endocytoplasmic reticulum with betanin, vigor and protein active that the while betanin methylates and improves desmoenzyme by albumen, promote that by methylation of nucleotides dUTP transforms to dTTP in the cell, can obtain enough thymus pyrimidines when making cellular replication and reduce cytogene variation and viral gene variation, strengthen the comprehensive antiviral effect of cell.

Embodiment four: betanin strengthens the experimentation that lamivudine suppresses hepatitis B virus duplication in the duck body

Principle: duck hepatitis-B is the hepatitis model of duck natural infection, can cause chronic hepatitis and liver cirrhosis, and known lamivudine is a nucleoside analog, can directly suppress the interior hepatitis B virus DNA of duck body and duplicate.By detecting Sanguis Anas domestica clear HBsAg, HBeAg and the content of HBV DNA, can measure the antiviral activity of medicine.Present embodiment with the lamivudine compatibility effect, is observed their coordinate repressions to hepatitis B virus with one of the chemical compound that active methyl or participation Methyl transporters can be provided betanin.

1, materials and methods

1.1 reagent: 32P-dCTP and nick translation test kit (Promega company product); Milt DNA, bovine serum albumin (Sigma company product); Betanin (Sigma company product); Lamivudine (GlaxoSmithKline PLC pharmaceutical Co. Ltd product).

1.2 animal: female sheldrake, 6, available from the free market; 1 age in days Beijing duck is available from GuangZhou, Guangdong Province city Li Kang agricutural industrial ﹠ commercial united corporation.

1.3 screening positive serum: 6 sheldrakes, under aseptic condition, draw blood separation of serum.Conventional PCR reaction, the result has 3 sheldrake pcr amplification results to be positive, and 3 positive bands occur, and positive band does not appear in negative control.Choose the higher sheldrake serum of virus titer as positive serum, standby.

1.4 preparation duck hepatitis B model: get the clear 1 age in days sheldrake that infects of positive Sanguis Anas domestica.Got 200 healthy ducklings the same day in hatching, wherein 195 every clear through the positive Sanguis Anas domestica of lower limb intravenous injection 100 μ l, do not handle as the normal control group for 5.The hepatitis B infected rate of duck detects: in infecting back the 2nd all venous blood collections, the PCR method detects DHBV.195 ducks that the result infects the DHBV positive serum have 120 positives, 5 normal control duck total negatives.Duck hepatitis B model modeling success.Select the serum virus titre to test greater than 60 sheldrakes of 10000.

1.5 lamivudine 100mg/kg body weight and the treatment of betanin 20mg/kg body weight compatible combination.60 positive ducks are divided into 4 groups, and the grouping situation is as follows:

Experiment grouping situation

Annotate: "+" expression adds corresponding medicine, and "-" expression does not add corresponding medicine.

In infecting back the 2nd all begin treatments, treat 8 all later half quantitative PCR methods continuously and detect dhbv dna titre situation of change.

1.6PCR reaction: get 5 μ l Sanguis Anas domestica and reset and add 50 μ l lysates, 100 ℃ are boiled 10min, centrifugal being placed on ice is stand-by as template fast.Conventional PCR reaction, with the positive contrast of positive serum, 5 positive controls are established in each reaction altogether, and blank contains the required all the components of RT-PCR, but does not add template.

1.7 gel electrophoresis analysis: the PCR product is separated with agarose gel, and the result carries out quantitative analysis in multi-functional imaging analysis system.

1.8 pathological examination: the conventional paraffin wax section of hepatic tissue, HE dyeing, tectology, inflammation degree and the denaturation degrees of observation liver.

1.9 all results of statistical analysis all carry out statistical analysis with the SPSS statistical package, P＜0.05 is judged as difference significance.

2, result

2.1 after lamivudine and the betanin treatment, compare with matched group, single betaine group and single lamivudine group hepatocellular degeneration and inflammatory all obviously alleviate, and the combination group hepatocellular degeneration is similar with the normal control group with inflammation, illustrate that the compatibility application can obviously improve the hepatocellular restore funcitons of pathological changes.

2.2 duck body inner virus titre result of variations sees Table 6 before and after lamivudine and the betanin treatment.

Table 6: the treatment of lamivudine and betanin is to the influence of hepatitis B virus titre in the duck body (n=15, x ± SD)

Lamivudine	-	+	-	+
Lamivudine	-	+	-	+	Betanin	-	-	+	+
The DHBV titre	17867.6±11576.4	3896.4.2±1108.7**	8654.6±2524.8*	1836.8±879.6**	Betanin	-	-	+	+

^*Expression p＜0.05; ^*Expression p＜0.05 (relatively) with physiology saline group.

The result shows: after treatment finished, simple lamivudine therapy group still had low titre DHBV, and after the treatment of associating betanin, the DHBV titre further descended during Sanguis Anas domestica was clear, and antiviral effect is better than simple lamivudine therapy group.Simple lamivudine therapy group DHBV titre (3896.4.2 ± 1108.7) significantly is lower than normal saline matched group (17867.6 ± 11576.4) (P＜0.01).After the treatment of associating betanin, further decline was similar with normal control group (2526.3 ± 1022.6) for DHBV titre (1836.8 ± 879.6) during Sanguis Anas domestica was clear, and antiviral effect is better than simple lamivudine therapy group.

3, conclusion

3.1 the pathological change of lamivudine and betanin treatment all can obviously alleviating duck hepatitis-B, both compatibilities use, better effects if.

3.2 betanin itself also is certain inhibitory action to dhbv dna.

3.3 betanin can obviously strengthen the inhibition effect of lamivudine to dhbv dna.

Embodiment five: betanin compatibility lamivudine suppresses the experimentation of In vitro culture encephalitis b virus

Principle: epidemic encephalitis B virus is as a representative member in the flaviviridae, utilize epidemic encephalitis B virus wild strain and human hepatoma cell strain KN73 to set up the external persistent infection model of encephalitis b virus, and, observe the effect that betanin compatibility lamivudine suppresses the In vitro culture encephalitis b virus by virus inhibition experiment.

1, material and method

1.1RPMI1640 culture fluid (sigma), 3%FBS (homemade, Hangzhou Ilex purpurea Hassk.[I.chinensis Sims company), 24 porocyte culture plates (Costar).Betanin (Sigma company product); Lamivudine (GlaxoSmithKline PLC pharmaceutical Co. Ltd product).

1.2 cell and viral human hepatoma cell strain KN73 and epidemic encephalitis B virus strain derive from Guangzhou and import and export central laboratory of inspection and quarantine bureau.

1.3 cell strain KN73 adds under 37 ℃ of 5%CO2 conditions of 10% hyclone with 1640, per 5～7 days, adopts 0.25% trypsin to go down to posterity.

1.4 epidemic encephalitis B virus persistent infection model epidemic encephalitis B virus infects the KN73 cell, 37 ℃ adsorbed 90 minutes, change culture fluid and cytopathy occurs, continue to cultivate remaining cell, carry out passage behind the cell monolayer to be formed up to 90% cell.

1.5 carrying out virus inhibition experiment, the application antiviral drugs goes down to posterity, cultivates persistent infection cell KN73, after forming cell monolayer, under the prerequisite of no cytotoxicity effect, add betanin 0-20 μ g/ml and lamivudine 0-1000 μ g/ml, collect after the medication 1 day cell culture fluid and the cell after 2 days, use the titration of virus method and detect culture fluid and intracellular virus amount.

Select the betanin 5 μ g/ml and the lamivudine 200 μ g/ml of suitable dose to carry out compatibility test, every group 4 hole.Experiment is grouped as follows:

Experiment grouping situation

1.6 virus titer assay method: adopt hamster kidney cell plaque test method.Collect different times virus infected cell supernatant and cell, multigelation between-80 ℃ and the room temperature 3 times, born of the same parents' inner virus is disengaged in cell because of after birth breaks.Handle through two anti-, make 10-1,10-2,10-3,10-4 respectively, 10-5......10-9 doubly dilutes, and is inoculated in sensitive cells according to a conventional method.Cultivated 3～6 days and observed CPE (comprising cell rounding, karyopyknosis and cytolysis).Measure the 50tissue infection dose (TCID50) of virus according to the method for Reech-Mud.The dyeing of 1% crystal violet solution, counting plaque number, unit: plaque forming unit (PFU)/ml.

2, result

2.1 betanin and lamivudine see Table 7 to the influence of In vitro culture epidemic encephalitis B virus titre.

Table 7: betanin and lamivudine are to the influence of In vitro culture epidemic encephalitis B virus titre

The result shows: 5-20 μ g/ml betanin has slight inhibitory action to the In vitro culture epidemic encephalitis B virus, and 50-1000 μ g/ml lamivudine then shows the obvious suppression effect.

2.2 lamivudine and the treatment of betanin compatibility see Table 8 to the influence of epidemic encephalitis B virus titre in the In vitro culture supernatant

Table 8: lamivudine and betanin compatibility are to the influence of encephalitis b virus titre (n=4, x ± SD)

^*, p＜0.05, ^*, p＜0.01, with matched group relatively.

Lamivudine and betanin compatibility suppress viral experimental result and show, behind the passage 2 days, lamivudine can suppress the encephalitis b virus in the cell culture very significantly, and betanin can make virus titer further descend.

3, conclusion

Chemical compound-the betanin that active methyl can be provided or participate in Methyl transporters can slightly suppress the epidemic encephalitis B virus in the cell culture, and with lamivudine coordinate repression is arranged.

The influence that embodiment six, betanin and ribavirin (ribavirin, ribavirin) use in conjunction is duplicated HCV among the human hepatoma cell line HepG2 of HCV (hepatitis C virus) RNA positive serum infection In vitro culture.

Principle: the ribavirin mechanism of action is not illustrated as yet.Its effect through cellular enzymes in host cell changes into single phosphoric acid, bis phosphoric acid and triphosphoric acid ribavirin.Single phosphoric acid ribavirin can suppress single inosinyl phosphate inosine dehydrogenase competitively, reduce the synthetic of triphosphoric acid guanylic acid (GTP), the triphosphoric acid ribavirin can suppress the virus mRNA cap formation that GTP relies on competitively, thereby suppresses duplicating of multiple RNA, DNA viruses.The HCVRNA positive serum of using hepatitis C patients infects the human hepatoma cell line HepG2 of In vitro culture, observes the influence that betanin and ribavirin duplicate in cell HCV.

1, material and method

1.1 material

1.1.1HCV positive serum: from the outpatient service hepatitis C patients, the course of disease is more than 3 months, and do not accept antiviral therapy, previously the blood transfusion history arranged, and liver function test ALT is unusual repeatedly.It is positive that RT-polymerase chain reaction (RTPCR) detects serum HCV RNA result.

1.1.2 cell strain: human hepatoma cell strain HepG2 is available from cell institute of the Chinese Academy of Medical Sciences.

1.1.3HCV PCR detection kit: be that Zhongshan University reaches peace gene diagnosis center product.RPMI1640 is a U.S. Sigma company product, and reverse transcriptase (MMLV) is a U.S. Promega company product, and TaqDNA polymerase and dNTP are Japanese precious biotech firm product.HCV RNA fluorescent quantificationally PCR detecting kit is produced by Guangzhou Huayin Pharmaceutical Technology Co., Ltd.

1.1.4 injection ribavirin (ribavirin, ribavirin), 0.25g, 5ml is produced by Zhejiang Asia-Pacific Pharmaceutical Co., Ltd.Betanin (Sigma company product).

1.2 experimental technique

1.2.1 cell culture: with the RPMI1640 culture medium that contains 10% calf serum, under 37 ℃ of 5%CO2 conditions, cultivate the HepG2 cell, change liquid, go down to posterity once every 3d.

1.2.2HCV Infection in Vitro HepG2 cell: go down to posterity with 1 * 105HepG2 cell culture, it is full 80% that cell grows to, and growth conditions adds HCV positive serum (infecting the serum final concentration is 10%) when good, shakes up gently, continues to cultivate.And, after the absorption culture supernatant, wash 5 times with 1640 liquid respectively at infecting back collection in the 2nd, 4,6 day part cell, 025% trypsinization is made cell suspension, and the centrifugal 5min of 1000r/min abandons supernatant, behind PBS centrifuge washing 5 times ,-20 ℃ of refrigerators are frozen to be detected.

1.2.3RT PCR detects cultured cell HCV RNA: adopt Zhongshan University to reach the HCVRNA detection kit that peace gene diagnosis center provides, the operation by specification carries out.With positive serum in the test kit as positive control.After detecting HCV RNA in the cultured cell, carry out follow-up test.

1.2.4 betanin and lamivudine are to the influence of HepG2 cell HCV RNA in cultivating.

The HepG2 cell inoculation that HCV infects in 1640 culture medium that contain 10% calf serum, cover with culture bottle after, change being inoculated in 24 well culture plates, 1 * 105 cell/ml of concentration, every hole 500 μ l. 37 ℃ of cultivations under 5% gas concentration lwevel.Use the pastille culture fluid in inoculation instead after 24 hours, every kind of drug level all adds 4 holes, and experiment is grouped as follows:

The betanin of A variable concentrations (0,0.5,1,2,5,10 μ g/ml) and

The ribavirin of variable concentrations (0,100,200,500,1000,2000 μ g/ml) joins respectively in the culture medium, establishes negative control group.

B chooses the ribavirin (500 μ g/ml) of suitable concentration and the betanin (5 μ g/ml) of suitable concentration, and compatible combination adds in the culture medium respectively, establishes negative control group.

Commutation every day culture fluid together once continues to cultivate 3 days termination tests.

1.2.5HCVRNA detect with fluorescent quantitation RT PCR method respectively, the operation by specification carries out.Cell HCV RNA amount is with 10 ⁶Cps (copy number)/hole is represented.Calculate suppression ratio respectively with following formula: suppression ratio=(the HCVRNA content of control wells-experimental port HCVRNA content)/control wells HCV rna content * 100%.

(1.2.6MTT tetramethyl azo azoles salt) method detects cell survival rate, referring to document (Zou Qingyan, etc.Piglets brain extract and cerebrolysin are to the neuronic influence of tire Mus cerebral cortex in cultivating.Tianjin medicine, 1994,11 (4): 661-664).The percentage ratio of calculating survivaling cell.Cell survival rate=experimental port OD value/control wells OD value.

2, result

Find 2.1HCV infect the characteristic test of HepG cell, behind the cell infection the 4th day, can measure HCV RNA in its Cell sap.

2.2 the influence of HCV RNA and cell survival in the medicine pair cell

Influence to HCV RNA titre and cell survival in the HepG cell sees Table 9.The result shows: ribavirin secretion to virus antigen when the above concentration of 100 μ g/ml can reach effective inhibitory action, and the pair cell activity does not have obvious toxic and side effects.And betanin has facilitation at 1 μ g/ml with the propagation of interior pair cell, but not obvious to the influence of viral RNA titre.Therefore we select for use ribavirin (500 μ g/ml) and betanin (5 μ g/ml) to work in coordination with inhibition test as compatible combination.

Table 9: medicine is to the influence (n=4) of viral RNA content and cell survival in the HepG cell

2.3 the coordinate repression of medicine

We select for use ribavirin (500 μ g/ml) and betanin (5 μ g/ml) to work in coordination with inhibition test as compatible combination, and the 3rd day cell after the thing effect of getting it filled carries out RNA extracting and RNA quantitative assay, the results are shown in Table 10.

Table 10: medicine is to the coordinate repression of HepG2 cell hepatitis C virus (n=6, x ± SD)

^*, p＜0.05, ^*, p＜0.01, with matched group relatively.

The result shows that betanin and ribavirin are compatible, and hepatitis C virus is shown the obvious synergistic inhibitory action.

3, conclusion

3.1 ribavirin in 1000 μ g/ml the time pair cell activity do not have the obvious suppression effect, and betanin in 1 μ g/ml the time cell growth slight facilitation is arranged, just demonstrate slight facilitation to 10 μ g/ml.

3.2 ribavirin when 100 μ g/ml in the pair cell hepatitis C virus show inhibitory action, IC50 is: 777.3 μ g/ml.And betanin does not show the obvious suppression effect at 10 μ g/ml with the hepatitis C virus in the interior pair cell.

3.3 ribavirin (500 μ g/ml) and betanin (5 μ g/ml) be as compatible combination, and the amount of viral RNA is shown the obvious synergistic inhibitory action.Its mechanism is relevant with methylation in the cell, and betanin can recover methylating of the interior STAT1 of cell and improve the signal conduction of alpha-interferon.

Embodiment seven: the clinical experimental study of betanin and lamivudine therapy chronic hepatitis B

One, clinical research plan

1. purpose and meaning detailed observation under the condition of contrast can provide curative effect, effective dose, indication and the untoward reaction of the chemical compound of active methyl or participation Methyl transporters.

2. case is selected

Intend clinical verification 50-100 example.Man, woman half and half are mainly HBeAg positive chronic hepatitis B patient, are tried case and must meet following condition:

2.1 diagnosis is clear and definite: the HBeAg positive chronic hepatitis B, with reference to " viral hepatitis is prevented and treated scheme " and 2005 " chronic hepatitis B is prevented guide " in 2000.

Diagnostic criteria: possess that 5 persons include range of observation in following 6: 1,18～65 years old age; 2, the HBsAg positive〉6 months; 3, serum HBV-DNA〉10^5copies/ml; 4, the positive and anti-HBe feminine gender of HBeAg; 5, Serum ALT continues or raises repeatedly; 6, the liver histological inspection has the hepatitis pathological changes.

2.2 can be with examining 1 year person.

2.3 be not in the mood for, kidney, endocrine, obviously hemopoietic system illness and other influence drug evaluation factor person.

2.4 generally select the inpatient, also can select the part outpatient, but must satisfy the every requirement in the EXPERIMENTAL DESIGN.

2.5 exclusion standard:

HIV, anti-HIV and anti-HCV positive person; Connective tissue disease, thyroid disease, patients with depression; Serious liver, renal insufficiency patient suffer from serious cardiac, cancer and other serious diseases patient; Using other treatment hepatitis medicament and the patient that may influence this product observation of curative effect.Drug allergy history or can not anti-receptor is arranged.The women of anemia of pregnant woman, age of sucking and child.The research doctor thinks have any discomfort to close those selected.

3. case culling level in the process of the test:

3.1 find not meet case choice criteria person in the process of the test.

3.2 have no adverse reaction in the medication process, because of other reason therapy discontinued person.

3.3 in the medication process, suffer from other disease (as pneumonia, nephritis etc.) because of non-medicine factor and end treatment.

3.4 duration of test is not taken medicine by this programme requirement, compliance is poor.

4. experimental group and matched group setting

4.1 the employing random packet, method is as follows:

4.1.1 clinical diagnosis unanimity

4.1.2 age unanimity: the age differs and is no more than 10 years old

4.1.3 sex unanimity

4.2 with lamivudine therapy 50 examples as the positive drug matched group.Experimental group 50-150 example adds on the basis of lamivudine therapy with active methyl being provided or participating in the chemical compound (betanin or other can provide active methyl or participate in the chemical compound of Methyl transporters, as methionine etc.) of Methyl transporters.

5. drug dose, the course of treatment and medication

5.1 general treatment: reasonable diet, suggestion low salt diet.Chronic hepatitis B treatment comprises mainly that antiviral, immunomodulating, antiinflammatory protect the liver, fibrosis and symptomatic treatment.

5.2 treatment group: the chemical compound of active methyl or participation Methyl transporters can be provided, each 0.3g, every day 3 times and conventional lamivudine therapy, be 12 months first course of treatment.Partly case can proceed for second course of treatment in case of necessity, and dosage strengthens the 1-4 multiple dose according to the situation of first course of treatment, and the time is 12 months.

5.3 matched group: conventional lamivudine therapy, 12 months each courses of treatment, can observe 1-2 the course of treatment.

Must not add arbitrarily during the treatment with other medicines (as other clinical research medication, the liver protecting and ALT lowering medicine, hypolipidemic and swash etc.), in principle with the chemical compound that active methyl or participation Methyl transporters can be provided, if treatment is more than 1 month, the state of an illness is improved not obvious, and when the state of an illness occurring and increasing the weight of, can use other medicine for treatment instead.Press invalid case summary during statistics.

6. the result observes and record

6.1 symptom: weak, poor appetite, pain, abdominal distention, feel sick, vomiting, low grade fever, arthritis, gingiva bleeding gingival hemorrhage, epistaxis etc.Before the treatment symptom have, be difficult ten ,-expression, treatment back transference cure, alleviate, constant, increase the weight of with ", ± ,+, ++ " expression.Before treatment, during the treatment weekly, treatment finishes, the treatment end was observed respectively and write down once in back 3 months.The clinical symptoms order of severity is divided in 0-3, and its standard is as follows: 0 minute, asymptomatic; 1 minute, slight, note a little feeling that symptom is arranged; 2 minutes, moderate, conscious have symptom but do not influence work; 3 minutes, obviously influence work and life.

6.2 sign: liver, splenomegaly, observation such as spider angioma and writing time are the same.

6.3 each important organ function such as the heart, liver, lung, kidney: serum alanine transaminase (sALT), SAST (sAST), alkali phosphatase, bilirubin, alpha-fetoprotein (AFP), total serum protein, albumin, globulin are before treatment, during the treatment every month, treatment finishes, treatment finish back 3 months each detect once.

6.4HBV serologic marker comprises HBsAg, anti--HBs, HBeAg, anti--HBe, anti--HBc and anti--HBc IgM, back each detection in 3,6,9,12 months is finished once in every month, treatment before treatment, during the treatment.

6.5HBV DNA is qualitative and quantitative, genotype and variation detect

7. efficacy determination: MAIN OUTCOME MEASURES: (HBsAg, HBeAg negative conversion rate, HBV DNA＜10 are replied in uniting of liver function index, nosetiology index ⁵Copy/ml and ALT are normal again).

Criterion of therapeutical effect: treatment is replied

(1) individual event is replied

1. virusology is replied: refer to the serum HBV DNA detection less than (PCR method) or be lower than the detection lower limit, or than baseline decline 〉=2log10.

2. serology is replied: refer to that serum HBeAg turns out cloudy or the HBeAg serology is changed or HBsAg turns out cloudy or HBsAg serology conversion.

3. biochemical replying: refer to that Serum ALT and AST recover normal.

(2) time sequencing is replied

1. initially or in early days reply: treatment replying during 12 weeks.

2. reply when treatment finishes: reply when treatment finishes.

3. reply lastingly: treatment is followed up a case by regular visits to more than 6 months or 12 months after finishing, and curative effect remains unchanged, and does not have recurrence.

4. keep and reply: during antiviral therapy, show as the HBV DNA detection less than (PCR method) or be lower than the detection lower limit, or ALT is normal.

5. bounce-back: reached initially and replied, but under the situation of not change treatment, the HBV dna level raises again, or transfers the positive again to after once turning out cloudy, can have or not have ALT and raise.Sometimes after referring to also that ALT and AST are normal again, under the situation of not change treatment, raise once again, raise but should get rid of the ALT and the AST that cause by other factors.

6. recurrence: reached when treatment finishes and replied, but HBV DNA raises or the sun commentaries on classics again after the drug withdrawal, also refers to ALT and the AST rising once again after drug withdrawal sometimes, raise but should get rid of the ALT and the AST that cause by other factors.

(3) unite and reply

1. reply fully: HBeAg positive chronic hepatitis B patient, treatment back ALT recovers normal, and the HBV DNA detection does not go out (PCR method) and HBeAg serology conversion; The negative chronic hepatitis B patient of HBeAg, treatment back ALT recovers normal, and the HBV DNA detection does not go out (PCR method).

The part reply: between reply fully and no response between.As HBeAg positive chronic hepatitis B patient, treatment back ALT recovers normal, HBV DNA＜105 copy/ml, but do not have HBeAg serology conversion.

3. no response: do not reach above respondent.

Two, clinical research preliminary conclusion

1, data and method

1.1 object of study: selecting 56 routine HBeAg positive chronic hepatitis B patients, all is to be in hospital and the part out-patient year August in October, 2004～2006.Diagnosis and Clinical typing standard meet the tenth national viral hepatitis meeting revised standard of JIUYUE in 2000.Wherein, male 32 examples, women 24 examples, 33 years old mean age.Hepatitis course of disease June-3 year, average 1.2 years, HBsAg, HBeAg and HBcAb were all positive; Liver function test ALT65U/L-276U/L, the total bilirubin 20.5 μ mol/L-291.6 μ mol/L that raise, albumin reduces 23.2-39.5g/L.Two groups are carried out paired comparison before treatment, the fellow lists experimental group and matched group at random respectively in.

1.2 medicine: lamivudine, GlaxoSmithKline PLC pharmaceutical Co. Ltd product.Can provide chemical compound (closing betanin, B2, B6, B12 and the folic acid) capsule of active methyl or participation Methyl transporters, self-control.

1.3 treatment group experiment group: active methyl can be provided or participate in chemical compound capsule 0.3g/ time of Methyl transporters, one day three times oral; Lamy stationary slice, 100mg/ time, oral once a day, 12 months is a course of treatment.Matched group: list is used lamivudine, and the course of treatment is with the treatment group.

1.4 lab testing: Serum ALT is pressed reitman-frankel method.HBsAg and HBeAg quantitative analysis: adopt the sub-enzyme of full-automatic fast trace to exempt from analytical system, instrument and reagent are U.S. Abbott product.HBV DNA detection by quantitative: adopt quantitative polyase chain reaction (PCR) to detect, AG-9600 fluorescent DNA analyzing and testing instrument is produced by U.S. Biotronics company.

1.5 statistical procedures: adopt the SPSS statistical software that data are carried out X 2 test or variance test.

2, result

2.1 4 examples recover normal in symptom and the sign splenomegaly treatment group, all the other change little.Other sings and symptoms treatment groups and matched group improvement situation do not have the significance difference.

2.2 active methyl can be provided or participate in of the influence of the chemical compound of Methyl transporters to hepatitis B patient serum liver biochemical indicator

Can provide the chemical compound of active methyl or participation Methyl transporters that hepatitis B patient is treated ALT, AST, ALP, serum albumin and bilirubinic influence in 1 year, the results are shown in Table 11.Experimental group and matched group liver function substantially all recover normal in treatment after 1 year, because of can't adding up and compare negative conversion rate a bit, but liver function recovery speed has tangible difference between the two.

Table 11: active methyl can be provided or participate in the compounds for treating 1 year of Methyl transporters influence (n=28, x ± SD) to the hepatitis B patient liver biochemical indexes

^*Expression p＜0.05; ^*Expression P＜0.01 (corresponding data relatively between two groups)

+, p＜0.05; ++ p＜0.01 is relatively preceding with treatment

The chemical compound that active methyl can be provided or participate in Methyl transporters is when promoting that liver function (ALT and AST) is recovered, and its speed is obviously faster than matched group.To some intractable jaundice patients, active methyl can be provided or participate in Methyl transporters chemical compound the jaundice effect is fallen also clearly, especially hepatitis is promoted that the albuminous synthetic effect of hepatocyte is good.

2.3 the influence that can provide the chemical compound of active methyl or participation Methyl transporters that the hepatitis B patient virus causing disease is learned index the results are shown in Table 12.

Table 12: active methyl can be provided or participate in the chemical compound of Methyl transporters to the influence of hepatitis B patient nosetiology index (n=28, x ± SD)

^*Expression p＜0.05; ^*Expression P＜0.01 (relatively) with matched group.

The result shows: the chemical compound that active methyl can be provided or participate in Methyl transporters can obviously improve HBeAg negative conversion rate and the frequence of seroconversion and the HBVDNA negative conversion rate of lamivudine.

3, conclusion

3.1 the chemical compound that active methyl can be provided or participate in Methyl transporters can be accelerated the recovery of hepatocyte function by protecting hepatocyte and promoting that obviously the hepatocyte albumin is synthetic.

3.2 share the effect that can obviously improve its anti-hepatitis virus with lamivudine.

Because antiviral compounds such as influenza virus, the also available interferons chemical compound of the present invention of bird flu virus, nucleoside analogues compounds are treated, by with embodiment one to seven similar experimental verification, also obtain good effect, show that the chemical compound of active methyl or participation Methyl transporters that provides of the present invention also can be used for preparing the medicine for the treatment of influenza or bird flu, can obviously improve the therapeutic effect of antiviral compounds such as interferons chemical compound, nucleoside analogues compounds.

Embodiment eight: utilize betanin to be major ingredient, can make injection, injectable powder, capsule and tablet.Can make oral or injectable drug is treated relevant viral disease.

1, injection prescription:

Betanin 1-10 part+distilled water (water for injection) 90-99 part (1%-10%) is transferred pH to 7.0 with 1mol/LHCl, makes 1ml to 100ml injection.Preferred 10% betanin is made the 2ml injection.

2, injectable powder prescription:

Betanin 1-10 part+distilled water (water for injection) 90-99 part (1%-10%), preferred 10% concentration is transferred pH to 7.0 with 1mol/LHCl, and making the ultimate density of mannitol with mannitol is 4%, (preferred 2ml) is added in the bottle with the 2-20ml amount, and lyophilization is standby.

3, capsule prescription:

3 parts of betanins, microcrystalline Cellulose 0-1 part, starch 0-0.5 part is adorned No. 0 or No. 2 capsules, and making capsule contain the betanin final quantity is 0.1g/ grain or 0.3g/ grain.This prescription does not limit capsule size and loading amount.

4, tablet formulation:

3 parts of betanins, microcrystalline Cellulose 0-1 part, amylum pregelatinisatum 0-2 part is made the sheet type of 0.1g-1g, and making every, to contain the betanin final quantity be 0.1g/ sheet-0.5g/ sheet.This prescription does not limit sheet type, tablet coating, size and loading amount.

5, oral liquid prescription:

Betanin 1-10 part, cyclamate is an amount of, and it be (1%-10%) that essence (lemon flavor, rose scent an amount of)+distilled water (water for injection) is transferred betanin concentration, carries out mouthfeel and debugs.Preferred 3% betanin is made the 10ml oral liquid.

6, chewable tablet prescription:

Betanin 80-90 part, sweeting agent are 600-680 part, and correctives is 10-14 part, and essence is 3-5 part, and pigment is 6-6.5 part, and lubricant is 10-14 part, and binding agent is an amount of.The mouthfeel debugging.

Embodiment nine: utilize betanin to be major ingredient, the method that is equipped with multivitamin and adjuvant reference example eight can be made oral liquid, capsule, or oral or chewable tablet (also can make food or health food).

Optimization formula: 500 parts of betanins, VB1 are 10 parts, and VB2 is 20 parts, and VB6 is 50 parts, and VB12 is 2 parts, and VE is 100 parts, and VA is 5 parts, and VC is 50 parts, 50 parts in nicotinic acid, 0.3 part of biotin, 1 part in pantothenic acid, 2 parts in folic acid; Trace element is an amount of.

Select prescription: can betanin be major ingredient optional above-mentioned two-ten several combine, the amount of combination can be with requiring and become, and can be 10-500 part as betanin, VB1 can be 0-50 part, VE can be 0-200 part etc.

Embodiment ten: utilize betanin, folic acid and vitamin B12 to be major ingredient, the method that is equipped with multivitamin and adjuvant reference example eight can be made oral liquid, capsule, or oral or chewable tablet (can make food or health food).

Optimization formula: 500 parts of betanins, 10 parts in folic acid, vitamin B12 is 1 part

Add VB1:10 part, VB2:20 part, VB6:50 part, VE:100 part, VA:5 part, VC:50 part, nicotinic acid: 50 parts, biotin: 0.3 part, pantothenic acid: 1 part; Trace element is an amount of.

Select prescription: can betanin, folic acid and vitamin B12 be major ingredient optional above-mentioned two-ten several combine, the amount of combination can be with requiring and become, and can be 10-500 part as betanin, VB1 can be 0-50 part, VE can be 0-200 part etc.

Embodiment 11: utilize betanin to be major ingredient, be equipped with antiviral agents (nucleoside analog such as lamivudine) and adjuvant and can make capsule and tablet.Can make the relevant viral disease of oral drug therapy.

1, capsule prescription:

Betanin: 500 parts, lamivudine: 100 parts, microcrystalline Cellulose 0-100 part, starch 0-50 part is adorned No. 0 or No. 2 capsules, and making capsule contain the betanin final quantity is 0.1g/ grain or 0.3g/ grain, and the lamivudine amount is 0.02g/ grain or 0.06g/ grain.This prescription does not limit capsule size and loading amount.

2, tablet formulation

Betanin: 500 parts, lamivudine: 100 parts, amylum pregelatinisatum 0-200 part is made the sheet type of 0.1g-1g, and making every, to contain the betanin final quantity be 0.1g/ sheet-0.5g/ sheet, and the lamivudine final quantity is 0.02g/ sheet-0.10g/ sheet.This prescription does not limit sheet type, tablet coating, size and loading amount.

Embodiment 12: utilize betanin, folic acid and vitamin B12 to be major ingredient, be equipped with antiviral agents (nucleoside analog such as lamivudine) and adjuvant and can make capsule and tablet.Can make the relevant viral disease of oral drug therapy.

1, capsule prescription:

Betanin: 500 parts, folic acid: 10 parts, vitamin B12: 1 part; lamivudine: 100 parts, microcrystalline Cellulose 0-100 part, starch 0-50 part; adorn No. 0 or No. 2 capsules, making capsule contain the betanin final quantity is 0.1g/ grain or 0.3g/ grain, and the lamivudine amount is 0.02g/ grain or 0.06g/ grain.This prescription does not limit capsule size and loading amount.

2, tablet formulation

Betanin: 500 parts, folic acid: 10 parts, vitamin B12: 1 part, lamivudine: 100 parts, amylum pregelatinisatum 0-200 part is made the sheet type of 0.1g-1g, making every, to contain the betanin final quantity be 0.1g/ sheet-0.5g/ sheet, and the lamivudine final quantity is 0.02g/ sheet-0.10g/ sheet.This prescription does not limit sheet type, tablet coating, size and loading amount.

Embodiment 13: utilize betanin to be major ingredient, be equipped with interferon and can make injection and injectable powder.Can make injectable drug and treat relevant viral disease.

1, injection prescription:

Betanin: 0.1-1.0g

Interferon: 5.0-50.0MU

Distilled water (water for injection): 2-20ml transfers pH to 7.0 with 1mol/LHCl, makes the 2-20ml injection.Preferred 2ml injection.

2, injectable powder prescription:

Betanin: 0.1-1.0g

Interferon: 5.0-50.0MU

Distilled water (water for injection): 2-20ml transfers pH to 7.0 with 1mol/LHCl, and making the ultimate density of mannitol with mannitol is 4%, and (preferred 2ml) is added in the bottle with the 2-20ml amount, and lyophilization is standby.

Claims

1. the chemical compound that active methyl can be provided or participate in Methyl transporters is characterized in that as the application of unique active component in the medicine of preparation treatment viral disease: the described chemical compound that active methyl is provided or participates in Methyl transporters is a betanin; Described viral disease is hepatitis B or encephalitis B.

2. a compositions that is used for the treatment of viral disease is characterized in that, said composition is made up of following:

A kind of chemical compound that active methyl is provided or participates in Methyl transporters; And

A kind of antiviral compound;

Wherein, the described chemical compound that active methyl is provided or participates in Methyl transporters is a betanin, and described viral disease is a hepatitis B, and described antiviral compound is interferon or lamivudine.

3. a compositions that is used for the treatment of viral disease is characterized in that, said composition is made up of following:

The chemical compound of active methyl or participation Methyl transporters can be provided; And

A kind of antiviral compound;

Wherein, the described chemical compound that active methyl is provided or participates in Methyl transporters is betanin, B2, B6, B12 and folic acid, and described viral disease is a hepatitis B, and described antiviral compound is a lamivudine.

4. a compositions that is used for the treatment of viral disease is characterized in that, said composition is made up of following:

A kind of antiviral compound;

Wherein, the described chemical compound that active methyl is provided or participates in Methyl transporters is a betanin, and described viral disease is an encephalitis B, and described antiviral compound is a lamivudine.

5. a compositions that is used for the treatment of viral disease is characterized in that, said composition is made up of following:

A kind of antiviral compound;

Wherein, the described chemical compound that active methyl is provided or participates in Methyl transporters is a betanin, and described viral disease is a hepatitis C, and described antiviral compound is a ribavirin.

6. a pharmaceutical composition that is used for the treatment of viral disease is characterized in that, said composition is made up of following:

A kind of chemical compound that active methyl is provided or participates in Methyl transporters of dose therapeutically effective;

A kind of antiviral compound of dose therapeutically effective; And

Acceptable carrier on the materia medica;

7. a pharmaceutical composition that is used for the treatment of viral disease is characterized in that, said composition is made up of following:

The chemical compound of the provided active methyl of dose therapeutically effective or participation Methyl transporters;

A kind of antiviral compound of dose therapeutically effective; And

Acceptable carrier on the materia medica;

8. a pharmaceutical composition that is used for the treatment of viral disease is characterized in that, said composition is made up of following:

A kind of antiviral compound of dose therapeutically effective; And

Acceptable carrier on the materia medica;

9. a pharmaceutical composition that is used for the treatment of viral disease is characterized in that, said composition is made up of following:

A kind of antiviral compound of dose therapeutically effective; And

Acceptable carrier on the materia medica;

10. according to the described pharmaceutical composition of one of claim 6 to 9, it is characterized in that: described pharmaceutical composition is mixed with the dosage form that is selected from one of injection, capsule, tablet, oral liquid.

11. pharmaceutical composition according to claim 10 is characterized in that: described pharmaceutical composition is mixed with injectable powder.

12. pharmaceutical composition according to claim 10 is characterized in that: described pharmaceutical composition is mixed with chewable tablet.