CN105147753B

CN105147753B - Application of the arasaponin in direct anti hepatitis C virus drug is prepared

Info

Publication number: CN105147753B
Application number: CN201510695240.2A
Authority: CN
Inventors: 何严萍; 冯悦; 杨军锋; 夏雪山; 姚赟; 李聪; 王月平
Original assignee: Yunnan University YNU; Kunming University of Science and Technology
Current assignee: Yunnan University YNU; Kunming University of Science and Technology
Priority date: 2015-10-23
Filing date: 2015-10-23
Publication date: 2018-06-22
Anticipated expiration: 2035-10-23
Also published as: CN105147753A

Abstract

Application of the arasaponin in direct anti hepatitis C virus drug is prepared, belongs to field of medicaments.Arasaponin anti-HCV activity and toxotest have been carried out using the method for cell in vitro level, the results showed that, arasaponin has good anti-HCV activity, and toxicity is low, hence it is evident that better than control group, clinical application safety.The present invention provides application of the arasaponin in direct anti hepatitis C virus drug is prepared for the first time, is expected to develop the drug of direct anti-hepatitis c virus.

Description

Application of the arasaponin in direct anti hepatitis C virus drug is prepared

Technical field

The invention belongs to field of medicaments, and in particular to new application of the arasaponin in pharmaceutical field.

Background technology

Hepatitis C caused by Hepatitis C Virus (HCV) is to seriously endanger the infectious diseases of human health, the disease The incidence of infection is high, and chronic rate is high, can prevent without vaccine, protracted course progress, be still so far global difficult medical problem and The heavy financial burden of society.Treatment chronic hepatitis C (CHC) is generally using antiviral therapy [interferon at present (IFN)+Ribavirin], curative effect is relatively low, main prevalence type HCV2a weak curative effects, continued viral response rate particularly to China (SVR) only about 50-60%, and the adverse reaction of interferon and Ribavirin also has an impact therapeutic dose and the course for the treatment of.Directly The appearance of antiviral drugs (DAA) provides more more options, but the Clinical practice limited experience of DAA, some problems for treatment CHC Such as to the tolerance of drug, be used in combination and drug resistance, it is expensive the problems such as make it that can not be widely used in clinic immediately.

Chinese Traditional Medicine has been widely used in the treatment of CHC, and the Chinese medicine and Chinese patent drug clinically applied at present are a lot of, Liver protection, anti-virus aspect achieve certain effect, also provide more more options for the treatment of CHC.It is clinical currently in use Kushenin is exactly the monomer oxidation matrine extracted from natural plants Sophora alopecuroide, is made in the clinic of chronic hepatitis B and CHC The effect of with having been achieved with generally acknowledging in the process.

Radix Notoginseng be Araliaceae herbaceos perennial, arasaponin (Panax notoglnseng saponins, PNS) It is the main active of Radix Notoginseng.It is many to the research of PNS pharmacological activity in recent years, it is had proven in central nervous system, the heart Cerebrovascular system, hematological system, immune system and anti-fibrosis, anti-aging, antitumor etc. are respectively provided with extensive physiology and live Property.Traditional Chinese medicine thinks that the pathogenesis of chronic hepatitis is epidemic disease caused by damp-heat pathogen poison retention blood vessels, gathers in liver body, leads to liver depression, and then spleen deficiency Or the deficiency of liver-yin and kidney-yin, eventually lead to qi depression to blood stasis, obstruction of collaterals by blood stasis.Arasaponin can be by promoting blood circulation and removing blood stasis, active change of promoting blood circulation Kind retention blood vessels, the deficiency of liver-yin and kidney-yin, and then reach qi and blood smoothness.Arasaponin is in terms of Protecting Hepatic Injury, anti-hepatic fibrosis Important function has obtained further confirmation.Tai Yong etc. treats virus hepatitis hyperbilirubinemia patient with arasaponin 【Tai Yong, Cui Wei wise man arasaponins treat virus hepatitis 35 observation of curative effect [J] clinic liver and bladder diseases of hyperbilirubinemia Magazine, 1999,15 (3)：184】；Xiong Lei etc. has found that arasaponin effect of anti hepatic fibrosis may be by inhibiting liver starlike thin Born of the same parents be proliferated and the generation of intraor extracellular type i collagen come realize (bear is built, and Liu Ping notoginsenosides generate hepatic stellate cells and collagen Influence [J] traditional Chinese and western medicine hepatopathy magazines, 1999,9 (3)：19)；Li Zhe etc. with causing Isolated Perfused Rat liver injury model, and Influences of the PNS to the model is observed, shows that PNS can significantly increase vascular flow, improves Microcirculation of Liver, mitigate hepatic injury (influence [J] Yunnan University of Traditional Chinese Medicine journal of the Li Zhe arasaponins to Isolated Perfused Rat glycosides, 1993,16 (2)：11)；It is remaining Ten thousand osmanthus can significantly improve hepatic ischemia/reperfusion injury anoxic with infrared human blood glucose Radix Notoginseng, protect liver cell.Treatment virus hepatitis differs Surely it is direct anti-hepatitis c virus, does not find that notoginsenoside is directly used in the report prepared in terms of HCV-Ab IgG virus drugs so far.

Invention content

The object of the present invention is to provide application of the arasaponin in direct anti hepatitis C virus drug is prepared.

Applicant has carried out external anti-HCV activity test, the results showed that the arasaponin apparent HCV-Ab IgG of energy is answered System, and no cytotoxicity.Specifically, the present invention is viral (HCVcc) using Huh7.5.1 cell lines and HCV cell culture, with profit Ba Weilin and interferon have carried out notoginsenoside external anti-HCV activity evaluation, the results showed that the total soap of Radix Notoginseng as positive control Glycosides has apparent HCV-Ab IgG virus activity and no cytotoxicity.

Drug is prepared described in the present invention and is meant that arasaponin can close acceptable auxiliary material system in pharmacy with appointing Into preparation, such as any conventional oral dosage formulations can be made：Powder, granule, tablet, capsule, pill, solution, suspension Liquid, syrup, buccal tablets, sublingual lozenge etc..

Following methods preparation can be used in the arasaponin that the present invention uses, but not limited to this：

Crude Panax notoginsensr material is taken, adds in water, biological enzyme extraction, alcohol precipitation or fining agent clarification, supernatant concentration a certain concentration Upper macroreticular resin further detaches afterwards, and eluent ion-exchange resin decolorization is collected in organic solvent elution, collects eluent, dense It contracts, be drying to obtain.

To more fully understand the present invention, below by viral using Huh7.5.1 cell lines and HCV cell culture (HCVcc), using interferon as positive control, the external anti-HCV activity of notoginsenoside progress, cytotoxicity is evaluated, come Illustrate the new application of arasaponin, i.e. application of the arasaponin in direct anti hepatitis C virus drug is prepared.

Experiment：The external anti-hepatitis c virus of arasaponin (HCV)

The preparation of notoginsenoside：Radix Notoginseng corase meal 200g is taken, adds water 1500ml, adds in appropriate biological enzyme, after adjusting pH, puts water Enzymolysis and extraction is bathed, extracting solution is spare.Residue is carried twice with the water of 5 times of amounts of first time crude drug weight, merges extracting solution three times, is added in Fining agent stands flocculation, centrifuges, and clarified solution is concentrated under reduced pressure, and upper D101 types resin is first eluted with water to no Molish and reacts, Reacted again with ethanol elution to no saponin(e, ethanol eluate decolourizes through anion exchange resin, concentrate, be spray-dried Radix Notoginseng is total Saponin(e.

Arasaponin sample 1,2,3 is prepared using the above method, preparation condition and total saponin content are as shown in table 1：

1 arasaponin sample of table, 1,2,3 preparation condition and total saponin content

Sample number

Biological enzyme

PH value

Extracting temperature

Color

Yield

Total saponin content

Sample 1

Alpha-amylase

5-6

60℃

White

12.1%

98.2%

Sample 2

Cellulase

4-5

50℃

White

11.6%

98.4%

Sample 3

Pectase

4-5

50℃

White

11.2%

98.1%

Sample and processing：It weighs appropriate amount of drug to be dissolved in 100% DMSO, is prepared into the storing liquid of 100mg/ml； 0.22 μm of membrane filtration degerming dilutes 7 gradients with DMSO successively.

Cell and virus：Human hepatoma cell strain Huh7.5.1 uses DMEM culture mediums, and addition 10%FBS and 1% is dual anti-, 5%CO₂, cultivate under the conditions of 37 DEG C；HCV cell culture virus (HCVcc) is by the HCV full-length genomes J6/JFH- comprising 2a hypotypes 1 plasmid is through in-vitro transcription to transfect Huh7.5.1 cells, high virulence, the HCV virus of high appeal obtained after culture after RNA Liquid.

Drug anti-HCV activity detects：It takes the logarithm the Huh 7.5.1 cells in growth period, with 1.5 × 10⁴Cells/well is spread After 96 orifice plates, adherent 5 hours, water gradient dilution drug, 5 times of dilutions are added in, 6 dilutions, each gradient sets three repetitions Hole, while virus is added in, if cell controls, virus control, HCV-Ab IgG positive control (interferon), water compare, 200 μ L/ of final volume well.Culture plate is placed in 37 DEG C, 5%CO₂Incubator is cultivated, and supernatant 3000rmp/min centrifugation 10min are collected after 72, The viral RNA that 100ul clarified supernatants is taken to extract respectively carries out HCV RNA copy numbers using fluorescent quantitative PCR technique and quantitatively examines It surveys.Using the virus load of blank control wells as control, the inhibiting rate (%) of various concentration drug is calculated respectively, is finally used GraphPad Prism5 softwares calculate drug EC₅₀(half effective concentration, the drug for inhibiting HCV virus carrying capacity 50% in cell are dense Degree) value, and draw amount-effect curve figure.

Mtt assay detects drug cytotoxicity：It takes the logarithm the Huh 7.5.1 cells in growth period, with 1 × 10⁴cells/well Cell is laid on 96 orifice plates, after adherent 5h, adds in 2 μ L DMSO-D₆The drug of gradient dilution, 10 times of dilutions, 6 dilutions, often A gradient sets three repeating holes, while sets blank control (containing only culture medium), cell controls, DMSO-D₆Control and HCV-Ab IgG Positive drug Ribavirin compares, 200 μ L/well of final volume.Culture plate is placed in 37 DEG C of 5%CO₂Incubator is cultivated. After 72h, the 5mg/mL MTT solution of 20 μ L, 37 DEG C of 5%CO are added in experimental port₂It is incubated 4 hours.It discards supernatant, adds in 150 μ The DMSO-D of L/well₆, after oscillation dissolving 10min, in measuring OD in microplate reader₄₉₀Value, using the OD values of blank control wells as Control calculates the inhibiting rate (%) of various concentration drug respectively, finally calculates drug CC using GraphPad Prism5 softwares₅₀ (CC₅₀：Drug concentration needed for cell growth inhibition 50%) value, and draw amount-effect curve figure.

Experimental result：

As stated above, Ribavirin and above-mentioned arasaponin sample 1,2,3 are carried out and external anti-HCV activity is real It tests, cytotoxicity (CC₅₀) and activity (EC₅₀) as shown in table 2.

2 arasaponin anti-HCV activity of table

1,2,3 sample of arasaponin has apparent HCV-Ab IgG virus activity, and no cytotoxicity, toxic side effect are much excellent In Ribavirin.

Beneficial effects of the present invention：Arasaponin is provided for the first time in direct anti hepatitis C virus drug is prepared Using.

Specific embodiment

Embodiment：Radix Notoginseng corase meal 200g is taken, adds water 1500ml, PH=5.5 is adjusted, adds in appropriate alpha-amylase, fully stir It mixes, puts 60 DEG C of water enzyme digestion extraction 2h, extracting solution is spare.Residue is carried twice with the water of 5 times of amounts of first time crude drug weight, merges three Secondary extracting solution adds in the flocculation of ZTC1+1 natural clarifying agents, filtering, and filtrate decompression is concentrated into 1/2 volume, filters, D101 on filtrate Type resin is first eluted with water to no Molish and reacts, then is reacted with 70% ethanol elution to no saponin(e, and ethanol eluate is through the moon Ion-exchange resin decolorization concentrates, is spray-dried to obtain arasaponin.Gained arasaponin is taken to add appropriate excipient by tablet The tablet that every sheet weight is 100mg or 50mg is made in conventional method, which is the drug of direct anti-hepatitis c virus, is used for Treat hepatitis C.

Claims

1. application of the arasaponin in direct anti hepatitis C virus drug is prepared.

2. application as described in claim 1, it is characterised in that arasaponin can with appoint close it is acceptable auxiliary in pharmacy Preparation is made in material.