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CN106074569A - Momordica grosvenori alcohol application in preparation antiviral drugs - Google Patents

Momordica grosvenori alcohol application in preparation antiviral drugs Download PDF

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Publication number
CN106074569A
CN106074569A CN201610808172.0A CN201610808172A CN106074569A CN 106074569 A CN106074569 A CN 106074569A CN 201610808172 A CN201610808172 A CN 201610808172A CN 106074569 A CN106074569 A CN 106074569A
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China
Prior art keywords
virus
momordica grosvenori
grosvenori alcohol
alcohol
related compound
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CN201610808172.0A
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Chinese (zh)
Inventor
罗明锋
谢海峰
胡云岭
谢期林
张朝凤
郭建龙
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Chengdu Universal Drug Development Co Ltd
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Chengdu Universal Drug Development Co Ltd
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Priority to CN201610808172.0A priority Critical patent/CN106074569A/en
Publication of CN106074569A publication Critical patent/CN106074569A/en
Priority to PCT/CN2017/081424 priority patent/WO2018045760A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Emergency Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

The invention provides the medical new application of momordica grosvenori alcohol, i.e. momordica grosvenori alcohol and related compound thereof for treatment or/and preventing viral infect medical application.Present invention also offers momordica grosvenori alcohol and related compound thereof in preparation treatment or/and application in preventing viral infection medicine, in particular it relates to momordica grosvenori alcohol and the anti-hepatitis C virus of related compound thereof and the application of anti HIV-1 virus.The present invention experiments prove that, momordica grosvenori alcohol can significantly activate the expression of body TLRs receptor, and then activates body innate immune system, plays antivirus action.Experimentation shows, momordica grosvenori alcohol all has stronger suppression scavenging action for hepatitis C virus and inhibition of HIV, is potential anti-hepatitis C or anti HIV-1 virus medicine.Momordica grosvenori alcohol derives from natural plants, and former plant distributions is extensive, aboundresources, preparation of industrialization low cost, beneficially popularization and application.

Description

Momordica grosvenori alcohol application in preparation antiviral drugs
Technical field
The invention belongs to natural drug preparation field, relate to a kind of can be used for preventing or/and treat the sky of viral infection So compound, in particular it relates to the antiviral medical application of momordica grosvenori alcohol and related compound thereof and preparing antiviral agents Application in thing.
Background technology
Virus by its shell whether be wrapped in the film rich in lipid can be simply divided into without togavirus with have togavirus Two classes.Mainly under clathrin mediates, infected cell is entered by pinocytosis without togavirus；There is togavirus Invasion mainly virus envelope and the fusion process of host cell membrane.According to the structure of cyst membrane surface glycoprotein during poisoning intrusion As change, togavirus can be divided into 3 classes: wherein orthomyxovirus section, Retroviridae, Filoviridae, paramyxovirus section Being classified as I class togavirus with coronaviridae, it represents virus for influenza virus, human immunodeficiency virus, Ebola virus With SARS virus；Flaviviridae and Togaviridae are classified as II class togavirus, and it represents virus and is Dengue virus and Japanese encephalitis virus；Herpetoviridae, Rhabdoviridae and Rhabdoviridae are classified as III class togavirus, It represents virus for herpes simplex virus and vesicular stomatitis virus.The research and development of anti-virus formulation at present are based primarily upon two sides Face is designed, and one is from virus dip-dye aspect, and two is from host cell defense aspect.List major part antiviral drugs at present It is virus itself based on virus itself, i.e. action receptor.The antiviral agents designed and developed based on host cell defense aspect Thing is the most rare, but in recent years deeply has been achieved for many gratifying results at present along with study, such as acts on The medicine of Toll receptor is continuously developed out.Based on host cell defense aspect design antiviral drugs mainly from improving body Immunity, activation body immune system, the raising aspect such as body Active Defending System Against and Passive Defence system connectivity are started with.
Interferon (IFN) is the generation antiviral drugs developed the earliest, is first iuntercellular element being found, because it has The ability of " interference " virus replication is had to name.Interferon be 1957 by Britain Alick Isaacs and Jean Two research worker of Lindenmann are found, when cell is infected with the virus, can produce interferon immediately to resist disease Poison, and the neighbouring normal cell of warning simultaneously, step up vigilance, in case poisoning intrusion.Interferon is largely divided into two big classes: I type and II type.The interferon of I type includes interferon-' alpha ', interferon-beta, interferon-ω, interferon Ct, and most cell can table Reach interferon-' alpha ' and interferon-beta；The interferon of II type includes interferon-γ, only expresses the immunocyte in part, such as sky So kill (NK) cell, CD4+Helper T cell 1 (TH1) and CD8+ Cytotoxic T lymphocyte.
During virus infects, the interferon of I type can quickly be induced to produce, then can be with the I type on target cell Interferon receptors combines, startup Jak-STAT signal pathway, unlatching interferon-stimulated gene (IFN-stimulatedgene, ISG) express.Interferon-stimulated gene protein is interferon strategy main to antiviral, has at present
Be defined out more than the interferon-stimulated gene protein of kind more than 500, these molecules participate in scope widely, From antiviral, apoptosis, protein degradation, inflammatory cell response to lipid metabolism, contain several functions.It is widely known by the people most Interferon-stimulated gene protein includes protein kinase R (PKR), acts on the ADA Adenosine deaminase of RNA, 2 ', 5 '-oligomerization adenosine Acid enzyme, RNase L and Mx albumen.PKR can the synthesis of blocking virus albumen, ADAR meeting by suppression eukaryotic initiation factor Suppression viral RNA editor, OAS, RNase L can degrade viral RNA, and Mx albumen then can suppress virus replication.
In addition to energy facedown virus, the interferon of I type also take part in immunoregulation, at congenital and acquired immunity Reaction plays key player.Drive survival and the propagation of natural killer cell except IL-15 can be produced, also can sting simultaneously Swash the expression of MHC, CD80, CD86 and CD40 molecule, and then promote dendritic cell maturation, additionally, the interferon of I type also can be induced Plasmoid dendritic cell differentiation is ripe antigen-presenting cell.In terms of acquired immunity, the interferon of I type is at CD8+ Cytotoxicity The activation of T lymphocyte, CD4+ Helper T cell 1 (TH1) and CD8+ The survival of cytotoxic T lymphocyte, B lymph Cell differentiation plays key player in propagation.
Owing to the interferon of I type has antiviral, cell growth control and immunoregulatory ability, the most successfully transport For clinical treatment, include through American and Britain, day, the indication of De Deng advanced country approval: the disease caused by (1) virus, example As, the Kaposi sarcoma etc. that chronic hepatitis b, chronic hepatitis c, cauliflower (tip condyloma latum), AIDS sufferer are common；(2) blood Liquid disease, such as, trichoblast leukemia, chronic lymphocytic leukemia, multiple myeloma, rudimentary non_hodgkin lymphoma Deng；(3) other tumor, such as, melanoma, renal cell carcinoma, basal cell carcinoma etc..
It has now been found that, the generation of interferon is due to immune messenger Toll sample receptor (Toll- Likereceptor, TLR) activate and induce it to produce.Toll is first to find in fruit bat body for about 1988, is feeding subsequently It has also been found that the receptor high with Toll similarity, referred to as Toll-like receptor in breast animal.Toll-like receptor (Toll-like Receptors, TLRs) it is a more conservative receptor family of ratio in evolution, at least include 13 members, Toll-like receptor can be special Different identification pathogen-associated molecular pattern (PAMP), all plays an important role in the natural immunity and acquired immunity, is even Connect the bridge of the natural immunity and acquired immunity.In recent years, the research to TLRs signal transduction, particularly degenerative to TLRs Research, is in progress very fast, and they play an important role in infection, and the balance of signal is adjusted by particularly negative feedback mechanism Joint plays an important role in anti-infectious immunity.The natural immunity is the important component part of immunity of organism, but at long time Interior being it is believed that is the rudimentary form of immunne response, does not has specificity and immunological memory.Along with to immune system research Deeply, the particularly discovery of pattern recognition receptors, if Toll-like receptor is exactly a kind of pattern recognition receptors.Identify pathogenic microorganism Standpatter in evolution, mainly includes LPS, Peptidoglycan, zymosan and pathogen nucleic acid etc., makes it was recognized that natural Immunity is not simple performance non-specific phagocytosis, scavenging action, and relates to the antigen recognition mechanism of complexity, with acquisition Property immunity as can be correct differentiation " oneself " and " non-oneself ".There is pattern recognition receptors in body, special identification cause of disease is micro- Antigen molecule conservative in biological evolution, i.e. pathogen-associated molecular pattern, thus effectively monitor the invasion of pathogenic microorganism And induction immune response.The reaction of TLRs mediate innate immune is mainly by the spy to pathogenic microorganism and product thereof Determining the identification of pathogen-associated molecular pattern, this is that body judges that natural immunity signal path is invaded and started to pathogenic microorganism, letter Number conducted by TLRs, cause interference with the generation of element-1, inflammatory cytokine, chemokines, induce body immune system pair The removing of pathogenic microorganism.Secondly, by TLRs, the effector lymphocyte such as macrophage identifies that pathogen-associated molecular pattern is activated, Then secretion inflammatory mediator such as cytokine etc. and some biocide molecule such as NO etc., inducing inflammatory reaction also plays bactericidal action, Signal is passed down by TLRs, inducing dendritic shape cell maturation, starts acquired immune system, assists opposing chronic disease Poison infects, and reaches to remove the final purpose of virus, in defense mechanism, it is provided that more powerful, more single-minded protection.
That rely primarily in antiviral immunity is reacted is the TLRs in natural immunity receptor family, and TLRs is by identifying disease The infection of poison mediates antiviral immunity, activation signal pathway, produces antiviral cell factor and chemotactic factor, at TLRs Identify virus in family mainly has TLR7/8, TLR3, TLR9 etc., and TLR7/8 mainly identifies ssRNA virus, and TLR3 identifies DsRNA virus, TLRs is directed to identify the chromogene of virus simultaneously, as TLR9 identifies CPG DNA sequence, TLR2 and TLR4 It is directed to virus identification, is primarily directed to the identification of viral envelope glycoprotein.Additionally some TLR passes through RNA helicase to carefully The identification of the viral dsRNA in kytoplasm, such as RIG-I is exactly the nucleotide identifying virus in this way, and virus is by drawing Send out host's innate immune reaction, reach to activate TLRs signal, cause antiviral response.Research shows that TLRs passes through endocytosis pair The virus entering kytoplasm is identified, the generation by the protein induced interferon of multi-signal of this paths, ultimately results in and transcribes Factor NF-kB, the activation of interferon regulatory factor (IRF) IRF3 and IRF5 and IRF7.
At present, the Toll sample receptor found in human body has 10 kinds, respectively from Toll-like receptor 1 to Toll-like receptor 10, Various different exotic can be identified, including: antibacterial, virus, mycete and protozoacide.Toll-like receptor structurally comprises Two parts: (1) born of the same parents are outer rich in leucic repetitive sequence (LRR), and it is responsible for the identification of exotic；(2) intracellular Toll-interleukin 1 receptor (TIR) domain, its adaptin effect with downstream, such as, myeloid differentiation factor 88 (MyD88), TIR are correlated with egg (TIRAP/MAL), the linker containing Toll/IL-1 receptor domain (TRIF/TICAM-1) of induction IFN-β and Toll are subject in vain Body correlation molecule (TRAM/TIRP/TICAM-2), and then the kinases (ERK) of activation extracellular signal regulation and control, p38, c-Jun N-end End kinases and NF-kB, inducible proinflammatory cytokine: IL-1, IL-6, interferon-' alpha ' and I type interferon produce.
In all of Toll-like receptor, Toll-like receptor 2 recognizes the most multiple PAMP, and major part is from antibacterial, bag Include: LAM, lipopolysaccharide, lipoteichoic acid, Peptidoglycan and other lipoprotein, glycolipid, glycoprotein.Toll-like is subject to Body 2 also can identify virus invasion: Measles virus, human cytomegalovirus, hepatitis C virus, defense mechanism plays Critically important role.
Toll sample receptor 7 is at immunocyte: mononuclear cell, B lymphocyte and dendritic cell camber are expressed, and once know After not going out the invasion of foreign substance, can produce larger numbers of I type interferon, particularly interferon-' alpha ', it is exempted from congenital Important pivotal player is play in epidemic disease.Toll sample receptor 7 mainly detecting is from the ssRNA rich in G/U of virus, bag Include: HIV (human immunodeficiency virus) and VSV, in the removing of virus, have very important power of influence.
In terms of drug development, have been enter into Toll-like receptor 9 antagonist of clinical experimental stage at present, dendron can be stimulated The interferon-' alpha ' of cell generation IL-12 and very a large amount, and induce B lymphopoiesis and antibody-secreting, at interferon-' alpha ' Good curative effect is had on patient HCV of Endodontic failure.Toll-like receptor 7 antagonist then can produce antiviral widely should Answering (antiviral response), discharge element between various kinds of cell, especially interferon-' alpha ', in the disease of different HCV genotype Obvious virus sweep rate is had on the person.
Existing multiple antiviral drugs is developed in recent years, and is widely used in the treatment of clinic.Such as: Li Ba Wei Lin (ribavirin), it is a kind of nucleoside analog, can suppress the growth of multiple virus experimentally, such as respiratory syncystial Virus, influenza virus (influenza virus), adenovirus (adenovirus), HIV and HCV etc.；Amantadine (amantadine), its M2 memebrane protein acting as suppressing influenza A virus, thus outside making influenza A virus to slough smoothly Shell, and then follow-up replication work can not be carried out；Zidovudine (zidovudine, AZT), Didanosine (didanosine, DdI), zalcitabine (zalcitabine, ddc), stavudine (stavudine, d4T) and lamivudine (lamivudine, 3TC) etc. then can suppress the reverse transcriptase of HIV so that the RNA of virus reverse transcription cannot become DNA, thus interrupts the continuation of DNA Synthesis.
Although the antiviral drugs of many is effectively used in clinic successively, but drug-resistant virus the most day by day floats in recent years Existing.The gene of Drug resistance Producing reason mainly virus produces sudden change and makes antiviral drugs lose the target institute of its effect Cause.Hereby give a few examples and be described as follows: the thymidine kinase gene of HSV is undergone mutation, it is impossible to by acyclovir (acyclovir) and more VCV (ganciclovir) etc. becomes effective composition at cellular transformation, therefore develops immunity to drugs those medicines；Influenza A The M2 protein gene mutation of virus, then can develop immunity to drugs to amantadine or rimantadine (rimantadine)；HIV reverses The variation of record enzyme or protease gene is also the main cause causing Drug resistance to produce；Unstructuredness 5A and the env gene 2-sugar of HCV The genovariation of albumen can make HCV develop immunity to drugs interferon.Increasing antiviral drugs is had to develop now, towards exempting from The direction of epidemic disease regulation and control strides forward, and posteriority reaction congenital by stimulation of host reaches to remove the purpose of external microorganism.
Fructus Momordicae is Cucurbitaceae (Cucurbitaceae) plant Fructus Momordicae (Siratia grosvenorii (Swingle) C. Jeffrey) mature fruit, main product is in counties such as Yongfu, Guangxi, Lingui and Long Sheng, for the famous special product in Guangxi.Fructus Momordicae Cool, sweet in the mouth, returns lung, large intestine channel, has nourishing the lung to arrest cough, clearing away summer-heat, relieving sore throat to recover voice, effect of removing heat from blood laxation, is that China is peculiar Integration of edible and medicinal herbs industrial crops, recorded in Pharmacopoeia of People's Republic of China simultaneously, used as conventional Chinese medicine, swallowed in treatment Laryngitis, pertussis, acute and chronic tracheitis, gastrointestinal disease aspect are evident in efficacy.Fructus Momordicae total glycosides is main effective in Fructus Momordicae Composition, has biological activity and pharmacology widely and is worth.Modern medicine study proves, Fructus Momordicae total glycosides not only has antitussive, puts down Breathe heavily, eliminate the phlegm, antiinflammatory, effect of regulation digestive tract function, moreover it is possible to enhancing immunity, the liver protecting and ALT lowering, treatment acute lung injury, antioxygen Change and anti-aging.Fructus Momordicae extract be mainly composed of Momordia grosvenori aglycone, the total glycosides in Fructus Momordicae is calabash alkane type triterpenoid glycosides Compounds, including mogroside Ⅴ (Mogroside V);Momordia grosvenori aglycone IVe (Mogroside IVe);Momordia grosvenori aglycone IIIe(Mogroside IIIe);Momordia grosvenori aglycone II A2 (Mogroside II A2);Momordia grosvenori aglycone III A1 (Mogroside III A1);Momordia grosvenori aglycone IVa (Mogroside IVa);Mogroside Ⅴ I (Mogroside VI);Match Door glycosides I (Siamenoside I);11-O-mogroside Ⅴ (11-Oxomogroside V) etc..Momordica grosvenori alcohol is Momordia grosvenori aglycone The aglycon of compounds, can be obtained by acid hydrolysis.
The present inventor finds in experimentation, momordica grosvenori alcohol can the innate immune system of excitating organism, improve machine Body immunity, further investigation finds that momordica grosvenori alcohol is probably and acts on Toll-like receptor and then activation body autoimmune further System, thus improve the immunity of body.Based on this, it is believed that momordica grosvenori alcohol is likely to be of good antivirus action, And mechanism of action differs from current most of antiviral drugs.Studying accordingly, the present inventor the most tentatively confirms momordica grosvenori alcohol Antiviral activity.Specifically, momordica grosvenori alcohol infects for such as influenza virus, hepatitis virus, inhibition of HIV etc. and has directly Antivirus action, also has certain treatment simultaneously for other related complication shapes caused because of virus or/and preventive effect.
Additionally, the present inventor further confirms that, momordica grosvenori alcohol and the existing antiviral drugs (Li Ba acting on virus itself Wei Lin, acyclovir etc.) combination, it is possible to mutually promote, given play to higher antiviral effect.Momordica grosvenori alcohol is main anti-with existing The virus drugs mechanism of action is different, and Papillary achieves the action effect of Synergistic.
Have no the momordica grosvenori alcohol clinical report for antiviral at present, also have no momordica grosvenori alcohol and existing antiviral agents Internet of Things Close be applied to antiviral therapy or/and prevention report.
Summary of the invention
First purpose of the present invention is to disclose the medical new application of momordica grosvenori alcohol, i.e. momordica grosvenori alcohol and related compound thereof Treatment or/and preventing viral infect medical application.
Second purpose of the present invention be to provide a kind of for treatment or/and preventing viral infect pharmaceutical composition, its Including at least momordica grosvenori alcohol or momordica grosvenori alcohol related compound, and at least one medicinal diluent, carrier or figuration Agent.
The 3rd purpose of the present invention is to provide momordica grosvenori alcohol and related compound thereof and existing antiviral drugs composition Compositions preparation for treatment or/and preventing viral infect medicine in application, said composition at least contains arhat Really alcohol or momordica grosvenori alcohol related compound and at least one the second antiviral drugs.
The present invention also provides for a kind of associating medicament comprising momordica grosvenori alcohol or its related compound for viral infection Prevention or/and treatment using method.Described associating medicament is except comprising momordica grosvenori alcohol or its related compounds beyond the region of objective existence also comprises at least A kind of compound having identical, similar effect or having auxiliary therapeutic action, the most existing antiviral drugs, improvement infect to be suffered from The medicine of person's complication, improve the medicine of patient's lipid metabolic disorder, the medicine etc. of enhancing human body immunity power, include but not limited to Ribavirin, acyclovir, acycloguanosine, interferon, antibiotic, lipid-lowering statins, antioxidant, immunostimulant, Huang Astragalus polysaccharides etc..Further, described associating medicament can give patient (or curee) together, it is also possible to separately suffers from Person.Further, first give patient's momordica grosvenori alcohol or its related compound preparation, give other the most again and there is identical, class Like effect or the preparation of the compound with auxiliary therapeutic action.
Momordica grosvenori alcohol of the present invention and related compound thereof are from cucurbitaceous plant, especially from cucurbitaceous plant Fruit.Further, above-mentioned plant is exactly Genus Siraitia Merr, and usual above-mentioned fruit refers to Fructus Momordicae.Further, on State momordica grosvenori alcohol and related compound can also be chemosynthesis.
Inventors expect that, when jointly giving halogen or halide salt, neutrophilic granulocyte and/or monocytic can be strengthened Produce or/and activate.Further, different halogen can be selected according to the demand strengthening specificity antivirus activity.
Further, can to above-mentioned momordica grosvenori alcohol and the like, derivant, precursor, metabolite, pharmaceutically active salt or Prodrug carries out halogenation.Further, compositions of the present invention comprises this halogen, and this halogen can give together with compositions, This halogen can also be given before or after said composition.
Momordica grosvenori alcohol related compound of the present invention refer to the analog of momordica grosvenori alcohol, derivant, precursor compound, Metabolite, pharmaceutically active salt or prodrug.
Viral infection of the present invention, includes but not limited to rhinovirus, adenovirus, respiratory tract and cellular virus, sidestream Influenza Virus, coronavirus, influenza virus, mumps virus, poliovirus, Cook Sa Ji virus, ECHO disease Poison, rotavirus, norwalk virus, Astrovirus, Calicivirus, hepatitis A virus, hepatitis B virus, hepatitis C virus Poison, hepatitis D virus, hepatitis E virus, Measles virus, rubella virus, virus of exanthem subitum, chickenpox, variola, simple bleb Exanthema virus, rabies virus, foot and mouth disease virus, encephalitis b virus, western equine encephalitis virus, eastern equine encephalitis virus, St. Louis encephalitis Virus, Venezuelan equine encephalitis virus, california antigenic group viruses, cytomegalovirus, acquired immunodeficiency disease, hemorrhage Fever virus, yellow fever virus, dengue virus, Colorado Ticks wear infection that any of which viruses such as fever virus cause or multiple The mixed infection that virus causes.Further, viral infection of the present invention refers to infection with hepatitis C virus or inhibition of HIV Infect.
Second antiviral compound of the present invention, refers to the existing approach infected for opposing or break virus, As directly suppressed or kill virus, viral interference absorption, stoping virus to penetrate cell, suppression viral organism synthesis, suppression virus The compounds such as release, include but not limited to virazole, amantadine, acycloguanosine, polyinosinic acid, interferon, rimantadine, In the middle of moroxydine, vidarabine, zidovudine, acyclovir, ribavirin, ganciclovir, deoxyribosylthymine, Sebivo etc. One or more.
The antivirus action that present inventor have determined that momordica grosvenori alcohol is by the Toll-like receptor effect with host cell And embody, momordica grosvenori alcohol can active cell innate immune response in this way, strengthen cell acquired immunity simultaneously Response.When momordica grosvenori alcohol uses with the existing antiviral drugs acting on virus itself simultaneously, owing to action target spot is different, machine Reason is different and produces cooperative effect, can effectively reduce poisonous side effect of medicine, reduce drug dose, minimizing drug resistance risk, improve Antiviral effect.
Detailed description of the invention
The present invention relates to the antiviral medical treatment new application of momordica grosvenori alcohol and related compound thereof, specifically, momordica grosvenori alcohol and Its related compound can prevent effectively or/and treat the such as viral infection such as hepatitis C and HIV.Further, Comprise momordica grosvenori alcohol or the compositions of at least one momordica grosvenori alcohol related compound formation, can effectively suppress viral infection. Specifically, present inventor have demonstrated that the antivirus action of momordica grosvenori alcohol is not direct and Virus Interaction, but make Toll-like receptor in host cell, excitating organism innate immune system thus play remove virus antiviral effect. This antiviral mode effectively avoids the generation of drug-resistant virus, decreases drug resistance risk.
Momordica grosvenori alcohol related compound of the present invention include the analog of momordica grosvenori alcohol, metabolite, precursor, derivant, Pharmaceutically active salt or the prodrug of momordica grosvenori alcohol.
Further, all compounds of the present invention can be with stereoisomer or/and the form of geometric isomer Exist, such as they can have one or more asymmetric and/or geometric center, it is possible to two or more The form of stereoisomer and/or geometric format exist.The present invention relates to all independent stereoisomers of described compound and several What isomer and purposes of its mixture, as long as described form remains suitable functional activity.
Further, present invention additionally comprises the purposes of the prodrug forms of the compounds of this invention, specifically, this prodrug refers to this The entity of sample, it has some blocking group, and itself can not have pharmacologically active, but is given under certain condition After, the compound with pharmacologically active forming the present invention can be metabolized in vivo.
Further, metabolite of the present invention refers to following any material, its from or be produced from curee couple Be given compound metabolism or digestion after retain product (especially by after hepatic metabolism produce).
According to preferred embodiment, In some embodiments of the present invention, momordica grosvenori alcohol and related compound thereof are also Can be their analogies.Described analogies refer to following any compound, and these compounds include but not limited to peptide, many Peptide, antibody or other organic compound, it has and has identical pharmacologically active or effect with the compounds of this invention or compositions.
Derivant of the present invention refers to be chemically modified described compound the product of formation, and it remains original Chemism desired by compound.Specifically, the means that can be by chemical modification strengthen or reduce chemical combination of the present invention Interaction of hydrogen bond between thing or medicament with action receptor, charge interaction, hydrophobic interaction, Van der Waals force phase interaction With or dipolar interaction, thus improve the pharmacologically active of the compounds of this invention or medicament.
Medical active salt of the present invention refers to the organism given does not causes significant stimulation and does not eliminate the life of compound The salt of the compound of thing activity and characteristic.In some embodiments, described salt is the acid-addition salts of compound.Salt pharmaceutically Can by compound and inorganic acid reaction are obtained, described mineral acid such as halogen acids (such as hydrochloric acid or hydrobromic acid), sulphuric acid, Nitric acid or phosphoric acid etc..Salt pharmaceutically obtains with organic acid reaction also by by compound, described organic acid such as aliphatic hydrocarbyl Or aromatic hydrocarbyl carboxylic acid or sulfonic acid, such as acetic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinic acid, Methanesulfonic acid, ethyl sulfonic acid, p-toluenesulfonic acid, salicylic acid or LOMAR PWA EINECS 246-676-2.Salt pharmaceutically is also by by compound and alkali reaction Form salt to be obtained, described salt such as ammonium salt, alkali metal salt such as sodium salt or potassium salt, alkali salt such as calcium salt or magnesium salt, organic Alkali salt such as dicyclohexyl amine salt, N-methyl-D-glucosamine, three (hydroxymethyl) methyl amine, C1-C7 alkylamine, cyclohexylamine, three second Hydramine, ethylenediamine and there is the salt of amino acid whose salt such as arginine, lysine etc..
Analog of the present invention refers to have structural similarity, the same or similar compound of functional activity.
The compounds of this invention can be isolatable from the fruit of natural plants especially Genus Siraitia Merr, chemical combination the most of the present invention Thing can also be prepared by chemical synthesising technology.It will be apparent to those skilled in the art that and synthesizing Cheng Zhong, it may be necessary to susceptible functionality is protected and deprotects.This can be realized by routine techniques.React at some During, some Stereocenter existed, under certain conditions, by epimerization, can pass through selective response order, bar Part, reagent, protection/deprotection scheme etc. avoid the generation of these type of potential problems.
Virus of the present invention infects and infects selected from one or more banzi virus or togavirus, includes but not limited to: California antigenic group viruses, Saint Louis' encephalitis virus, western language equine encephalitis virus, eastern equine encephalitis virus, Colorado Ticks Fever virus, La Crosse virus, Japanese encephalitis virus, yellow fever virus, Venezuelan equine encephalitis virus, GB virus, step on Leather fever virus, sindbis alphavirus etc..
Virus of the present invention can also derive from rubella virus, such as people rubella virus, Pestivirus；Bovine diarrhoea virus, example Such as bovine viral diarrhea virus, hog cholera virus and border disease of sheep poison；Retrovirus, such as human immunodeficiency virus, including HIV1 and HIV2, simian immunodeficiency virus, recombined human simian immunodeficiency virus, feline immunodeficiency virus, cat or the white blood of Mus Virus, cat or murine sarcoma virus, the infection of Rote virus and Marburg virus etc..
Further, of the present invention virus infect be hepatites virus infections, its selected from hepatitis A, hepatitis B, third Type hepatitis, hepatitis D, hepatitis E, own type hepatitis, hepatitis G, pungent type hepatitis and lupoid hepatitis virus infect institute Cause hepatitis.Preferably, it is simply that the virus infection caused by hepatitis C virus.
Further, virus of the present invention infects and can also is that the AIDS syndrome caused by HIV.
Additionally, the compounds of this invention and/or compositions can suppress or/and prevent sending out of the disease relevant with hepatitis C Make, or/and development, to include but not limited to liver cirrhosis, compensatory hepatic disease, hepatoma carcinoma cell, hepatic fibrosis etc..
Curee of the present invention or patient refer to animal, preferred mammal and especially people.Model of the present invention In enclosing, curee includes the mammal such as mankind, primates and domestic animal (sheep, pig, cattle, horse, donkey etc.)；Laboratory test animal Such as mice, rabbit, rat, Cavia porcellus etc.；And house pet such as cat, Canis familiaris L. etc..For the present invention, mammal is preferably people.
According to the preferred embodiment of the present invention, in some embodiments, curee can be that immunity is suppressed Animal or people.In some embodiments, curee is the AIDS patient that weakens of immunity or infection has retrovirus, As demonstrated the inhibition of HIV of AIDS related syndromes.
Some preferred embodiment in, curee is neonate, can neonate give a birth before and/or newly It is administered during raw youngster's childbirth.
In some preferred implementation, in addition to giving the present composition, it is also possible to give the second antiviral chemical combination Thing, includes but not limited to: nucleoside analog, non-nucleoside reverse transcriptase inhibitor, protease inhibitor, ribavirin, diamantane (obsolete) Amine, Saquinavir, cidofovir, adefovirdipivoxil, famotine hydrochloride, acyclovir, vidarabine, interferon etc..
The present composition can give curee by means of any suitable approach, is generally selected and can strengthen dissolubility And the transmission system of maximum bioavailability is provided.
Some preferred embodiment in, can be by said composition being attached to liposome or carbohydrate carrier In, then said composition is delivered to infected cell.By the antibody for virus antigen is placed on liposome or carrier Surface, thus liposome or carbohydrate carrier can be by the infected host cells of targeting specifically.Preferred at some Embodiment in, it is provided that such liposome, it is by the one in the momordica grosvenori alcohol of high concentration or its related compound Or multiple it is transported to infected cell.
Some preferred embodiment in, route of administration can include but not limited to parenteral route (include subcutaneous to Medicine, intravenous administration, intramuscular injection, patch etc.), oral, anal route (suppository), nasal-cavity administration, topical be (as Sublingual is given Medicine), transfusion, vagina administration, intradermal injection, intraperitoneal routes, intracranial route, intrathecal drug delivery and epidural route.
Following example are intended to further illustrate the present invention rather than limit the present invention.Without prejudice to the present invention spirit and On the premise of principle, any change or the change that carry out the indivedual technical steps of invention fall within scope.
Specific embodiment
The impact on infecting rotavirus neonatal rat TLRs expression of receptor of embodiment 1 momordica grosvenori alcohol
1. experiment material
LLC-MK2 cell (Virus culture cell)；Monkey rotavirus SA11 strain；Naturally neonatal rat of giving birth to (meets cleaning grade requirement, puts down All body weight 1.88g, breast feeding after life, laboratory environment: SPF)；1640 culture medium；Hyclone；Pancreatin；Dual anti-；Colyliform is sick Poison detection kit；Trizol total RNA extraction reagent box；SYBR Green qPCRSuperMix；Cell grown cultures liquid is (tight Under lattice aseptic condition, take RPMI-1640 90ml and add fresh hyclone 10ml, 4 DEG C of preservations)；Virus maintains liquid (1640 trainings Supporting and add trypsin in base, concentration is 1 μ g/ml)；Water isolation type constant incubator；Ultra cold storage freezer；Optical microscope；Electronics Balance；Liquid nitrogen container；Refrigerated centrifuge；Fluorescent PCR instrument.Target gene sequence used is closed by Jin Sirui bio tech ltd Become.
2. test method
2.1 Virus culture: LLC-MK2 cell (RhMK) is cultivated and cultivates at the hyclone RPMI1640 containing 10% In base, after growing up to monolayer, after washing with PBS liquid, add the RV through 10 μ g/ml trypsin pretreatment, 37 DEG C of absorption 60min, adds the RPMI1640 virus without hyclone and maintains liquid static gas wave refrigerator (37 DEG C, 5%CO2), treat that cell is the most sick Results virus during change.By the virus liquid multigelation 3 times of results, 8000r/min is centrifuged 30min, collects supernatant ,-80 DEG C of guarantors Deposit, use TCID50Measure virus titer, treat that virus titer reaches 1x107TCID50Shi Jinhang zoopery.
2.2 animal models are prepared and draw materials: selected Kunming neonatal rat animal feces RV on pretreatment used detection is the moon Property, the birth Kunming neonatal rat of latter 2 days is randomly divided into Normal group, model group, momordica grosvenori alcohol group, often group 20.Model group Every neonatal rat contains 1x10 with 300 μ L7TCID50RV virus liquid gavage；Momordica grosvenori alcohol group bushing stomach 300 μ L contains 1x107TCID50 RV Outside virus liquid, every day also takes momordica grosvenori alcohol, and dosage is 300mg/kg body weight；Blank group gavage equivalent is without RV virus Cell culture fluid.Prohibit milk 4 hours before all neonatal rat gavages, after gavage, prohibit milk 2 hours.Within 3rd, 6 days, use cervical dislocation after infection Method puts to death neonatal rat, takes out rapidly small intestinal 10% formaldehyde and fixes and make paraffin section and carry out pathological observation.Separately take 50-100mg small intestinal Mucous membrane tissue is used for extracting total serum IgE, is stored in-80 DEG C, detects for reverse transcriptase polymerase chain reaction (RT-PCR).
2.3 clinicing symptom observations: experiment starts to observe neonatal rat fed state, stool, body weight growth pattern rear every day Deng, count neonatal rat death quantity, and press reagent operation instructions detection neonatal rat stool RV antigen.The criterion of diarrhoea is divided into 6 Level: 1 grade is without stool；2 grades be yellow molding just；3 grades be yellow paste just；4 grades is yellow water sample Mucous Stool；5 grades is yellow Egg flower sample is just；6 grades is complete yellow watery stool.3 grades and the above diarrhoea that is just judged as, 3,4,5 grades of diarrhoea are for light-duty, and 6 grades are Heavy.
2.4 neonatal rat mucous membrane of small intestine TLR2-9 and the expression of IFN-γ mRNA: extract by Trizol reagent description
Total tissue RNA, measures through ultraviolet spectrophotometer, measures A260 /A280Ratio is more than 1.8, then through reverse transcription amplification cDNA.The cDNA of synthesis detects for RT-PCR, uses two step method, adds each genes of interest primer sequence, overall reaction system 20 μ L, is placed in the reaction of RT-PCR instrument enterprising performing PCR, and cycling condition is 95 DEG C of denaturations 2min, 40 PCR cycle (95 DEG C of degeneration 15 S, anneals and extends 32s, collecting fluorescence).In order to set up the solubility curve of PCR primer, after amplified reaction terminates, continue from 60 DEG C It is heated slowly to 95 DEG C.Genes of interest and the CT value of house-keeping gene of each sample is obtained according to amplification curve.Use 2-△ △ Ct Formula calculates the relative expression quantity of each genes of interest.
3 experimental results
3.1 clinicing symptom observations: whole 20 neonatal rats of model control group obvious abdomen occur after inoculating RV SA-11 strain 24 hours Swollen, spirit is not good enough, but there is no diarrhoea and manifest；Yellow paste occurs just after 48 hours, diarrhea rate 100%, tired mind, activity Few, skin has fold, and body weight increases inconspicuous, diarrhoea peak period within the 3rd day, occurs, within the 6th day, starts to take a turn for the better, the course of disease the most about 7- 10 days, being light-duty diarrhoea, there are 3 neonatal rat death period.Momordica grosvenori alcohol group has part neonatal rat Huang occur after inoculating 48 hours Color mushy stool, diarrhea rate 75%, spirit is not good enough, and activity reduces, and body weight increases inconspicuous, and the neonatal rat that starts for the 5th day to suffer from diarrhoea has Turning, the course of disease is about about 7 days, and period is dead without neonatal rat.Normal group is without symptom of diarrhea, and the mental status is good, skin light Pool, body weight increases substantially.Comparing model group, momordica grosvenori alcohol group neonatal rat symptom of diarrhea is considerably lighter, and the course of disease is considerably shorter, display Momordica grosvenori alcohol improves virus infection state, improves the anti-virus ability of neonatal rat.
3.2 small intestines pathological change: the model group cellular swelling is obvious, it is seen that a large amount of cavity sample degeneration, a little inflammation is thin Born of the same parents infiltrate, and have part hypertrophy, necrosis and Villus atrophy to come off, and pit cell has no substantially change.Momordica grosvenori alcohol group cell has gently Micro-swelling, it is seen that a small amount of cavity sample degeneration and a little inflammatory cell, has no that obvious hyperplasia, necrosis and Villus atrophy are de- Fall.Normal group has no any obvious pathological change.
3.3 small intestinal mucosa TLR2-9 and INF-γ mrna expression: the PCR primer solubility curve of each TLR and GAPDH is analyzed Display, solubility curve is simple spike, and solution temperature is homogeneous, and peak character is sharp keen, illustrates that PCR primer specificity is high, without miscellaneous band.With phase To quantitative method 2-Δ Δ CtValue respectively group TLR and INF-γ mrna expression amount, statistical result is shown in Table 1.Now there are some researches show, with Virus identifies that the TLRs relevant with Immune responses of the antivirus mainly has TLR2,3,4,7,8,9, and wherein TLR3,7,8,9 are positioned Intracellular, mainly identify viral nucleic acid, and TLR2,4 be positioned at after birth, identify the glycoprotein of virus.As it can be seen from table 1 model Group is relative to normal group, and after infecting virus, internal corresponding TLRs expression of receptor amount has to a certain degree increases, but there is no notable Difference, not statistically significant, therefore neonatal rat infects after RV virus and un-activation body natural immune system, therefore causes viral Diarrhoea occurs.Relative to after model group, normal group virus after infection, internal TLRs expression of receptor amount presents bright momordica grosvenori alcohol group Aobvious growth trend, wherein TLR3,4,7, IFN-γ mrna expression amount dramatically increase, display momordica grosvenori alcohol may pass through TLR3, 4,7 receptor activations body natural immune system, and then cause the internal IFN-γ mrna expression amount to rise, play the natural immunity Effect, is conducive to removing virus, shows antiviral activity.In numerous TLR receptors, the expression of TLR4 receptor increases the most Significantly, momordica grosvenori alcohol most possibly mainly acts on TLR4 receptor and plays its antiviral activity.
Table 1 neonatal rat small intestinal mucosa TLR2-9 and the expression of IFN-γ mRNA
Note: compare with normal group,*P ＜ 0.05,**P ＜ 0.01；Compare with model group,#P ＜ 0.05,##P ＜ 0.01.
The impact on hepatitis C virus cytotoxic activity of embodiment 2 momordica grosvenori alcohol
1. experiment material and instrument
Huh7.5.1 cell；Plasmid pJFH1 containing HCV2a type JFH1 Strain genom sequence；With reporter gene Viral JFH1-5AGFP；DMEM culture medium (purchased from GIBCO company)；MTT detectable (purchased from biomol company)；Limit Property restriction endonuclease (purchased from NEB company)；Zeiss senior inverted microscope Axio observer A1 (Carl Zeiss)； PerkinElmer multi-tester；Momordica grosvenori alcohol (self-control, purity 98%).
Experimental technique and result
2.1 plasmid pJFH1-5AGFP build and the preparation of virus JFH1-5AGFP
PJFH1 carries out plasmid transformation, chooses one restriction enzyme site Xho(nt7523-nt7528 of NS5A coding region C end, Aa419-aa420) EGFP gene (being amplified from PEGFP-N1 plasmid) is inserted. after plasmid construction success, order-checking is identified.With XbaI Plasmid PJFH1-5AGFP after linearisation is template, and in vitro transcription obtains virus genome RNA, and then electricity turns Huh-7.5.1 Cell.After 9-10 days, will appear from obvious cytopathy, then collect culture medium supernatant, after subpackage-80 DEG C frozen.In order to obtain Substantial amounts of viral stock, the virus of the infection multiplicity with 0.02 infects Huh7.5.1 cell, obvious cytopathy to appear After, collect infectious supernatant, store stand-by.
The cytotoxicity detection of 2.2 momordica grosvenori alcohols
37 DEG C, 5%CO2Humidified incubator is cultivated.Use the penicillin containing 10%FBS, 100U/mL and the DMEM of streptomycin Culture medium.Cell passes on to 90% degree of converging, and passes on ratio 1/4 1/6.Huh7.5.1 cell presses 8 × 103Individual cells/well connects Plant in 96 porocyte culture plates, standby after cell attachment.With culture medium by momordica grosvenori alcohol from the beginning of 100 μMs, 2 times of gradients are successively It is diluted to 8 concentration, the multiple hole of every concentration 3.In every hole, add 5mg/mlMTT20 μ l after cultivating 72h, put cell culture incubator Middle continuation is cultivated；Cultivate after 4h, abandon culture fluid supernatant, every hole add 100 μ l/ hole three lysates (lysate by SDS10g, Isobutanol 5ml, 10M HCl0.1ml, dissolves with distilled water and is made into 100ml), 37 DEG C of incubators dissolve multifunctional examining after overnight Surveying light absorption value at instrument detection 570nm wavelength, tuning wavelength is 630nm, and calculates drug level cell survival rate.Result such as table 2 Shown in, when momordica grosvenori alcohol concentration is 100 μMs (micromole), cell survival rate still reaches more than 85%, concentration be 50 μMs and Time following, cell survival rate is close to 100%.This shows,
Momordica grosvenori alcohol cytotoxicity is smaller, when 50 μMs and following concentration, to cell almost without toxicity.
2.3 virus activity detections
Luciferase (Luciferase) Reporter System is to be that substrate is to detect fluorescein with fluorescein (luciferin) A kind of reporting system of enzymatic activity.Luciferase can be oxidized to oxyluciferin with catalytic fluorometry element, in the mistake of fluorescein oxidation Cheng Zhong, can send bioluminescence (bioluminescence).May then pass through fluor tester and be also referred to as Chemiluminescence Apparatus (luminometer) bioluminescence or discharged in liquid flashing determining instrument mensuration fluorescein oxidizing process.Fluorescein and luciferase This bioluminescence system, can detect the expression of gene the sensitiveest, efficiently.JFH1-prepared by the present inventor's laboratory With Luciferase reporter gene in Luc-5AGFP Strain, pharmaceutical that therefore can be the sensitiveest is to viral gene The impact expressed.By Huh7.5.1 cell by 8 × 103 Cells/well is inoculated in 96 porocyte culture plates, 37 DEG C of cell culture incubators After middle cultivation 14-18h, standby after cell grows up to monolayer.By culture medium by momordica grosvenori alcohol from maximum concentration with 2 times of gradient dilutions Become 10 concentration, often 3 repetitions of group.Joining in culture plate after 2h by various dose medicine, the infection multiplicity by 0.2 adds JFH1-Luc-5AGFP virus, after infection, 72h carries out luciferase detection, and thin PBS to be measured washes 2 times, then uses Renilla The method of Luciferase Assay Reagent box description adds lysate makes cell fully crack, and then lysate sample is added dilution In good substrate, luciferase detector detects.Result is with Relative fluorescence units numerical value (relative light Units, RLU) display.Experimental data is expressed as the meansigma methods of three independent experiments, and error represents with standard deviation.
This experimental result is as shown in table 1, and is not added with medicine, and the control wells only infecting virus is compared, and momordica grosvenori alcohol dosage depends on Inhibit HCV in intracellular propagation badly.When drug level is 50 μMs, cell is close to 100%
Survival, and the suppression ratio that drug on viral replicates has reached 98%.This shows, momordica grosvenori alcohol is true to the suppression of HCV Rather than cause virus replication to be suppressed due to the toxicity to cell.
The impact on HCV virus activity of table 2 momordica grosvenori alcohol
Drug level (μM) 100 50 25 12.5 6.25 3.12 1.56 0.78
Mean percent cell survival 81.32 98.29 101.82 103.28 102.56 100.39 99.98 103.22
Average virus suppression ratio 99.76 98.13 91.89 89.74 63.56 51.68 28.31 9.59
The impact on HCV virus infection process of 2.4 momordica grosvenori alcohols
4 DEG C of pre-coolings of Hu7.5.1 cell 1 hour.With culture medium by momordica grosvenori alcohol from the beginning of 50 μMs, 2 times of gradients are diluted to 8 successively Concentration and be cooled in advance 4 DEG C standby.Then, in viruses adsorption is tested, the above-mentioned culture medium containing variable concentrations medicine is added In the 96 each holes of orifice plate, HCV infection (JFH1) 50 μ L/ hole simultaneously, continue 4 DEG C and place 3 hours, allow virus the most fully be adsorbed onto On target cell, wash away the virus do not adsorbed afterwards with pre-cooling PBS；In Virus entry is tested, first it is not added with medicine, makes HCV straight Connect and hatch 3 hours with 4 DEG C of cell, after pre-cooling PBS washes away the virus do not adsorbed, rapidly join above-mentioned containing variable concentrations medicine Culture medium and transfer at once 37 DEG C continue cultivate 1 hour, then remove pastille culture medium, wash away residual drug with PBS. Finally, the cell through different disposal is all replaced with fresh culture, and 37 DEG C are further cultured for about 48 hours.Virus replication is tested In, Huh7.5.1 cell (10% FBS, DMEM) is with 105 The concentration of individual/ml adds (Costar3904) in 96 orifice plates, every hole 100μl；After 24 hours, by culture supernatant sucking-off, add the vial supernatant 50 μ l of MOI=0.1；After 8 hours, add 50 μ l Variable concentrations medicine to be detected, adds 100 μ l culture fluid, cultivates 72 hours；Sucking-off supernatant detects.Above by reporter gene The detection of luciferase, calculates the impact that HCV is adsorbed, invades, replicates by momordica grosvenori alcohol, is specifically shown in Table 3.
The impact on HCV virus infection process of table 3 momordica grosvenori alcohol
Drug level/μM 50 25 12.5 6.25 3.13 1.56 0.78 0.39
Absorption average inhibition 76.57 65.29 61.35 48.72 34.65 26.58 21.34 17.49
Invade average inhibition 62.33 56.41 48.74 35.29 28.33 21.47 12.52 8.37
Replicate average inhibition 74.65 68.27 54.38 49.23 41.46 36.19 28.47 19.36
From table 3 it can be seen that be not added with medicine only infect virus culture hole for comparison (suppression ratio is considered as 0%),
Duplication after momordica grosvenori alcohol is adsorbed onto target cells for HCV, invades target cell and invade cell has different journey The inhibitory action of degree, and there is dosage according to patience.Above-mentioned experiment shows that momordica grosvenori alcohol has stronger vitro inhibition HCV virus Effect.
Embodiment 3 momordica grosvenori alcohol HIV (human immunodeficiency virus)-resistant activity is tested
This experiment uses cytopathic effect method (CPE) and the RNA reverse transcription fluorescence quantitative PCR method In Vitro Anti to momordica grosvenori alcohol HIV effect is evaluated.
1 material and reagent
CEMxl74 cell and HIV-l virus (derive from U.S.'s Aarond Diamond acquired immune deficiency syndrome (AIDS) research center, by middle traditional Chinese medical science Institute of lab animals of subject institute presents)；DMSO(Military Medical Science Institute import subpackage)；RPMI Medium 1640 Basic (1 ×), F etalBovine Serum(Shanghai Li Fei Bioisystech Co., Ltd)；Zidovudine (AZT, 3 '- Azido-3 '-deoxythymidine) purchased from Sigma company.Each gene mRNA quantification PCR primer, by the handsome biology in Shanghai Technology Co., Ltd. synthesizes.Momordica grosvenori alcohol (self-control, purity is more than 98%).
2. test method
2.1 cells are cultivated
It is inoculated in RPMll640 culture medium (1% mycillin, 10% hyclone) after being recovered by CEMxl74 cell strain, puts full With humidity, 37 DEG C, containing 5%C02Cultivate in incubator.Treating that cell grows to logarithmic (log) phase, cell counting, with 1-2*105/ ml passes on, Again pass on for pharmacodynamic experiment after about 3 days.
2.2 couples of HIV induce the cytopathic inhibitory action of CEMxl74
CEMxl74 is with 2.0*105Density 0.1mL of individual/mL is inoculated in 96 orifice plates, by virus HIV 10 times of gradient dilutions 8 Concentration, the multiple hole of each concentration 8, observation of cell pathological changes after cultivating 3 days, calculate TCID50.Cell blank group, virus control are set Group, AZT positive drug control group, momordica grosvenori alcohol group (high, normal, basic three administration concentration are set), momordica grosvenori alcohol-AZT group.Administration group is first Start test using non-toxic concentrations as final concentration, first with RPMll640, medicinal liquid is diluted to 100 times of final concentration as being administered Concentration.By CEMxl74 cell with 2.0*105The density of individual/mL is inoculated in 24 orifice plates, and every hole is 0.75mL, cell controls group Adding 0.25mL culture fluid, remaining test group adds the inhibition of HIV 0.25mL of 10*TCID50 respectively.AZT positive drug control group adds Enter the AZT 1O μ l of lOO μM of concentration, momordica grosvenori alcohol group be separately added into 100 μMs (high dose group), 50 μMs (middle dosage group), 25 μMs The momordica grosvenori alcohol solution 10 μ l of (low dose group) concentration, each sample concentration is all provided with 3 parallel holes.Momordica grosvenori alcohol-AZT organizes addition The AZT of 100 μMs of concentration and the mixed solution 10 μ l of momordica grosvenori alcohol.All test group put 37 DEG C, 5%CO2Cultivate in incubator, 3 After it, observation of cell pathological changes (CPE) under inverted microscope, treats pathological changes out, changes liquid, 5-7 days, treat virus control group occur CPE "+ ++ ~ ++++" time observed and recorded result, and collect cell and supernatant.
CPE criterion: without Syncytium formation "-", each hole have 29 syncytial cells "+", there is 10-20 in each hole Individual syncytial cell " ++ ", each hole has > 20 syncytial cells " +++ one ++++".
2.3 fluorescence quantitative PCR method detection HIV-RNA expressions
After above-mentioned observation CPE result, the cell of collection and supernatant, process further, extract RNA and carry out fluorescent quantitative measurement.Adopt Cell HIVRNA expression is detected with nucleotide colloid (SYBR) dyestuff quantitative real-time PCR.Collect cell in centrifugal Guan Zhong, every Kong Yiguan, about 1-2 × 106Individual/pipe, extracts cell total rna by Trizol (500 μ 1) method.Take cell total rna 2 μ g (5.5 μ l), is reversed by the RevertAid Reverse Transcriptase explanation of Thermo Scientific company Record is cDNA.After diluting ten times, with GAPDH320 as reference gene, take equivalent cDNA (2 μ 1) and carry out dye method SYBR fluorescence calmly Amount PCR reaction.Reaction condition is 95 DEG C of denaturations 1min；95 DEG C of 15s, 60 DEG C of lmin, 40 circulations；95 DEG C of 15s of solubility curve, 60 DEG C of 30s, 95 DEG C of 15s.Genes of interest/house-keeping gene ratio represents result.
Employing sonde method fluorescence quantitative PCR method detection supernatant HIVRNA expression collection supernatant is in centrifuge tube, often Kong Yiguan, extracts viral RNA with RNA extracting solution 400 μ l:lOO μ l supernatant by about 1 milliliter.Directly press Thermo Scientific The RevertAid Reverse Transcriptase of company illustrates reverse transcription, system 8 μ l, 42 DEG C of 60min.Take 2 μ l to visit Skill of handling needles quantitative fluorescent PCR reacts.Reaction condition is 50 DEG C of 2min；95 DEG C of denaturations lOmin, 95 DEG C of 15s, 60 DEG C of lmin, 45 Circulation, does standard curve quantitative, represents result with the expression i.e. virus load of virus.
3 experimental results
HIV-1 is induced the cytopathic inhibitory action of CEMxl74 by 3.1 momordica grosvenori alcohols
When basis of microscopic observation virus control combination cell space formational situation be " +++ " time, cell blank group, AZT positive controls, Momordica grosvenori alcohol-AZT group, momordica grosvenori alcohol high dose group do not observe plasmodial formation, dosage group and low dosage in momordica grosvenori alcohol Group has a small amount of Syncytium formation, but it is few to compare virus control group quantity, as shown in table 4.Result illustrates, momordica grosvenori alcohol pair HIV-1 induction CEMxl74 cytopathy has inhibitory action, and has doses according to patience.Experimental observation is to momordica grosvenori alcohol simultaneously Having obvious cooperative effect with existing inverase AZT combination, effect ratio is used alone momordica grosvenori alcohol or AZT is more aobvious Write.
Table 4 momordica grosvenori alcohol acts on the impact of the CEMxl74 cell syncytia (CPE) of HIV
The impact that inhibition of HIV is expressed by 3.2 momordica grosvenori alcohols
From experimental result (table 5) it can be seen that momordica grosvenori alcohol each dosage group is sick for intracellular HIVRNA expression and supernatant Poison carrying capacity has certain inhibitory action, and wherein the inhibitory action of momordica grosvenori alcohol high dose group is extremely notable, with positive controls Effect is suitable.In addition test and also find that suppression intracellular HIVRNA that momordica grosvenori alcohol and AZT combination are embodied is expressed and born of the same parents are outside upper The effect of clear liquid virus load is significantly stronger than the two and is used alone, and shows extremely strong synergism.
The impact on HIV-1 virus expression of table 5 momordica grosvenori alcohol
Group Intracellular HIVRNA relative quantity Supernatant virus load relative value
Cell blank group 0.0000062±0.0000028<sup>***</sup> 3.28±2.87<sup>***</sup>
Virus group 3.358905±0.557812 8.41±7.98
AZT positive controls 0.004128±0.000309<sup>***##</sup> 5.39±5.12<sup>***##</sup>
Momordica grosvenori alcohol high dose group 0.031789±0.008764<sup>***##</sup> 5.75±4.28<sup>***##</sup>
Dosage group in momordica grosvenori alcohol 2.425807±0.328617<sup>**</sup> 7.23±5.21<sup>**</sup>
Momordica grosvenori alcohol low dose group 2.879213±0.187526 8.01±4.87<sup>*</sup>
Momordica grosvenori alcohol high dose-AZT group 0.001246±0.000087<sup>***</sup> 4.29±3.58<sup>***</sup>
Note: compare with virus group,***P ＜ 0.001,**P ＜ 0.01,*P ＜ 0.05；With momordica grosvenori alcohol high dose-AZT group ratio Relatively,##P ＜ 0.01.

Claims (26)

1. momordica grosvenori alcohol and related compound application in treatment and/or preventing viral are infected thereof.
Application the most according to claim 1, wherein said momordica grosvenori alcohol related compound refers to the similar of momordica grosvenori alcohol Thing, metabolite, precursor compound, derivant, pharmaceutically active salt or prodrug.
Application the most according to claim 1, wherein said momordica grosvenori alcohol is from cucurbitaceous plant.
Application the most according to claim 3, wherein said cucurbitaceous plant is Genus Siraitia Merr.
5., according to the application described in claim 3-4, wherein said momordica grosvenori alcohol comes from Fructus Momordicae.
Application the most according to claim 1, wherein said momordica grosvenori alcohol and related compound thereof are chemosynthesis.
Application the most according to claim 6, wherein said momordica grosvenori alcohol and related compound thereof can be by halogenations.
Application the most according to claim 1, wherein said viral infection, include but not limited to rhinovirus, adenovirus, Respiratory tract and cellular virus, parainfluenza virus, coronavirus, influenza virus, mumps virus, poliovirus, Cook Sa Ji virus, echo, enteric cytopathogenic human orphan virus, rotavirus, norwalk virus, Astrovirus, Calicivirus, hepatitis A virus, B-mode Hepatitis virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, Measles virus, rubella virus, roseola infantum disease Poison, chickenpox, variola, herpes simplex virus, rabies virus, foot and mouth disease virus, encephalitis b virus, western equine encephalitis virus, east horse Encephalitis, St. Louis encephalitis virus, Venezuelan equine encephalitis virus, california antigenic group viruses, cytomegalovirus, acquisition Property immunodeficiency virus, hemorrhagic fever virus, yellow fever virus, that dengue virus, Colorado Ticks wear any of which such as fever virus is sick What poison caused infects or the mixed infection that causes of multiple virus.
Application the most according to claim 8, wherein said viral infection is the hepatitis C that flavivirus causes Or the infection that HIV causes.
10., for treatment or/and a pharmaceutical composition for preventing viral infection, it includes at least momordica grosvenori alcohol, or sieve One or more in Chinese fruit alcohol related compound, and at least one medicinal diluent, carrier or excipient.
11. pharmaceutical compositions according to claim 10, wherein said momordica grosvenori alcohol closes from Fructus Momordicae or by chemistry One-tenth means obtain, and momordica grosvenori alcohol can also be by halogenation further.
12. pharmaceutical compositions according to claim 10, wherein said momordica grosvenori alcohol related compound includes but does not limits In analog, metabolite, precursor compound, derivant, medical active salt or the prodrug of momordica grosvenori alcohol, further, described sieve Chinese fruit alcohol related compound can be obtained by chemosynthesis or biosynthesis, metabolism, and described related compound can also enter one Step is by halogenation.
13. is pharmacy according to the pharmaceutical composition described in claim 10 to 12, wherein said diluent, carrier or excipient The common medicinal supplementary material of upper acceptance.
14. according to the pharmaceutical composition described in claim 10 to 13, wherein said viral infection include but not limited to by Simple infection that one or more viruses cause or mixed infection, further, described viral infection refers to hepatitis C virus The infection that poison or inhibition of HIV cause.
15. 1 kinds are used for treatment or/and the pharmaceutical composition of preventing viral infection, and it includes at least momordica grosvenori alcohol, or arhat Really one or more in alcohol related compound, and at least one second antiviral drugs, the most also comprise at least one medicine With diluent, carrier or excipient.
16. pharmaceutical compositions according to claim 15, wherein said second antiviral drugs refers to existing conventional Antiviral drugs, includes but not limited to virazole, amantadine, acycloguanosine, polyinosinic acid, interferon, rimantadine, disease Poison spirit, vidarabine, zidovudine, acyclovir, ribavirin, ganciclovir, deoxyribosylthymine, Sebivo etc..
17. pharmaceutical compositions according to claim 15, wherein said momordica grosvenori alcohol closes from Fructus Momordicae or by chemistry One-tenth means obtain, and momordica grosvenori alcohol can also be by halogenation further.
18. pharmaceutical compositions according to claim 15, wherein said momordica grosvenori alcohol related compound includes but does not limits In analog, metabolite, precursor compound, derivant, medical active salt or the prodrug of momordica grosvenori alcohol, further, described sieve Chinese fruit alcohol related compound can be obtained by chemosynthesis or biosynthesis, metabolism, and described related compound can also enter one Step is by halogenation.
19. is pharmacy according to the pharmaceutical composition described in claim 15 to 18, wherein said diluent, carrier or excipient The common medicinal supplementary material of upper acceptance.
20. according to the pharmaceutical composition described in claim 15 to 19, wherein said viral infection include but not limited to by Simple infection that one or more viruses cause or mixed infection, further, described viral infection refers to hepatitis C virus The infection that poison or inhibition of HIV cause.
21. 1 kinds for prevention or the/treatment associating medicament of viral infection and using method thereof, its comprise momordica grosvenori alcohol or Momordica grosvenori alcohol related compound preparation and at least one auxiliary therapy medicament.
22. associating medicaments according to claim 21, its described momordica grosvenori alcohol or momordica grosvenori alcohol related compound preparation are Refer to comprise the pharmaceutical preparation of one or more in momordica grosvenori alcohol, momordica grosvenori alcohol related compound, further, described pharmaceutical preparation Include but not limited to injection, capsule, tablet, infusion solution, pill, microcapsule, targeting preparation etc..
23. associating medicaments according to claim 21, its described auxiliary therapy medicament refers to have identical or be similar to anti- Virus activity medicine and can improve the pharmaceutical preparation of viral infection symptom medicine, the most existing antiviral drugs, improves sense Dye patient's complication medicine, improve the medicine of patient's lipid metabolic disorder, the medicine etc. of enhancing human body immunity power, including but not It is limited to ribavirin, acyclovir, acycloguanosine, interferon, antibiotic, lipid-lowering statins, antioxidant, immunostimulant The pharmaceutical preparation such as agent, astragalus polysaccharides.
24. associating medicaments according to claim 21, its described using method is for simultaneously or separately to give patient Fructus Momordicae Alcohol or its related compound preparation and auxiliary therapy medicament.
25. usings method according to claim 24, its using method is for first giving patient's momordica grosvenori alcohol or its relevantization Compound preparation, gives auxiliary therapy medicament the most again.
26. according to the associating medicament described in claim 21-25, wherein said viral infection refer to hepatitis C virus or The infection that inhibition of HIV causes.
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