CN106085868B - One plant of Aspergillus and its application - Google Patents
One plant of Aspergillus and its application Download PDFInfo
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- CN106085868B CN106085868B CN201610424586.3A CN201610424586A CN106085868B CN 106085868 B CN106085868 B CN 106085868B CN 201610424586 A CN201610424586 A CN 201610424586A CN 106085868 B CN106085868 B CN 106085868B
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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
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
-
- 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
- A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P15/00—Preparation of compounds containing at least three condensed carbocyclic rings
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
Abstract
The invention discloses one plant of Aspergillus and its applications.The bacterial strain number of the Aspergillus is CPCC400735, is CGMCC No.12376 in the number of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.The methanolic extract of aspergillus CPCC400735 fermentation culture medium is 99.9% to the inhibiting rate of HIV-1 when concentration is 4mg/mL.1 to 4 compound represented of formula is when concentration is 100 μM to the inhibiting rate of HIV-1 in 96% or more, IC502.4-32.6 μM of value.The methanolic extract and formula 1 of aspergillus CPCC400735 fermentation culture medium of the invention have stronger inhibitory activity to HIV-1 to 4 compound represented of formula, and toxicity is smaller.
Description
Technical field
The present invention relates to one plant of Aspergillus and its applications.
Background technique
HIV (Human Immunodeficiency Virus) virus, Chinese name human immunodeficiency virus belong to inverse
One kind of Retroviral.After inhibition of HIV invades host cell, host body immune function can be destroyed, seriously so as to cause serious
Opportunistic infections so that causing the secondary immunodeficiency characterized by malignant disease, as acquired immunodeficiency syndrome
(acquired immunodeficiency syndrome, AIDS).Patient With Aids case was found in 1981 in the U.S.,
It then begins to propagate rapidly in the world, has become one of the significant threat of human health at present.Inhibition of HIV can be divided into
HIV-1 type and HIV-2 type, the two genome have very big difference, and wherein HIV-1 is the master for causing global spread of aids
Want pathogen.
Chemotherapy is still currently the most important ones HIV-1 treatment means, has 26 kinds of chemical entities to enter clinic so far
Using.Classify by its mechanism of action, is mainly reverse transcriptase inhibitor and protease inhibitors in these marketed drugs, adds up
To 22 kinds.And only 4 kinds of other mechanism of action, including 2 kinds of integrase inhibitors and 2 kinds of viral entry inhibitors.With anti-
The continuous research and development application of HIV drug is especially treated AIDS patient by Cocktail treatment mode, can be had
Effect reduces virus load, significant to extend the survival of patients time, but the problems such as drug resistance, adverse reaction and the compliance of patient still
Often cause the failure of clinical treatment.Therefore, it is still necessary to which the new anti-HIV-1 medicines of Persisting exploitation are for clinical application.
Microbe species are huge, the metabolite with enriched types, are find high-efficiency low-toxicity HIV-resistant activity substance one
A important sources.It is quickly screened by establishing screening model and produces the bacterial strain of HIV-resistant activity substance, then from its metabolite
Finding compound with HIV-resistant activity or having certain active compound is guide, carries out that structure is simplified and modification, announcement
The mechanism of action and structure-activity relationship are the effective ways for finding new inverase.
Summary of the invention
The technical problem to be solved by the present invention is to how inhibit HIV.
In order to solve the above technical problems, the present invention provides one plant of aspergillus.
Aspergillus (Aspergillus sp.) provided by the present invention, bacterial strain number are CPCC400735 (also known as CPCC
It 400735) is, CGMCC in the number of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center
No.12376。
Above-mentioned aspergillus (Aspergillus sp.) CPCC400735 can with conidium, mycelia or containing conidium and/
Or the mycelial form of mycelia exists.
Above-mentioned aspergillus (Aspergillus sp.) CPCC400735 is preparing the application in hiv inhibitor, above-mentioned aspergillus
(Aspergillus sp.) CPCC400735 answering in preparation treatment or/and prevention acquired immunodeficiency syndrome drug
With, the hiv inhibitor that is obtained by above-mentioned aspergillus (Aspergillus sp.) CPCC400735, by above-mentioned aspergillus
The treatment or/and prevention acquired immunodeficiency syndrome drug that (Aspergillus sp.) CPCC400735 is prepared are equal
It belongs to the scope of protection of the present invention.
The culture of above-mentioned aspergillus (Aspergillus sp.) CPCC400735 also belongs to protection scope of the present invention.
The culture of above-mentioned aspergillus (Aspergillus sp.) CPCC400735 is by above-mentioned aspergillus (Aspergillus
Sp.) the substance in the culture vessel that CPCC400735 is cultivated in microbiological culture media.
In the culture of above-mentioned aspergillus (Aspergillus sp.) CPCC400735, the microbiological culture media can be song
Mould culture medium (culture medium that can cultivate aspergillus) can be solid medium, semisolid culturemedium or fluid nutrient medium.It is described micro-
Biological medium can be rice culture medium, potato dextrose agar or other fungies well known to those skilled in the art
Fermentation medium etc..In the culture of above-mentioned aspergillus (Aspergillus sp.) CPCC 400735, the rice culture medium can
Be made, can also be made of rice, water and inorganic salts of rice and water, can also be made of rice, water and nitrogen source, can also by rice,
Water, nitrogen source and inorganic salts are made.The potato dextrose agar can be made of potato, glucose, agar and water,
It can also be made, can also be made of potato, carbon source, agar, water and nitrogen source of potato, glucose, agar, water and inorganic salts,
It can also be made of potato, carbon source, agar, nitrogen source and inorganic salts.Other described fungi fermentations well known to those skilled in the art
Culture medium can by it is a certain or it is several it is quick-acting, late imitate carbon source, a certain kind or it is several it is quick-acting, imitate nitrogen source, water and/or inorganic salts etc. late
It is made.
Above, the rice can be rice or brown rice.Rice or brown rice are the products of paddy.Paddy, which refers to, not to be gone
Except the fruit of the husk of paddy rice, paddy is made of husk, pericarp, kind skin, perisperm, aleurone, endosperm and embryo.Brown rice refers to paddy
Husk is sloughed, the product of the other each sections of paddy is retained；Rice, which refers to, only retains endosperm, and paddy rest part is all sloughed
Product.Carbon source is that microorganism grows a kind of nutrients, is carbon compound, including carbohydrate, grease, organic acid and organic acid esters
With small molecular alcohol etc. it is quick-acting, imitate carbon source late.The substance of nitrogen needed for nitrogen source refers to offer microbial nutrition, including peanut cake
Powder, soybean cake powder, yeast powder, peptone, ammonium hydroxide, ammonium salt and nitrate etc. are quick-acting, imitate nitrogen source late.
In the culture of above-mentioned aspergillus (Aspergillus sp.) CPCC400735, the temperature of the culture can be 20-30
DEG C, incubation time can be 10-40 days.
The present invention also provides a kind of hiv inhibitors.
A kind of active constituent of hiv inhibitor provided by the present invention is to use methanol from above-mentioned aspergillus (Aspergillus
Sp. the obtained substance for being dissolved in methanol) is extracted in the culture of CPCC400735.
The present invention also provides the preparation methods of the hiv inhibitor, including with methanol from above-mentioned aspergillus (Aspergillus
Sp. the substance for obtaining being dissolved in methanol) is extracted in the culture of CPCC400735, using the substance as the activity of hiv inhibitor
Ingredient.
The present invention also provides a kind for the treatment of or/and prevention acquired immunodeficiency syndrome drugs.
The active constituent for the treatment of or/and prevention acquired immunodeficiency syndrome drug provided by the present invention is to use methanol
The obtained substance for being dissolved in methanol is extracted from the culture of above-mentioned aspergillus (Aspergillus sp.) CPCC400735.
The present invention also provides the treatment or/and the preparation methods of prevention acquired immunodeficiency syndrome drug, including
It is extracted from the culture of above-mentioned aspergillus (Aspergillus sp.) CPCC400735 with methanol and obtains the substance for being dissolved in methanol,
Using the substance as the active constituent for treating or/and preventing acquired immunodeficiency syndrome drug.
The present invention also provides another hiv inhibitors.
The active constituent of another kind hiv inhibitor provided by the present invention, is II compound represented of formula:
The R is
The present invention also provides the methods for preparing hiv inhibitor, including with methanol from above-mentioned aspergillus (Aspergillus
Sp. the substance for obtaining being dissolved in methanol) is extracted in the culture of CPCC400735, then is extracted from the substance and obtained II institute of formula
The compound shown, using II compound represented of formula as the active constituent of hiv inhibitor.
The present invention also provides another kind treatment or/and prevention acquired immunodeficiency syndrome drugs.
The treatment or/and prevention acquired immunodeficiency syndrome drug, active constituent is II compound represented of formula.
The present invention also provides the methods for preparing the treatment or/and prevention acquired immunodeficiency syndrome drug, including
It is extracted from the culture of above-mentioned aspergillus (Aspergillus sp.) CPCC400735 with methanol and obtains the substance for being dissolved in methanol,
It is extracted from the substance again and obtains II compound represented of formula, obtained II compound represented of formula as treatment or/and prevention
Obtain the active constituent of property immunologic deficiency syndrome drug.
The present invention also provides the methods of II compound represented of preparation formula.
The method of II compound represented of preparation formula provided by the present invention, including with methanol from above-mentioned aspergillus
The obtained substance for being dissolved in methanol is extracted in the culture of (Aspergillus sp.) CPCC400735, then from the substance
Extraction obtains II compound represented of formula.
The present invention also provides 9) -12) method:
9) treat or/and prevent acquired immunodeficiency syndrome method, including to receptor application methanol from
The substance for obtaining being dissolved in methanol is extracted in above-mentioned aspergillus (Aspergillus sp.) CPCC400735, is treated or/and is prevented
Acquired immunodeficiency syndrome；
10) inhibit the method for HIV infection animal, including to receptor application methanol from above-mentioned aspergillus
The substance for obtaining being dissolved in methanol is extracted in (Aspergillus sp.) CPCC400735 culture to inhibit HIV infection animal；
11) method for treating or/and preventing acquired immunodeficiency syndrome, including to shown in receptor application formula II
Compound treated or/and prevented acquired immunodeficiency syndrome；
12) inhibit the method for HIV infection animal, including to receptor application II compound represented of formula to inhibit HIV
Infection animal.
In the above method, from extracting in the substance, to obtain II compound represented of formula specifically may include A1)-A4) in
Corresponding steps:
A1) that the substance is water-dispersible, it is then extracted with ethyl acetate, collects ethyl acetate phase, remove ethyl acetate
Ethyl acetate in phase, obtains medicinal extract；
A2 the medicinal extract) is subjected to silica gel column chromatography separation, elution program used in silica gel column chromatography is first to use chloroform
Elute 2 column volumes；Carry out the following linear gradient elution of 13 column volumes again: mobile phase used is by chloroform and methanol
The mixed liquor of composition, the volume ratio of chloroform and methanol is linearly down to 7:1 by 20:1 in linear gradient elution mobile phase；Finally use again
Methanol elutes two column volumes；The liquid that the 0.9th to the 3.6th column volume elutes is collected, Fr.4-12 is named as, is received
Collect the liquid that the 4.5th to the 5.1st column volume elutes, is named as Fr.16-17；
A3 Fr.4-12) is subjected to silica gel column chromatography, elution program used in the silica gel column chromatography is to carry out 6 cylinders
Long-pending following linear gradient elution: the mixed liquor that mobile phase used is made of petroleum ether and acetone, in linear gradient elution
Mobile phase in petroleum ether and the volume ratio of acetone 5:1 is linearly down to by 8:1；Collect the 3.2nd to the 4.5th column volume elution
Liquid out is named as Tubes 27-37；
A4 Tubes 27-37) is subjected to preparative high performance liquid chromatography separation, which separates institute
Filler is octadecylsilane chemically bonded silica filler, and the partial size of filler is 5 μm, which separates institute
Chromatography column diameter is 8mm, a height of 250mm；Mobile phase is the mixing liquid of first alcohol and water composition, first alcohol and water in mobile phase
Volume ratio be 4: 1；Flow velocity is 4.0mL/min, collects the eluting peak that retention time is 23.9min, is named as tubes
27-37-P2；Tubes 27-37-P2 is subjected to preparative high performance liquid chromatography separation；Preparative high performance liquid chromatography separation
Filler used is octadecylsilane chemically bonded silica filler, and the partial size of filler is 5 μm, preparative high performance liquid chromatography separation
Chromatography column diameter used is 8mm, a height of 250mm；Mobile phase is the mixing liquid of first alcohol and water composition, in mobile phase methanol and
The volume ratio of water is 4: 1；Flow velocity is 4.5mL/min, collects the eluting peak that retention time is 23.9min, obtaining R in formula II isCompound (i.e. 1 compound represented of formula)；
A5 Tubes 27-37) is subjected to preparative high performance liquid chromatography separation, which separates institute
Filler is octadecylsilane chemically bonded silica filler, and the partial size of filler is 5 μm, which separates institute
Chromatography column diameter is 8mm, a height of 250mm；Mobile phase is the mixing liquid of first alcohol and water composition, first alcohol and water in mobile phase
Volume ratio be 4: 1；Flow velocity is 4.0mL/min, collects the eluting peak that retention time is 44.6min, is named as tubes
27-37-P6；Tubes 27-37-P6 is subjected to preparative high performance liquid chromatography separation；Preparative high performance liquid chromatography separation
Filler used is octadecylsilane chemically bonded silica filler, and the partial size of filler is 5 μm, preparative high performance liquid chromatography separation
Chromatography column diameter used is 8mm, a height of 250mm；Mobile phase is the mixing liquid of first alcohol and water composition, in mobile phase methanol and
The volume ratio of water is 4: 1；Flow velocity is 5mL/min, collects the eluting peak that retention time is 37.7min, obtaining R in formula II isCompound (i.e. 2 compound represented of formula)；
A6 Fr.16-17) is subjected to silica gel column chromatography, elution program used in the silica gel column chromatography be with by chloroform and
The mixing liquid of the volume ratio 10:1 of the chloroform and methanol of methanol composition is eluted as mobile phase, collects the 0th to 0.2 column
The liquid that volume elutes is named as tubes 1-3, collects what the 0.2nd column volume was eluted to the 0.9th column volume
Liquid is named as Tubes 4-13；Tubes 4-13 is subjected to open ODS column chromatography for separation, opening ODS column chromatography point
It is octadecylsilane chemically bonded silica filler from filler used, the partial size of filler is 50 μm, opening ODS column chromatography for separation institute
Chromatography column diameter is 4cm, a height of 16cm, and chromatography column volume is 201mL；Elution used in opening ODS column chromatography for separation
Program is the elution of two steps, and first step elution is made with the mixing liquid that the volume ratio for the first alcohol and water being made of first alcohol and water is 11:9
4 column volumes are eluted for mobile phase, complete first step elution；Second step elution: after completing first step elution, use methanol as stream
Dynamic phase is eluted, and is collected the liquid that the 0th to the 0.5th column volume that methanol elutes elutes, is named as Tubes
4-13-ODS-9.Tubes 4-13-ODS-9 and tubes 1-3 are merged and carry out entitled 4 compound represented -5 of formula preparation point
From preparative high performance liquid chromatography separation, the preparative efficient liquid phase of entitled -5 preparative separation of 4 compound represented of formula
Filler used in chromatographic isolation is octadecylsilane chemically bonded silica filler, and the partial size of filler is 5 μm, shown in the entitled formula 4
The used chromatography column diameter of preparative high performance liquid chromatography separation of -5 preparative separation of compound be 8mm, a height of 250mm；Stream
The mixing liquid of Xiang Weijia alcohol and water composition is moved, the volume ratio of first alcohol and water is 3: 1 in mobile phase；Flow velocity is 4.0mL/min；It receives
Integrate retention time and obtain P2 as the eluting peak of 18.2min, collect the eluting peak that retention time is 22.2min and obtain P3, by P2 and
P3 carries out the preparative high performance liquid chromatography separation of -5 preparative separation of 4 compound represented of entitled formula, the preparation of P2 again
The eluting peak that collection retention time is 18.2min in type high performance liquid chromatography separation obtains R in formula II and is(i.e. 4 compound represented of formula)；When collecting reservation in the preparative high performance liquid chromatography separation of P3
Between be 22.2min eluting peak obtain R in formula II and beCompound (i.e. 3 compound represented of formula).
Above-mentioned II compound represented of formula or its pharmaceutically acceptable salt, II compound represented of formula or its pharmaceutically may be used
The salt of receiving prepare the application in hiv inhibitor, with II compound represented of formula or its pharmaceutically acceptable salt for activity
The HIV inhibitor of ingredient all belongs to the scope of protection of the present invention.
Above, the HIV can be the HIV of HIV-1 type, and the acquired immunodeficiency syndrome can be drawn by HIV-1
It rises.
Above, the animal can be mammal, such as people.
Above, the hiv inhibitor and the treatment or/and prevention acquired immunodeficiency syndrome drug, except containing
Outside active ingredient, also containing suitable carrier or excipient.Here carrier material includes but is not limited to water-solubility carrier
Material (such as polyethylene glycol, polyvinylpyrrolidone, organic acid), (such as ethyl cellulose, cholesterol is hard for slightly solubility carrier material
Resin acid ester etc.), enteric solubility carrier material (such as the first and second cellulose of cellulose acetate phthalate and carboxylic).Wherein it is preferred that water-soluble
Property carrier material.A variety of dosage forms, including but not limited to tablet, capsule, dripping pill, aerosol, ball can be made using these materials
Agent, pulvis, solution, suspension, emulsion, granule, liposome, transdermal agent, buccal tablet, suppository, freeze drying powder injection etc..It can be with
It is ordinary preparation, sustained release preparation, controlled release preparation and various particulate delivery systems.It, can in order to which tablet is made in unit dosage forms for administration
Various carriers well known in the art are widely used.Example about carrier is, such as diluent and absorbent, such as starch, paste
Essence, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, white bole, microcrystalline cellulose, alumina silicate
Deng；Wetting agent and adhesive, as water, glycerol, polyethylene glycol, ethyl alcohol, propyl alcohol, starch slurry, dextrin, syrup, honey, glucose are molten
Liquid, mucialga of arabic gummy, gelatine size, sodium carboxymethylcellulose, lac, methylcellulose, potassium phosphate, polyvinylpyrrolidone etc.；
Disintegrating agent, such as dry starch, alginate, agar powder, laminaran, sodium bicarbonate and citric acid, calcium carbonate, polyoxy second
Alkene, sorbitan fatty acid ester, dodecyl sodium sulfate, methylcellulose, ethyl cellulose etc.；Disintegration inhibitor, such as sugarcane
Sugar, glyceryl tristearate, cocoa butter, hydrogenated oil and fat etc.；Sorbefacient, such as quaternary ammonium salt, lauryl sodium sulfate etc.；Lubrication
Agent, such as talcum powder, silica, cornstarch, stearate, boric acid, atoleine, polyethylene glycol etc..It can also be by piece
Coating tablet, such as sugar coated tablet, thin membrane coated tablet, enteric coated tablets or double-layer tablets and multilayer tablet is further made in agent.In order to incite somebody to action
Pill is made in unit dosage forms for administration, and various carriers well known in the art can be widely used.Example about carrier is, such as dilute
Agent and absorbent are released, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, height
Ridge soil, talcum powder etc.；Adhesive such as Arabic gum, bassora gum, gelatin, ethyl alcohol, honey, liquid sugar, rice paste or batter etc.；Disintegration
Agent, such as agar powder, dry starch, alginate, dodecyl sodium sulfate, methylcellulose, ethyl cellulose.In order to will be single
Suppository is made in position form of administration, and various carriers well known in the art can be widely used.Example about carrier is, such as poly- second
Glycol, lecithin, cocoa butter, higher alcohol, the ester of higher alcohol, gelatin, semi-synthetic glyceride etc..In order to by unit dosage forms for administration system
All diluents commonly used in the art can be used such as solution, emulsion, freeze drying powder injection and suspension at injection preparation,
For example, water, ethyl alcohol, polyethylene glycol, 1,3- propylene glycol, the isooctadecanol of ethoxylation, polyoxygenated isooctadecanol, polyoxy second
Alkene Span etc..In addition, suitable chlorination can be added into injection preparation in order to prepare isotonic injection
Sodium, glucose or glycerol, further, it is also possible to add conventional cosolvent, buffer, pH adjusting agent etc..In addition, if desired,
Colorant, preservative, fragrance, corrigent, sweetener or other materials can be added into pharmaceutical preparation.It can using above-mentioned dosage form
With administrated by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary administration etc.；Cavity/canal drug administration, such as per rectum and yin
Road；Respiratory tract administration, such as via intranasal application；Mucosa delivery.Above-mentioned administration route is preferably drug administration by injection.
It is demonstrated experimentally that the methanolic extract of aspergillus (Aspergillus sp.) CPCC400735 fermentation culture medium is in concentration
It is 99.9% to the inhibiting rate of HIV-1 when for 4 mg/mL.Chemical combination shown in the formula 1 to formula 4 separated from above-mentioned methanolic extract
Object concentration be 100 μM when be respectively 99.84%, 99.99%, 98.60% and 96.82% to the inhibiting rate of HIV-1, formula 1 to
The IC of 4 compound represented of formula50Value is respectively 2.4 μM, 4.5 μM, 22.1 μM and 32.6 μM.Aspergillus of the invention
The methanolic extract and formula 1 of (Aspergillus sp.) CPCC400735 fermentation culture medium are to 4 compound represented of formula to HIV-
1 has stronger inhibitory activity, and toxicity is smaller.
Preservation explanation
Strain name: aspergillus (Aspergillus sp.)
Strain number: CPCC400735
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism abbreviation: CGMCC
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date: on May 5th, 2016
Collection is registered on the books number: CGMCC No.12376
Detailed description of the invention
Fig. 1 is the structure of 4 compound represented of formula.
Fig. 2 is the structure of 2 compound represented of formula.
Fig. 3 is the structure of 1 compound represented of formula.
Fig. 4 is the structure of 3 compound represented of formula.
Fig. 5 is 4 compound represented of formula1H-NMR spectrum.
Fig. 6 is 4 compound represented of formula13C-NMR spectrum.
Fig. 7 is the hsqc spectrum of 4 compound represented of formula.
Fig. 8 is that the HMBC of 4 compound represented of formula is composed.
Fig. 9 is 4 compound represented of formula1H-1H COSY spectrum.
Figure 10 is that the NOESY of 4 compound represented of formula is composed.
Figure 11 is the Rh of 4 compound represented of formula2(OCOCF3)4Induce CD spectrum.
Figure 12 is the Mo (Ac) of 4 compound represented of formula4Induce CD spectrum.
Figure 13 is 2 compound represented of formula1H-NMR spectrum.
Figure 14 is 2 compound represented of formula13C-NMR spectrum.
Figure 15 is the hsqc spectrum of 2 compound represented of formula.
Figure 16 is that the HMBC of 2 compound represented of formula is composed.
Figure 17 is 2 compound represented of formula1H-1H COSY spectrum.
Figure 18 is that the NOESY of 2 compound represented of formula is composed.
Figure 19 is 1 compound represented of formula1H-NMR spectrum.
Figure 20 is 1 compound represented of formula13C-NMR spectrum
Figure 21 is the hsqc spectrum of 1 compound represented of formula.
Figure 22 is that the HMBC of 1 compound represented of formula is composed.
Figure 23 is 1 compound represented of formula1H-1H COSY spectrum.
Figure 24 is that the NOESY of 1 compound represented of formula is composed.
Figure 25 is the relative configuration of the hexa-atomic ether ring in C-23 to the C-33 structure fragment of 1 compound represented of formula.
Figure 26 is 3 compound represented of formula1H-NMR spectrum.
Figure 27 is 3 compound represented of formula13C-NMR spectrum.
Figure 28 is the hsqc spectrum of 3 compound represented of formula.
Figure 29 is that the HMBC of 3 compound represented of formula is composed.
Figure 30 is 3 compound represented of formula1H-1H COSY spectrum.
Figure 31 is that the NOESY of 3 compound represented of formula is composed.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified
Conventional method.The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Potato dextrose agar (PDA) culture medium as used in the following examples: peeling potatoes, 200g are cut into small
Block adds boiling to boil 30min, and 4 layers of filtered through gauze add 20g glucose, agar 17g, and distilled water constant volume to 1000mL boils mixing,
It sterilizes 20 minutes under the conditions of 121 DEG C, obtains PDA culture medium.
PNL4-3Luc (R-E-) and pHIT/G (Zhang et al.Retrovirology (2016) in following embodiments
13:13) public can obtain the biomaterial from NIH or applicant, the biomaterial only attach most importance to duplicate invention related experiment institute
With not can be used as other purposes and use.
The separation and identification of embodiment 1, aspergillus (Aspergillus sp.) CPCC400735
1.1 strain isolation
Bacterial strain CPCC400735 is isolated from the stem of kadsura longepedunculata (Kadsura longipedunculata).Acquisition south five
Taste plant is packed into valve bag, takes back laboratory treatment.1g kadsura longepedunculata stem is weighed, rinses 30s, sterile water with 75% alcohol
Cleaning 3 times, is shredded stem with sterile scissors, puts into sterilized mortar and quartz sand is added to be ground.Lapping liquid is added
In centrifuge tube equipped with 9mL sterile water, sample concentration 10-1, after shaking up, successively it is made into 10-2、10-3、10-4、10-5Gradient is dilute
Release liquid.Different 200 μ L of gradient dilution liquid are drawn respectively, are respectively coated on PDA plate, are inverted in 25 DEG C of incubators after drying
Culture 3-5 days, the fungi being separated to is transferred on the inclined-plane PDA and is cultivated, and after length is good, is placed in refrigerator and is saved.The number is taken to be
The bacterial strain of CPCC400735 carries out following identifications.
The identification of 1.2 bacterial strains
1.2.1 strain morphology is observed
Bacterial strain CPCC400735 is chosen with transfer needle to PDA culture medium plate center, is cultivated in 28 DEG C of incubators.Bacterial strain
CPCC400735 well-grown in PDA culture medium, bacterium colony is in light yellow at culture 5-7 days, quality velvet shape to cotton-shaped, compared with
Thickness has or does not have radial rill；Bacterium colony reverse side yellowish-brown, conidium is more or few, is just fawn, it is brown to be bordering on light powder
Color deepens after old, is bordering on pink cinnamon, and it is in loose cylindricality, the top capsule of conidial head that conidial head is just radiation shape afterwards
Spherical, top capsule is covered by the fusion overlapping layer that stigma fused layer and bottle obstruct of coming into being, and bottle stalk generates conidia chain, conidium
Spherical or subsphaeroidal, wall is smooth.In view of above-mentioned morphological feature, Preliminary Identification bacterial strain CPCC400735 is aspergillus fungi.
1.2.2 molecular biology identification
The genomic DNA of bacterial strain CPCC400735, PCR amplification 18S rRNA gene are extracted, and is sequenced.By gained sequence
(sequence 1 in sequence table) is compared with sequence in GenBank database, the results showed that bacterial strain CPCC400735 and aspergillus
The similitude of (Aspergillus sp.) is 99%, therefore, in conjunction with morphological feature before, determines that bacterial strain CPCC400735 is
Aspergillus fungi.
Aspergillus (Aspergillus sp.) CPCC400735 has been preserved in Chinese microorganism strain guarantor on May 5th, 2016
Administration committee's common micro-organisms center is hidden, deposit number is CGMCC No.12376.Hereinafter referred aspergillus (Aspergillus
sp.) CPCC400735。
Embodiment 2 prepares HIV-1 inhibitor using aspergillus (Aspergillus sp.) CPCC400735
The present embodiment is prepared for 5 kinds of HIV-1 inhibitor using aspergillus (Aspergillus sp.) CPCC400735, wherein
A kind of HIV-1 inhibitor be that the methanolic extract of aspergillus (Aspergillus sp.) CPCC400735 fermentation culture medium (uses first
Alcohol extracts the obtained substance for being dissolved in methanol from the culture of aspergillus (Aspergillus sp.) CPCC400735), in addition 4
Kind HIV-1 inhibitor is general formula compound shown in formula II
Formula II, structural formula is specifically as shown in formula 1 to formula 4 (table 1).
The substituent group of general formula compound shown in table 1, formula II
Above-mentioned 5 kinds of HIV-1 inhibitor specific the preparation method is as follows:
1, the culture of aspergillus (Aspergillus sp.) CPCC400735 is prepared
Aspergillus (Aspergillus sp.) CPCC400735 is cultivated 5 days for 28 DEG C on the inclined-plane PDA, and then picking mycelia block connects
Kind in equipped with 100ml seed culture medium (consisting of: glucose 2%, sucrose 1%, soybean powder 0.2%, peptone 1%, phosphoric acid
Hydrogen dipotassium 0.03%, polyethylene glycol 0.25%, sodium nitrate 0.3%, ammonium sulfate 0.3%, surplus are water, pH 6.0.121 DEG C of sterilizings
20 minutes, obtain seed culture medium) 500ml triangular flask in, 28-30 DEG C shake culture 2 days be used as seed liquor.Then, it will plant
Sub- liquid 10ml access equipped with rice medium 500ml triangular flask (500ml triangular flask is equipped with 80g rice, be added 100ml go from
Sub- water impregnates, and 121 DEG C of sterilizings obtain rice medium in 20 minutes) in, 28 DEG C are cultivated 40 days, and aspergillus (Aspergillus is obtained
Sp.) CPCC400735 solid fermentation culture.
2, the methanolic extract of aspergillus (Aspergillus sp.) CPCC400735 fermentation culture medium is prepared
Take aspergillus (Aspergillus sp.) the CPCC400735 solid fermentation culture glass bar of 2000g step 1 will
It is blended, and 4L methanol is added, and is extracted 3 times, each 30min at 28 DEG C, merges methanol extract liquid, with Rotary Evaporators (temperature: 40
DEG C, revolution: 70 turns) rotary evaporation remove methanol extract liquid in methanol, obtained substance is aspergillus (Aspergillus sp.)
The methanolic extract of CPCC400735 fermentation culture medium.
3, preparation formula 1 is to 5 compound represented of formula
3.1, the methanolic extract of the aspergillus of step 2 (Aspergillus sp.) CPCC400735 fermentation culture medium is used
Then water dispersion is extracted with ethyl acetate, collect ethyl acetate phase, with Rotary Evaporators (temperature: 40 DEG C, revolution: 70 turns) rotation
Turn the ethyl acetate in evaporation removing ethyl acetate phase, obtains medicinal extract.Silica gel column chromatography is carried out after taking 50g medicinal extract to be dissolved with methanol
Separation.Silica gel used in silica gel column chromatography is silica gel H, and the specification of silicagel column used is 6.5 × 20cm, and column volume is
663mL.Elution program used in silica gel column chromatography is first to elute 2 column volumes with chloroform；Carry out again 13 column volumes as
Lower linear gradient elution: the mixed liquor that mobile phase used is made of chloroform and methanol, chlorine in linear gradient elution mobile phase
Imitative and methanol volume ratio is linearly down to 7:1 by 20:1；Last again with methanol elutes two column volumes.Since elution program not
Interruption collects the liquid eluted, and every pipe collects 200ml (200ml/ fraction), continuously collects 56 fractions, is denoted as: Fr.1,
Fr.2, Fr.3, Fr.4 ..., Fr.56, TLC detection guidance merges identical fraction, finally obtains 12 merging components: Fr.1-
3, Fr.4-12 (this 9 fractions merge to obtain by Fr.4 to Fr.12, are the liquid that the 0.9th to the 3.6th column volume elutes),
(by Fr.16 and Fr.17, this 2 fractions merge to obtain, and are the 4.5th to the 5.1st cylinders by Fr.13-14, Fr.15, Fr.16-17
The liquid that product elutes), Fr.18-21, Fr.22-25, Fr.26-31, Fr.32-34, Fr.35-42, Fr.43-54, Fr.55-
56。
3.2, the Fr.4-12 for obtaining step 3.1 carries out pressurized silica gel column chromatography.Silicon used in pressurized silica gel column chromatography
Glue is silica gel H, and the specification of silicagel column used is 4 × 13cm (diameter 4cm, a height of 13cm), column volume 163mL.Pressurization
Elution program used in silica gel column chromatography be carry out 6 column volumes following linear gradient elution: mobile phase used be by
The mixed liquor of petroleum ether and acetone composition, the volume ratio of the petroleum ether and acetone in mobile phase in linear gradient elution is by 8:1
Linearly it is down to 5:1.The liquid eluted is uninterruptedly collected since elution program, every pipe collects 20ml (20ml/ fraction), continuously
Collect 43 fractions, be denoted as: Tube.1, Tube.2, Tube.3 ..., Tube.43.By Tube.27 to Tube.37 this 11
Fraction mixing, obtains the component (being the liquid that the 3.2nd to the 4.5th column volume elutes) of entitled Tubes 27-37.
Tubes 27-37 is subjected to preparative high performance liquid chromatography separation (RP-C18 liquid phase preparative separation).The preparative
Filler used in high performance liquid chromatography separation is octadecylsilane chemically bonded silica filler, and the partial size of filler is 5 μm, the preparative
Chromatography column diameter used in high performance liquid chromatography separation is 8mm, a height of 250mm.Mobile phase is the mixed liquor of first alcohol and water composition
Body, the volume ratio of first alcohol and water is 4: 1 in mobile phase；Flow velocity is 4.0mL/min.6 preparation components are obtained, title is respectively
tubes 27-37-P1(tR(retention time) is 6.0min), tubes 27-37-P2 (tRFor 23.9min), tubes 27-37-
P3(tRFor 27.0min), tubes 27-37-P4 (tRFor 31.2min), tubes 27-37-P5 (tRFor 34-42 min),
tubes 27-37-P6(tRFor 44.6min).
Tubes 27-37-P2 is subjected to preparative high performance liquid chromatography separation (RP-C18 liquid phase preparative separation).The preparation
Filler used in type high performance liquid chromatography separation is octadecylsilane chemically bonded silica filler, and the partial size of filler is 5 μm, the preparation
Chromatography column diameter used in type high performance liquid chromatography separation is 8mm, a height of 250mm.Mobile phase is the mixing of first alcohol and water composition
Liquid, the volume ratio of first alcohol and water is 4: 1 in mobile phase；Flow velocity is 4.5mL/min.Collect tR(retention time) is 23.9 min
Eluting peak, remove first alcohol and water with Rotary Evaporators (temperature: 40 DEG C, revolution: 70 turns) rotary evaporation, obtain the formula of 5.0mg
1 compound represented.
Tubes 27-37-P6 is subjected to preparative high performance liquid chromatography separation (RP-C18 liquid phase preparative separation).The preparation
Filler used in type high performance liquid chromatography separation is octadecylsilane chemically bonded silica filler, and the partial size of filler is 5 μm, the preparation
Chromatography column diameter used in type high performance liquid chromatography separation is 8mm, a height of 250mm.Mobile phase is the mixing of first alcohol and water composition
Liquid, the volume ratio of first alcohol and water is 4: 1 in mobile phase；Flow velocity is 5.0mL/min.Collect tR(retention time) is 37.7 min
Eluting peak, remove first alcohol and water with Rotary Evaporators (temperature: 40 DEG C, revolution: 70 turns) rotary evaporation, obtain 14.7 mg's
2 compound represented of formula.
3.3, the Fr.16-17 for obtaining step 3.1 carries out pressurized silica gel column chromatography.Used in pressurized silica gel column chromatography
Silica gel is silica gel H, and the specification of silicagel column used is 4 × 12cm (diameter 4cm, a height of 12cm), column volume 151mL.Add
Elution program used in pressure silica gel column chromatography is mixing with the volume ratio 10:1 for the chloroform and methanol being made of chloroform and methanol
It closes liquid to be eluted as mobile phase, the liquid eluted is uninterruptedly collected since elution program, every pipe collects 10ml
(10ml/ fraction), continuously collect 18 fractions, be denoted as: Tube.1, Tube.2, Tube.3 ..., Tube.18.TLC guidance
Merging obtains four merging components: (by Tube.1 to Tube.3, this 3 fractions mix tubes 1-3, obtain entitled tubes
The component of 1-3 is the liquid that the 0th to 0.2 column volume elutes), tubes 4-13 is (by Tube.4 to Tube.13 this 10
The mixing of fraction fraction, obtains the component of entitled tubes 4-13, is that the 0.2nd column volume to the 0.9th column volume elutes
Liquid), tubes 14-18.Tubes 4-13 is carried out to open ODS column chromatography for separation again.Opening ODS column chromatography for separation institute
Filler is octadecylsilane chemically bonded silica filler, and the partial size of filler is 50 μm, used in opening ODS column chromatography for separation
Chromatography column diameter is 4cm, and a height of 16cm, chromatography column volume is 201mL.Elution program used in opening ODS column chromatography for separation
It is the elution of two steps, first step elution uses mixing liquid that the volume ratio for the first alcohol and water being made of first alcohol and water is 11:9 as flowing
It is dynamic mutually to elute 4 column volumes, complete first step elution；Second step elution: after completing first step elution, use methanol as mobile phase
It is eluted, 1-100ml this 100ml liquid for collecting that the second step elutes since second step elution (uses methanol
The liquid that the 0th to the 0.5th column volume eluted elutes), it is named as Tubes 4-13-ODS-9.By Tubes 4-
13-ODS-9 and tubes 1-3, which merges, carries out preparative high performance liquid chromatography separation (RP-C18 liquid phase preparative separation).The preparation
Filler used in type high performance liquid chromatography separation is octadecylsilane chemically bonded silica filler, and the partial size of filler is 5 μm, the system
Chromatography column diameter used in standby type high performance liquid chromatography separation is 8mm, a height of 250mm.Mobile phase is the mixed of first alcohol and water composition
Liquid is closed, the volume ratio of first alcohol and water is 3: 1 in mobile phase；Flow velocity is 4.0mL/min.Obtain two preparation component P2 (tRFor
18.2min) and P3 (tRFor 22.2min).Then, it is efficient to carry out preparative under the identical liquid phase preparation condition respectively by P2 and P3
Liquid chromatogram separation is further purified.
The preparative high performance liquid chromatography of P2 separates and collects tR(retention time) is the eluting peak of 18.2min, is steamed with rotation
Hair instrument (temperature: 40 DEG C, revolution: 70 turns) rotary evaporation removing first alcohol and water, obtain 4 compound represented of formula of 52.0mg.
The preparative high performance liquid chromatography of P3 separates and collects tR(retention time) is the eluting peak of 22.2min, is steamed with rotation
Hair instrument (temperature: 40 DEG C, revolution: 70 turns) rotary evaporation removing first alcohol and water, obtain 3 compound represented of formula of 52.0mg.
3.4 formulas 1 to 4 compound represented of formula physicochemical property
Formula 1 to 4 compound represented of formula is brown solid, is soluble in methanol, ethyl alcohol, in DMSO equal solvent, indissoluble
Yu Shui.Formula 1 is to 5 compound represented of formula in silica gel thin-layer chromatography chloroform-methanol-water (volume ratio 70:15:2) dicyandiamide solution
Expansion Rf value is 0.5-0.7, obvious in 254nm and 365nm fluorescence developing, and vanillin-sulfuric acid, which develops the color, shows brownish red.
3.5 formulas 1 to 4 compound represented of formula structure elucidation
HRESIMS (anion) quasi-molecular ions of 4 compound represented of formula: m/z 623.2900 [M-H]-Prompt its molecular formula
For C35H44O10.Comprehensive analysis1H NMR (Fig. 5),13C NMR (Fig. 6) and hsqc spectrum (Fig. 7), thus it is speculated that two should be contained in compound
A ketone carbonyl, an ester carbonyl group, 16 olefinic carbons (wherein 12 are quaternary carbon, and 4 are tertiary carbon, and there are three carbon to connect oxygen atom) and
The carbon (wherein three connection oxygen atoms) of 16 sp2 hydridization.The visible features signal in hydrogen spectrum: δ 14.29 (1H, s), 13.07
(1H, s), 11.97 (1H, brs), 10.69 (1H, brs), 6.79 (1H, s), 6.52 (1H, t), 4.90 (1H, t), 4.79 (1H,
T), 2.80 (3H, s), 2.52 (1H, d), 2.11 (3H, s), 1.39 (3H, s), 1.24 (3H, s), 0.98 (3H, s), 0.92
(3H, s).From features above hydrogen signal, it can be found that following coherent signal in HMBC spectrum (Fig. 8): 14.29 (13- of δ
OH) related with 165.8 (C-13), 102.0 (C-3), 200.6 (C-2, weak)；δ 2.11 (H-14) and 107.2 (C-12), 162.9
(C-11), 165.8 (C-13) are related；δ 13.07 (C-7-OH) and 163.3 (C-7), 118.0 (C-8), 106.1 (C-5),
202.8 (C-2, weak) are related；δ 6.79 (8-H) and 106.1 (C-5), 112.8 (C-10), 26.1 (C-15), 163.3 (C-7),
202.8 (C-6, weak) are related；δ 2.80 (H-15) and 112.8 (C-7), 118.0 (C-8), 149.0 (C-9) are related；δ2.52(H-
16) with 81.2 (C-1), 200.6 (C-2), 202.8 (C-6), 115.4 (C-17), 139.6 (C-18) are related；δ4.79 (H-
17) with 15.6 (C-35), 39.2 (C-19), 41.2 (C-16) are related；δ 1.24 (H-35) and 39.2 (C-19), 115.4 (C-
17), 139.6 (C-18) are related；δ 4.90 (H-17) and 15.3 (C-34), 25.9 (C-20), 38.0 (C-19) are related；δ1.39
(H-34) with 38.0 (C-23), 124.2 (C-21), 133.7 (C22) are related；δ 6.51 (H-25) and 23.9 (C-27), 24.4
(C-24), 38.0 (C-23), 168.6 (C-33) are related；δ 3.00 (H-29) and 23.9 (C-27), 71.5 (C-30), 26.2 (C-
31), 24.6 (C-32) are related；δ 0.98 (H-31) and 24.6 (C-32), 71.5 (C-30), 77.3 (C-29) are related；δ0.92
(H-32) with 26.2 (C-31), 71.5 (C-30), 77.3 (C-29) are related.?1H-1It can be found in H COSY spectrum (Fig. 9) following
Coherent signal: δ 2.52 (H-16) is related to 4.79 (H-17)；δ 1.60 (H-19) is related to 1.51 (H-20)；δ1.51(H-20)
It is related to 4.90 (H-21)；δ 1.93 (H-23) is related to 2.18 (H-24)；δ 2.18 (H-24) is related to 6.51 (H-25)；δ
3.00 (H-24) are related to 1.10 (H-28b)；δ 1.52H (H-28a) and 2.38 (H-27a), 2.09 (H-27b) are related；δ
1.10H (H-28b) and 2.38 (H-27a), 2.09 (H-27b) are related.According to information above, chemical combination shown in formula 4 can be determined
The planar structure of object, hydrocarbon attribution data are shown in Table 2.NOESY composes coherent signal in (Figure 10): δ 4.79 (H-17) and 1.60 (H-
19) related；δ 4.90 (H-21) is related to 1.93 (H-23)；δ 2.18 (H-24) is related to 2.09 (H-27b), determines in structure
Three double bonds are trans- (E) configuration.The configuration of two chiral carbons in the position C-1 and C-29 is utilized respectively Rh in structure2(OCOCF3)4
Induce CD spectrum (Figure 11) and Mo (Ac)4Induce CD spectrum (Figure 12) identification.In the Rh of 4 compound represented of formula2(OCOCF3)4Induction
In CD spectrum, the Cotton effect that is negative is shown at 347nm, according to document (Lorenzo DB, Gennaro P, Carmela P, et
al.Determination of Absolute Configuration of Acyclic 1,2-Diols with Mo2
(OAc)4.1.Snatzke ' s Method Revisited [J] .J.Org.Chem. 2001,66:4819-4825) report structure
Type decision rule, C-1 absolute configurations should be R.In the Mo (Ac) of 4 compound represented of formula4It induces in CD spectrum,
The Cotton effect that is negative is shown at 317nm, according to document[32](Jadwiga Frelek, Wojciech J.Szczepek.[Rh2
(OCOCF3)4]as an auxiliary chromophore in chiroptical studies on steroidal
Alcohols [J] .Tetrahedron:Asymmetry, 1999,10:1507-1520) the configuration decision rule of report, C-29
Absolute configuration should be R.4 compound represented of final formula is accredited as the structure as shown in Fig. 1.
HRESIMS (anion) quasi-molecular ions of 2 compound represented of formula: m/z 575.7247 [M-H]-Prompt its molecular formula
For C35H44O7.2 compound represented of formula1H and13C H NMR spectroscopy difference is as shown in Figs. 13 and 14.2 compound represented of formula and formula
The nuclear magnetic data of C-1~C-22 structure fragment of 1 compound represented is almost the same, and the nuclear-magnetism of C-23~C-33 structure fragment
Data have larger difference.(Figure 16): δ 5.02 (H-25) and 38.0 (C-23), 25.3 (C-24), 34.5 (C-27) in HMBC spectrum,
58.0 (C-33) are related；δ 3.86 (H-33) and 125.2 (C-25), 139.0 (C-26), 34.5 (C-27) are related；δ5.02(H-
29) and 34.5 (C-27), 17.5 (C-32) are related；δ 1.58 (H-31) and 124.4 (C-29), 134.0 (C-30) are related, δ
1.50 (H-32) and 124.4 (C-29), 134.0 (C-30) are related.?1H-1H COSY 5.02 (H- of (such as Figure 17): δ into spectrum
25) related to 2.00 (H-24)；δ 2.00 (H-24) is related to 1.82 (H-23)；δ 5.02 (H-29) is related to 1.98 (H-28)；
δ 1.98 (H-28) is related to 1.96 (H-27), can determine the structure of C-23 to C-33 segment according to information above.Comprehensive analysis1H-1H COSY, HSQC (Figure 15) and HMBC spectrum, confirm the planar structure of 2 compound represented of formula, and return to hydrocarbon signal
Belong to (table 2).NOESY composes coherent signal in (Figure 18): δ 4.77 (H-17) is related to 1.59 (H-19)；δ 4.84 (H-21) and 1.82
(H-23) related；δ 2.00 (H-24) and 3.86 (H-33), 17 (18) double bonds of prompt are trans- (E) configuration, and 21 (22) double bonds are
Trans- (E) configuration, 25 (26) double bonds are cis- (Z) configuration.Speculated according to biosynthesis pathway, 2 compound represented of formula
The absolute configuration of C-1 should be identical as 4 compound represented of formula, is configured as R.2 compound represented of formula1H NMR (figure
19),13C NMR (Figure 22), hydrocarbon attribution data are shown in Table 2.Therefore, 2 compound represented of formula be accredited as it is as shown in Figure 2
Structure.
HRESIMS shown in formula 1 (anion) quasi-molecular ions: m/z 653.7920 [M-H]-Its molecular formula is prompted to be
C37H50O10.1 compound represented of formula1H and13C H NMR spectroscopy as shown in Figure 19 and 20 and table 2, is changed shown in contrast 1 respectively
Close object and 2 compound represented of formula1H and13The discovery of C NMR data, the two C-1 is identical to C-22 structure fragment, and C-23 is extremely
C-33 structure fragment differs greatly.(Figure 22): δ 0.95 (H-32) and 70.4 (C-30) in HMBC spectrum, 83.6 (C-29) phases
It closes；δ 1.01 (H-31) and 70.4 (C-30), 83.6 (C-29) are related；δ 3.24 (H-36) and δ 3.23 (H-37) respectively with
106.0 (C-33 is related)；δ 4.12 (H-33) and 77.2 (C-25), 43.0 (C-26), 23.0 (C-27) are related；δ2.76(H-
29) related to 23.0 (C-27)；δ 3.02 (H-25) is related to 34.6 (C-23).?1H-1(Figure 23): δ 4.12 in H COSY spectrum
(H-33) related to 1.37 (H-26), δ 1.37 (H-26) respectively with 3.02 (H-25) and 1.75 (H-27a), 1.22 (H-27b)
It is related；3.02 (H-25) are related to 1.25 (H-24b)；1.70 (H-24a) are related to 1.98 (H-23a).Not according to compound
Saturation degree and molecular formula speculate, C-23 into C-33 structure fragment there are a cyclic structure, according to HMBC compose in 2.76 (H- of δ
29) related to 77.2 (C-27) and δ 3.02 (H-29) and 83.6 (C-29) relevant informations, determine in the structure fragment be C-25 and
C-29 connect to form ether ring by single oxygen key.The structure of C-23 to C-33 segment can be determined according to information above.Comprehensive point
Analysis1H-1H COSY, HSQC (Figure 21) and HMBC spectrum determine the planar structure of compound shown in formula 1, and carry out to hydrocarbon signal
Belong to (table 2).NOESY composes coherent signal in (Figure 24): δ 4.77 (H-17) is related to 1.60 (H-19)；δ 4.86 (H-21) with
1.88 (H-23b) are related, prompt 17 (18) double bonds and 21 (22) double bonds are trans- (E) configuration.Speculated according to biosynthesis pathway,
The absolute configuration of the C-1 of 1 compound represented of formula should be identical as 4 compound represented of formula, is configured as R.C-25 in structure,
C-26 and C-29 tri- are chiral carbon, during absolute structure is calculated by ECD.According to NOESY compose in coherent signal, really
Determine the relative configuration (Figure 25) of hexa-atomic ether ring of the C-23 into C-33 structure fragment.Finally, 1 compound represented of formula is identified
For structure as shown in Figure 3.
HRESIMS (anion) quasi-molecular ions of 3 compound represented of formula: m/z 609.3094 [M-H]-Prompt its molecular formula
For C35H46O9.3 compound represented of formula1H and13C H NMR spectroscopy difference is as shown in figures 26 and 27.With 4 compound represented phase of formula
An oxygen atom is lacked compared with it than, 3 compound represented of formula, more 2 hydrogen atoms.Both comparisons carbon modal data, shown in formula 3
Compound ester carbonyl group carbon atom signal fewer than 4 compound represented of formula, more saturated carbon atoms of company's oxygen atom
Signal δ 58.2, and other carbon modal datas are almost the same, the position C-33 of 3 compound represented of prompting type may be a methylol.
Comprehensive analysis1H-1H COSY (Figure 30), HSQC (Figure 28) and HMBC compose (Figure 29), identify the planar junction of compound shown in formula 3
Structure, and (table 2) is belonged to hydrocarbon signal.Coherent signal in NOESY (Figure 31) spectrum: δ 4.78 (H-17) and 1.59 (H-19)
It is related；δ 4.85 (H-21) is related to 1.83 (H-23)；δ 2.00 (H-24) and 3.85 (H-33), it is anti-for prompting 17 (18) double bonds
Formula (E) configuration, 21 (22) double bonds are trans- (E) configuration, and 25 (26) double bonds are cis- (Z) configuration.It is pushed away according to biosynthesis pathway
It surveys, the absolute configuration of the C-1 and C-29 of 1 compound represented of formula should be identical as 4 compound represented of formula, and configuration is R.Cause
This, 3 compound represented of formula is accredited as structure as shown in Figure 4.
Table 2, formula 1 to 4 compound represented of formula nuclear magnetic data (1H NMR 600MHz；13C NMR 150MHz；DMSO-
d6)
4, to the inhibitory activity of HIV-1
(1) cell culture and transfection: 293T cell culture is in the DMEM culture medium containing 10%FBS.The every hole inoculation of 6 orifice plates
Cell number is 4 × 105A, culture transfects afterwards for 24 hours, and transfection reagent Lipofectamine 2000 is turned according to operation instruction
Dye；SupT1 cell (SUP-T1 cell, human T lymphoma cell) is incubated in 1640 culture medium of RPMI containing 10%FBS, is placed in
37 DEG C of 5%CO2Stationary culture in incubator.
(2) preparation of HIV-1 pseudovirus: being 1.5 × 10 by cell concentration5The amount of a/ml is by 293T cell before transfection
It is inoculated in 6 orifice plates within one day, cultivates in 2mL culture medium and transfected for 24 hours.Plasmid dosage are as follows: every hole transfection in six orifice plates
PNL4-3Luc (R-E-) 300ng and pHIT/G plasmid 210ng, transfection reagent Lipofectamine2000, by transfection reagent
Illustrate to be transfected.Supernatant is collected after 48 hours, crosses 0.45 μm of filter membrane, is collected virus liquid and is obtained the coated HIV-1 of VSV-G
Pseudovirus.
(3) measurement of viral infection: the every hole inoculation 5 × 10 of 96 orifice plates5The 200 μ L of SupT1 cell of a/ml.Take 10 μ l
Gained virus liquid infects SupT1 cell.After cultivating 48h, 50 μ l cell suspensions are taken out, isometric 2 × Luciferase is added
Cell Culture Lysis lytic cell.Use the fluorescence of kit Luciferiase Assay System measurement lysate
Plain enzyme (Luciferase) activity.The amount of infectious virus is obtained according to the active size of reporter gene Luciferase.
(4) Anti-HIV-1 Active measures: SupT1 cell concentration is 5 × 105A/ml, directly 96 orifice plates of paving, every 200 μ L of hole,
The HIV-1 pseudovirus of above-mentioned preparation is added simultaneously (the viral volume ratio with cell is 1:20).Sample is added, and (sample is dissolved in
In DMSO), each sample sets 3 repetitions, while blank control and negative control (DMSO) is arranged, 37 DEG C of incubation 48h.Take 50 μ
2 × Luciferase Cell Culture Lysis lysate, 50 μ L is added in L cell suspension, after 37 DEG C of incubation half an hours,
Wherein 6 μ L cell pyrolysis liquids addition, 40 μ L luciferase substrates are taken, 960 microplate reader of Centro XS3LB is utilized to measure fluorescein
Enzyme values (RLUs) calculate sample to the inhibiting rate of HIV.
HIV inhibiting rate calculation formula is as follows:
Wherein, sample is the methanol of aspergillus (Aspergillus sp.) CPCC400735 fermentation culture medium prepared by step 2
Formula 1 prepared by extract and step 3 is any one of to 5 compound represented of formula this 6 kinds of HIV-1 inhibitor.Pass through sample pair
The IC of formula 1 to 5 compound represented of formula is calculated in the inhibiting rate of HIV-150(refer to the drug for inhibiting HIV-1 duplication to reduce 50%
Concentration).
(5) drug toxicity detection detects drug toxicity: SupT1 using Cell Counting Kit-8 (CCK-8 kit)
Cell concentration is 5 × 105A/ml, directly 96 orifice plates of paving, every 100 μ L of hole.It is added sample (sample is dissolved in DMSO), each
Sample sets 3 repetitions, while blank control and negative control (DMSO) is arranged, 37 DEG C of incubation 48h.96 orifice plates are taken out, every hole adds
Enter 10 μ L CCK-8,37 DEG C are continued after being incubated for 1 hour, are detected each hole using 2300 multi-function microplate reader of Enspire and are existed
Absorbance value (OD) at 450nm wavelength, calculates the cell survival rate of sample well.Cell survival rate calculation formula:
Wherein, sample is the formula 1 for preparing of step 3 any one of to 4 compound represented of formula this 4 kinds of HIV-1 inhibitor.
By the cell survival rate of sample well, the CC of formula 1 to 4 compound represented of formula is calculated50(referring to causes 50% cell death
Drug concentration).
The result shows that the methanol of aspergillus (Aspergillus sp.) CPCC400735 fermentation culture medium prepared by step 2 mentions
Taking object is 99.9% to the inhibiting rate of HIV-1 when concentration is 4mg/mL.Formula 1 is 100 μM in concentration to 4 compound represented of formula
When be respectively 99.84%, 99.99%, 98.60% and 96.82% to the inhibiting rate of HIV-1, formula 1 to 4 compound represented of formula
IC50Respectively 2.4 μM, 4.5 μM, 22.1 μM and 32.6 μM, the CC of formula 1 to 3 compound represented of formula50100 μM are all larger than,
The CC of 4 compound represented of formula50For 78.6 μM (tables 3).Illustrate aspergillus (Aspergillus sp.) prepared by step 2
The methanolic extract and formula 1 of CPCC400735 fermentation culture medium live to HIV-1 with stronger inhibition to 4 compound represented of formula
Property, and toxicity is smaller.Wherein, 1 compound represented of formula is most strong to the inhibitory activity of HIV-1,2 compound represented pair of formula
The inhibitory activity of HIV-1 is weaker than 1 compound represented of formula but is better than 4 compound represented of formula 3 and formula, 3 compound represented of formula
4 compound represented of formula is better than to the inhibitory activity of HIV-1.
The Anti-HIV-1 Active of table 3, formula 1 to 4 compound represented of formula
| Compound number | Average inhibition % (100 μM, n=3) | IC50(μM) | CC50(μM) |
| Formula 1 | 99.84±0.12 | 2.4 | >100 |
| Formula 2 | 99.99±0.01 | 4.5 | >100 |
| Formula 3 | 98.60±0.53 | 22.1 | >100 |
| Formula 4 | 96.82±0.11 | 32.6 | 78.6 |
Claims (4)
1. aspergillus (Aspergillus sp.), bacterial strain number is CPCC400735, is entrusted in Chinese microorganism strain preservation management
The number of registering on the books of member's meeting common micro-organisms center is CGMCC No.12376.
2. the culture of aspergillus described in claim 1 (Aspergillus sp.) is by aspergillus described in claim 1
The substance in culture vessel that (Aspergillus sp.) is cultivated in microbiological culture media.
3. culture according to claim 2, it is characterised in that: the microbiological culture media is aspergillus culture medium.
4. the method for II compound represented of preparation formula, including extracted from culture described in claim 2 or 3 with methanol
To the substance for being dissolved in methanol, then from the substance extract obtain II compound represented of formula；
The R is
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| CN108822119B (en) * | 2018-06-25 | 2020-08-28 | 中国医学科学院医药生物技术研究所 | Erythronolide ketone compound with autophagy activation activity, preparation method and pharmaceutical application thereof |
| CN110251656B (en) * | 2019-07-04 | 2022-06-10 | 自然资源部第三海洋研究所 | Preparation method of polypeptide compound, application of polypeptide compound and HIV latent activation medicine |
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| CN113651683A (en) * | 2021-08-06 | 2021-11-16 | 中国医学科学院医药生物技术研究所 | Perylene ketone compound and application thereof |
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Non-Patent Citations (2)
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| Metabolites from the plant endophytic fungus Aspergillus sp. CPCC 400735 and their anti-HIV activities;Xu Pang等;《Journal of Natural Products》;20171010;第80卷;2595-2601 * |
| 植物内生真菌来源的抗菌活性物质研究进展;施琦渊等;《中国药学杂志》;20070630;第42卷(第11期);804-807 * |
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| CN110305798B (en) * | 2016-06-15 | 2021-11-19 | 中国医学科学院医药生物技术研究所 | Application of aspergillus, compound prepared from aspergillus and application of compound |
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