CN108546247A - A kind of alkaloid compound purposes in preparing antiadipositas drug object - Google Patents

A kind of alkaloid compound purposes in preparing antiadipositas drug object Download PDF

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CN108546247A
CN108546247A CN201810595611.3A CN201810595611A CN108546247A CN 108546247 A CN108546247 A CN 108546247A CN 201810595611 A CN201810595611 A CN 201810595611A CN 108546247 A CN108546247 A CN 108546247A
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aspergillus terreus
extract
formula
fermentate
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CN108546247B (en
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刘晓萌
吴长景
杨明生
崔香
毛建航
李凤
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Zhoukou Normal University
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    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
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    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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Abstract

Purposes that the present invention relates to a kind of alkaloid compounds in preparing antiadipositas drug object.The alkaloid compound has structure or its pharmaceutically acceptable salt shown in Formulas I；Its analogue individually or the purposes with composition forms in preparing antiadipositas drug object.By marine source Aspergillus terreusAspergillus  terreusML 44 carries out fermented and cultured, obtains fermentate containing compound of formula I wherein, fermentate is isolated and purified, obtain compound of formula I.Wherein, Aspergillus terreus ML 44, deposit number are CGMCC No.15664, and the deposit date is on 04 17th, 2018, preservation place was China General Microbiological culture presevation administrative center（CGMCC）.Present invention firstly discovers that the compound and its analogue have anti-obesity activity, lead compound is provided to develop novel antiadipositas drug object.

Description

A kind of alkaloid compound purposes in preparing antiadipositas drug object
Technical field
Purposes that the present invention relates to a kind of alkaloid compounds in preparing antiadipositas drug object.
Background technology
With the development of the social economy, China's incidence of obesity cumulative year after year, patient numbers increase from 2,400,000 in 1975 To 2014 more than 9,000 ten thousand, account for the 14.3% of whole world population of being obese（NCD Risk Factor Collaboration, Trends in adult body-mass index in 200 countries from 1975 to 2014: a pooled analysis of 1698 population-based measurement studies with 19•2 million participants. The Lancet, 2016, 387, 1377–1396）.Fat and type-2 diabetes mellitus, cardiovascular and cerebrovascular disease A series of generation of chronic diseases such as disease, cancer is closely related（Smith and Minson, Obesity and adipokines: effects on sympathetic overactivity. J Physiol, 2012, 590, 1787– 1801; Singer and Lumeng, The initiation of metabolic inflammation in childhood obesity. J Clin Invest, 2017, 127, 65–73）.It is fat（Including child form obesity）It is fast The exhibition of hailing can not only bring huge financial burden to society, certainly will also jeopardize the long-term sustainable development of Chinese society economy （Wolfenstetter, Juvenile obesity and comorbidity type 2 diabetes mellitus (T2 DM) in Germany: development and cost-of-illness analysis. Gesundheitswesen, 2006, 68, 600–612）.Therefore, fat prevention has become the significant problem of medical research field highest attention.
Fat method is treated mainly including reducing the intake of energy and food, the consumption of increase energy, inhibiting fat carefully The differentiation of born of the same parents and proliferation, the generation for inhibiting fat, the decomposition of increase fat and oxidation etc..In drug treatment, learn both at home and abroad Person develops a series of drugs around fat pathogenesis for different target spots, such as uses fat-reducing medicament, inhibits in enteron aisle Fat absorption（Orlistat）, promote fat metabolism etc.；Or directly act on central nervous system with adrenergic System plays appetite-suppressing, reduces appetite, the effect of losing weight.But these drug generally existing side effects are apparent, cure Rate is low, and rebound after drug withdrawal outstanding problems, the clinical value such as apparent are limited.Therefore, screening finds more preferably antiadipositas drug Object has important practical significance.
It is current field of obesity research that screening, which has the small-molecule substance for adjusting lipid generation and being metabolized, from nature One of hot spot, natural products provide colourful Chemical Diversity, can provide guide to develop new natural slimming drugs Compound（Fu et al., Natural Products with Anti-obesity Effects and Different Mechanisms of Action. J Agric Food Chem, 2016, 64, 9571–9585; Sun et al., Natural Dietary and Herbal Products in Anti-Obesity Treatment. Molecules, 2016, 21, 1351–1365）.Microbial secondary metabolite is the important sources of new drug and its guide structure, and from micro- life Screening obtains research of the regulating lipid metabolism in relation to bioactive natural product and is rarely reported in object.With pluripotency mescenchymal stem cell C3H10T1/2 is that cell model is screened, it is found that the fermentate of three plants of marine-derived fungals has and inhibit C3H10T1/2 at fat The activity of differentiation, wherein Aspergillus terreus ML-44（Aspergillus terreusML-44）Fermentate have and lower minimum have Imitate concentration.Further tracking activity separation separation from ML-44 bulk fermentation products identifies an alkaloid compound methyl 3,4,5-trimethoxy-2-(2-(nicotinamido)benzamido)benzoate（Formulas I）.It is currently known report There are five such compound in road is total, by the acidum nicotinicum of ortho-aminobenzoic acid of two molecules or derivatives thereof and a molecule （Niacin）It is keyed by amide, or further intramolecular cyclization forms（Arai et al., Metabolic products of Aspregillus terreus. VI. Metabolites of the strain IFO 8835. (3). The isolation and chemical structures of colorless metabolites. Chem Pharm Bull, 1981, 29, 1005–1012.; Wang et al., Three new compounds from Aspergillus terreus PT06-2 grown in a high salt medium, Mar Drugs, 2011, 9, 1368–1378; He et al., Asperterrestide A, a Cytotoxic Cyclic Tetrapeptide from the Marine- Derived Fungus Aspergillus terreus SCSGAF0162. J Nat Prod, 2013, 76, 1182– 1186; Chen et al., Structurally diverse secondary metabolites from a deep- sea-derived fungus Penicillium chrysogenum SCSIO 41001 and their biological evaluation. Fitoterapia, 2017, 117: 71–78）.The current document report in relation to such chemical combination microbic activity Road is seldom, and especially the bioactivity in terms of regulating lipid metabolism, anti-obesity there is no research to report.
Invention content
Purposes that the object of the present invention is to provide a kind of alkaloid compounds in preparing antiadipositas drug object.The alkaloids Compound has significant external inhibition Adipocyte Differentiation and internal anti-obesic action, i.e. the compound of the present invention has good Anti- function of obesity.
The structure of alkaloid compound provided by the invention is shown in formula I：
I
The present invention also provides compound of formula I analogues：terremide A、terremide B、terremide C、 Terremide D or its pharmaceutically acceptable salt are individually or the purposes with composition forms in preparing antiadipositas drug object.
The invention further relates to Aspergillus terreus（Aspergillus terreus）ML-44, deposit number are CGMCC No. 15664, the deposit date is on 04 17th, 2018, depositary institution was China General Microbiological culture presevation administrative center （CGMCC）, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101. The bacterial strain is detached from the alimentary canal sample of Pacific oyster, and categorized research is accredited as Aspergillus terreus（Aspergillus terreus）.
The preparation method of compound of formula I provided by the invention includes the following steps：
By above-mentioned Aspergillus terreus（Aspergillus terreus）ML-44 carries out fermented and cultured, obtains containing compound of formula I wherein Fermentate isolates and purifies fermentate, obtains the compound.It can also use the other of aspergillus that can generate formula I The production bacterium of compound carries out fermented and cultured.
It is described to isolate and purify the conventional method purified including the use of Separation of Natural Products well known to those skilled in the art, such as Extraction, column chromatography, efficient liquid phase preparation etc..
A kind of specific preparation method of compound of formula I provided by the invention includes the following steps：
1) by above-mentioned Aspergillus terreus（Aspergillus terreus）ML-44 carries out rice solid fermentation culture, is fermented Object；
2) by gained fermentate 50%-90%（v/v）Aqueous acetone solution ultrasound extraction, filtering, filtrate is through being concentrated under reduced pressure into not Containing acetone, remaining suspension is extracted with ethyl acetate, and obtains the acetic acid ethyl ester extract of fermentate；
3) step 2) is obtained acetic acid ethyl ester extract to be concentrated to dryness, obtains ethyl acetate extract；
4) the total medicinal extract of ethyl acetate is used into silica gel column chromatography successively（Petroleum ether dichloromethane methanol gradient elution）、 Sephadex LH-20 column chromatographies（Methanol elutes）With ODS column chromatographies（The methanol elution gradient of water → 100%）Separation, is contained The column chromatography component of the compound；
5) by the column chromatography group lease making HPLC separation containing the compound.
Wherein, the aqueous acetone solution in step 2) is 70%-90%（v/v）, preferably 75%-85%（v/v）, more preferably 80%（v/v）.
The present invention provides the extract of aspergillus fermentus, which contains the compound of formula I and its knot of the present invention Structure analog.Specifically, the extract be aspergillus fermentus the total medicinal extract of acetic acid ethyl ester extract, ethyl acetate or Chromatographic fraction.The corresponding steps that the extract is referred to the preparation method of the compound of the present invention above are made.Specifically, The extract can be prepared by extraction, column chromatography, efficient liquid phase.
The present invention provides purposes of the extract of aspergillus fermentus in preparing antiadipositas drug object.
The present invention provides purposes of the above-mentioned Aspergillus terreus in preparation of compounds of formula I or fermentation broth extract.
In short, the present invention provides have the medicine of structure or its pharmaceutically acceptable salt shown in Formulas I containing described Object, the drug include pharmaceutically acceptable adjuvant；The drug is injection, tablet, pill, capsule, suspending agent Or emulsion etc..
Term " pharmaceutically acceptable salt " in the present invention can be acceptable inorganic or organic salt.Formula I chemical combination There is basic group can form pharmaceutical salts, such as sulfate, hydrochloride, hydrobromate, phosphate with inorganic acid in object；Also may be used Pharmaceutical salts are formed with organic acid, such as acetate, oxalates, citrate, gluconate, succinate, tartrate, right Toluene fulfonate, mesylate, benzoate, lactate, maleate etc..
The compound of formula I and its above structure analog of the present invention can be with various pharmaceutically acceptable carriers, excipient Or antiadipositas drug object is made in supplementary product compatibility, the treatment for obesity and associated metabolic syndrome.
The compounds of this invention can be administered individually or in the form of pharmaceutical composition.Administration route can be take orally, non-bowel Or local administration.Pharmaceutical composition can be made into various suitable dosage forms according to administration route.
The pharmaceutical composition of the compounds of this invention can be applied with following any way：Oral, spraying sucking, rectum is used Medicine, nasal cavity applied medicine, cheek medication, local application, non-bowel medication, such as subcutaneous, vein is intramuscular, intrathecal in peritonaeum, intra-ventricle, In breastbone and intracranial injection or input, or by a kind of explant reservoir medication.It wherein preferably takes orally, peritonaeum is interior or intravenous administration Mode.
When oral medication, the compounds of this invention, which can be made into, arbitrarily takes orally acceptable dosage form, including but not limited to Tablet, capsule, aqueous solution or water slurry.Wherein, the carrier that tablet uses generally comprises lactose and cornstarch, in addition also may be used Lubricant such as magnesium stearate is added.The diluent that capsule preparations use generally comprises lactose and dried corn starch.Water slurry Preparation is typically then to be used in mixed way active constituent and suitable emulsifier and suspending agent.Optionally, the above oral dosage form In some sweeteners, aromatic or colorant can also be added.
When topical application, the compounds of this invention can be made into ointment, lotion or cream formulation form appropriate, wherein Active constituent is suspended or dissolved in one or more carriers.Carrier workable for ointment formulation includes but not limited to：Mineral Oil, Albolene, albolene, propylene glycol, polyethylene glycol oxide, polypropylene oxide, emulsifying wax and water；Lotion or creme can make Carrier includes but not limited to：Mineral oil, sorbitan monostearate, polysorbate60, cetyl ester wax, hexadecene Fragrant and mellow, 2- octyldodecanols, benzyl alcohol and water.
The compounds of this invention can the medication in the form of aseptic injection preparation, including aseptic injection water or oil suspension or sterile Inject solution.Wherein, workable carrier and solvent include water, Ringer's solution and isotonic sodium chlorrde solution.In addition, sterilizing Fixed oil also is used as solvent or suspension media, such as monoglyceride or two glyceride.
Furthermore, it should be pointed out that the compounds of this invention dosage and application method depend on factors, including patient Age, weight, gender, natural health situation, nutrition condition, the activity intensity of compound, Time of Administration, metabolic rate, illness Severity and diagnosis and treatment doctor subjective judgement.Preferred dosage is between 0.01-100 mg/kg body weight/days.
The present invention provides the preliminary cell activity test of compound of formula I, which can inhibit mice embryonic Mesenchyma C3H10T1/2 cell differentiations are adipocyte, significantly inhibit intracellular fat generation；It is aobvious in body activity preliminary experiment result Show that it can obviously inhibit the generation of high fat diet induced male drosophila obesity, makes drosophila weight maintenance in reduced levels.Thus originally The compound of invention has good anti-function of obesity, and lead compound is provided to develop novel antiadipositas drug object.
Description of the drawings
Fig. 1 compounds I significantly inhibits breaking up at fat for C3H10T1/2 cells;A) blank control, b) compound I（20 μ M）.
The Male Drosophila that Fig. 2 compounds I significantly inhibits induction high in fat is fat.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is The conventional products obtained can be bought by market.
In the examples below, compound shown in alleged compound I, that is, Formulas I is of the invention from marine-derived fungalAspergillus terreusIt is isolated in ML-44 fermentate ethyl acetate extracts, and its anti-fat work(is found for the first time Energy.
Arabic numerals indicate corresponding mark.
I
In the structural research of embodiment below, ESI-MS uses Thermo Scientific companies of U.S. LCQ Fleet liquid matter Combined instrument measures, NMR spectra 500 type NMR spectrometer with superconducting magnet of Bruke companies of Switzerland Avance III（500 MHz1H- NMR, 125 MHz13C-NMR）It measures.
Embodiment 1：The preparation of microbial fermentation culture and compound
1. the extraction process of fermented and cultured and fermentate
1) bacterial strain is produced
Producing strains in the present embodiment for fermenting and producing compound I are to be isolated from the gastral aspergillus terreus of Pacific oyster （Aspergillus terreus）ML-44 plants, it is preserved in China General Microbiological culture presevation administrative center（CGMCC）, preservation Number is CGMCC No. 15664.
2) fermented and cultured
By the conventional method of microculture, Aspergillus is taken out from 4 DEG C of refrigerators（Aspergillus terreus）ML-44 test tubes Inclined-plane aseptically scrapes appropriate spore with oese, and streak inoculation is in the 5 PDA solid mediums newly prepared（Group At：Glucose 2%, agar 2%, NaCl 1.5% are prepared with the water cooking liquid of 20% potato）On tablet, training is activated in 28 DEG C of incubators It supports 5 days, takes spore with the sterile washings of 10ml respectively, and all spores merging is scattered in 100 ml sterile waters, obtain spore Suspension.The inoculum concentration that the spore suspension is pressed to every bottle of 4 ml, is inoculated in respectively equipped with rice solid medium（Every bottle of 80 g are commercially available Rice adds 150 ml natural sea-waters, is made in 121 DEG C of 30 min of sterilizing after placement is overnight）24 1000 ml conical flasks in, It is cultivated 35 days in being stored at room temperature.
2. the preparation of extraction process and ethyl acetate extract
The aqueous acetone solution for being 80% by fermentate volume fraction（Every bottle of 500 ml）It impregnates and is suspended, be ultrasonically treated 1h, room temperature Extraction is for 24 hours afterwards with 4 layers of filtered through gauze, repetitive operation 3 times.Merging filtrate is concentrated under reduced pressure into without after acetone, remaining aqueous layer about 3L It is extracted 3 times with isometric ethyl acetate, obtains fermentate acetic acid ethyl ester extract, be concentrated to dryness, obtain ethyl acetate extract 66.2 g。
3. the preparation of the column chromatography for separation of ethyl acetate extract and the column chromatography component containing target compound I-V
By 66.2 g methylene chloride-methanols of above-mentioned ethyl acetate extract（v/v 1:1）200ml dissolves, and filters out insoluble matter, is added 90 g chromatographic silica gels（200-300 mesh）Sample is mixed in absorption, is added to prepackage silica gel glass decompression column after evaporated under reduced pressure（120 g silica gel, 4.8 × 18.0 cm of column bed）On, gradient elution chromatography is carried out with petroleum ether-methylene chloride-methanol solvent system, is obtained several Flow point.Merge corresponding flow point according to silica gel thin-layer analysis result, 8 components are obtained by elution order of polarity：Fr-1（12.4 g, Petroleum ether）、Fr-2（6.3 g, petroleum ether-dichloromethane 1:1 elution）、Fr-3（4.5 g, petroleum ether-dichloromethane 1:2 wash It is de-）、Fr-4（11.6 g, dichloromethane eluent）、Fr-5（15.8 g, methylene chloride-methanol 99:1 elution）、Fr-6（6.2 g, Methylene chloride-methanol 95:5 elutions）、Fr-7（1.4 g, dichloromethane 9:1 elution）、Fr-8（1.9 g, methylene chloride-methanol 7: 3 elutions）.
By Fr-4（11.6 g）Upper Sephadex LH-20 columns after being dissolved with about 50 ml methanol, methanol elution pick up 18 Fraction（Respectively meet 30 ml）；According to HPLC analysis results, component ML-4 gel chromatography fractions 1-18 is merged into 8 components：Fr- 4-1-Fr-4-8.To component Fr-4-5（About 1.8 g）After being dissolved with methanol and used 0.45 μm of membrane filtration, using in QuikSep Low pressure chromatography system（Intelligent moral is easy, Beijing, China）With 80% methanol（10 ml/min of flow velocity, room temperature）In the glass for being preinstalled with ODS Column（Column bed 1.8cm × 30cm）On detached.7 components are picked up according to ultraviolet testing result：ML-4-5-1-ML-4-5-7. Wherein, target compound I is contained in Fr-4-5-2.
4. prepared by the HPLC of compound I
By the component Fr-4-5-2 containing compound I（180 mg）It is dissolved with 5 ml methanol, after 0.45 μm of membrane filtration, is used 2545 type HPLC systems of Waters, column is prepared using Gemini C18（21.2 mm × 250 mm）Carry out HPLC separation（Column 26 DEG C of temperature, using 68% methanol as mobile phase, 10 ml/min of flow velocity, 0.5 ml of each sample introduction, Detection wavelength are 210 and 254 nm), Compound I is made（62 mg,t R= 19.6 min）.
The physicochemical constant and spectral data of compound I
Compound I is colorless needle crystals（MeOH）, m.p. 137-139oC.Cation ESI-MS：m/z 466 [M + H ]+, 488 [M+Na]+, 931 [2M+H]+；Anion ESI-MS：m/z 464 [M - H]-, 490 [M+Cl]-。1H NMR（500 MHz, CDCl3）δ：12.20 (1H, s, 2-NH), 9.20 (1H, br s, 2ʹʹ-NH), 9.19 (1H, d, J = 1.90 Hz, H-2ʹ), 8.79 (1H, br d, J = 7.85, H-3), 8.67 (1H, dd, J = 4.80, 1.90 Hz, H-4ʹ), 8.21 (1H, dt, J = 7.90, 1.90 Hz, H-6ʹ), 7.87 (1H, dd, J = 7.85, 1.10 Hz, H-6), 7.56 (1H, td, J = 7.85, 1.10 Hz, H-4), 7.33 (1H, dd, J = 7.90, 4.80 Hz, H-5ʹ), 7.24 (1H, s, H-6ʹʹ), 7.19 (1H, br t, J = 7.85 Hz, H- 5), 3.94 (3H, s, 5ʹʹ-OCH3), 3.874 (3H, s, 4ʹʹ-OCH3), 3.867 (3H, s, 3ʹʹ-OCH3), 3.78 (3H, s, 7ʹʹ-OCH3)。13C NMR（125 MHz, CDCl3）δ：168.2 (C-7ʹʹ), 167.2 (C-7), 164.0 (C-7ʹ), 152.6 (C-4ʹ), 151.5 (C-5ʹʹ), 149.2 (C-4ʹʹ), 148.9 (C-2ʹ), 146.9 (C-3ʹʹ), 140.4 (C-2), 135.2 (C-6ʹ), 133.6 (C-4), 130.6 (C-1ʹ), 127.9 (C-6), 125.7 (C-2ʹʹ), 123.8 (C-5), 123.6 (C-5ʹ), 121.8 (C-3), 120.4 (C-1), 119.1 (C- 1ʹʹ), 108.8 (C-6ʹʹ), 61.24 (4ʹʹ-OCH3), 61.23 (3ʹʹ-OCH3), 56.5 (5ʹʹ-OCH3), 52.7 (7ʹʹ-OCH3).The above NMR data is belonged to through being compareed with data in literature.
Embodiment 2：Compound I inhibits C3H10T1/2 cells to break up at fat
1）Growth inhibition effects of the compound I to C3H10T1/2 cells
Influence using mtt assay detection compound I to C3H10T1/2 cell viabilities.The C3H10T1/2 of logarithmic growth phase is thin Born of the same parents, it is 2 × 10 to prepare cell density with fresh DMEM medium5The cell suspension of a/ml is inoculated in 96 orifice plates, per hole 200 Sample sets add each 2 μ l of sample liquid per hole after μ l, 37 DEG C of 12 h of culture, and blank control group then adds each 2 μ l of methanol per hole, in 37 DEG C Handle 48 h.Add the MTT solution of the 5mg/ml of precooling per hole（It is prepared with PBS solution）Each 20 μ l, after 37 DEG C are incubated 4 h, in 4 DEG C, 2000 rpm centrifuge 10 min, suck supernatant, add 150 μ l DMSO per hole, being placed in microplate reader fully oscillation makes MTT purple products are completely dissolved, and measure the OD values at per 570 nm of hole.Sample and blank control group respectively set three in experiment Parallel hole takes OD average values, by IR%=(ODBlank-ODSample)/ODBlank× 100% formula calculates sample to C3H10T1/2 cells Inhibiting rate（IR%）.In mtt assay test, in conjunction with micro- sem observation inspection, compound I is right under 100 μM of activities C3H10T1/2 cells do not show any cytotoxicity and growth inhibition effect.
2）Compound I inhibits C3H10T1/2 cell differentiation adipocytes
It is inoculated in C3H10T1/2 cells to 6 orifice plates, after culture is paved with 80% ware bottom to cell, fresh inducing culture is added （+ 0.1+1.0 μM of dexamethasone of mM Indomethacins of+0.5 mM isobutyl methylxanthines of+10% fetal calf serum of DMEM culture mediums+ + 1% dual anti-solution of mycillin of 10 μ g/mL insulin）Culture 2 days；Change differential medium（+ 10% fetal calf serum of DMEM culture mediums+ + 1% dual anti-solution of mycillin of 1.0 μ g/mL insulin）Continue culture 2 days；Change growth medium（+ 10% tire of DMEM culture mediums The dual anti-solution of+1% mycillin of cow's serum）Continue culture 4 days（Change within every 2 days time fresh culture）.In each culture medium of processing group Each 2 μ l of sample solution are added, control group is not added with same method operation.In culture the 8th day, oil red O stain, cell microscopy were carried out respectively. Coloration result shows that blank group most cells are successfully divided into mature fat cell, and intracellular oil droplet is more and big, and processing group Into the cell almost without oil droplet（Fig. 1）, show compound I can pole significantly inhibit breaking up at fat for C3H10T1/2 cells.
Embodiment 3：Compound I inhibits the generation of induced male drosophila obesity high in fat
3 day age Male Drosophila is taken, is divided into 3 groups, control group is cultivated with minimal medium, and HFD induction groups give culture high in fat Base（Minimal medium adds 15% lard）, processing group then gives the culture medium high in fat of addition compound I（0.1 mg/ of final concentration ml）, continuous processing 20 days is detected each group drosophila weight.By testing result in Fig. 2 as it can be seen that with medium culture high in fat Drosophila weight can be made to dramatically increase, form fat drosophila；The drosophila that compound I processing can obviously inhibit HFD to induce is given simultaneously It is fat.
Although the specific implementation mode of the present invention has obtained detailed description, it will be understood to those of skill in the art that root According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these change the guarantor in the present invention Within the scope of shield.The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims (10)

1. a kind of alkaloid compound being used to prepare antiadipositas drug object, which is characterized in that the alkaloid compound With structure shown in Formulas I or its pharmaceutically acceptable salt：
I。
2. compound of formula I described in claim 1 and its analogue terremide A, terremide B, terremide C, terremide D or its pharmaceutically acceptable salt be individually or the purposes with composition forms in preparing antiadipositas drug object.
3. Aspergillus terreus ML-44, deposit number is CGMCC No.15664.
4. the preparation method of compound of formula I described in claim 1, it is characterised in that include the following steps：By marine source soil AspergillusAspergillus terreusML-44 carries out fermented and cultured, fermentate containing compound of formula I wherein is obtained, by fermentate It is isolated and purified, obtains compound of formula I.
5. preparation method according to claim 4, it is characterised in that it is described isolate and purify including extraction, column chromatography and efficiently It is prepared by liquid phase.
6. a kind of preparation method of compound of formula I described in claim 1, it is characterised in that include the following steps：
1) by Aspergillus terreus（Aspergillus terreus）ML-44 carries out rice solid fermentation culture, obtains fermentate；
2) by gained fermentate 50%-90%（v/v）Aqueous acetone solution ultrasound extraction, filtering, filtrate is through being concentrated under reduced pressure into not Containing acetone, remaining suspension is extracted with ethyl acetate, and obtains the acetic acid ethyl ester extract of fermentate；
3) step 2) is obtained acetic acid ethyl ester extract to be concentrated to dryness, obtains ethyl acetate extract；
4) the total medicinal extract of ethyl acetate is used into silica gel column chromatography, petroleum ether dichloromethane methanol gradient elution, Sephadex successively LH-20 column chromatographies, methanol elution and ODS column chromatographies, the methanol elution gradient of water → 100%, separation are obtained containing the compound Column chromatography component；
5) by the column chromatography group lease making HPLC separation containing the compound；
Wherein, the aqueous acetone solution in step 2) is preferably 75%-85%（v/v）, more preferably 80%（v/v）.
7. a kind of Aspergillus terreus（Aspergillus terreus）The extract of ML-44 fermentates, it is characterised in that the extract Contain compound of formula I described in claim 1.
8. purposes of the extract in preparing antiadipositas drug object described in claim 7.
9. the Aspergillus terreus described in claim 3（Aspergillus terreus）ML-44 is preparing Formulas I described in claim 1 The purposes in extract described in compound or claim 7.
10. a kind of anti-obesity drug, it is characterised in that there is structure or its medicine shown in Formulas I containing described in claim 1 Acceptable salt and pharmaceutically acceptable adjuvant on；The drug is injection, tablet, pill, capsule, hangs Floating agent or emulsion.
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Citations (2)

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JPS56161362A (en) * 1980-04-03 1981-12-11 Kyoto Yakuhin Kogyo Kk Anthranilic acid derivative
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS56161362A (en) * 1980-04-03 1981-12-11 Kyoto Yakuhin Kogyo Kk Anthranilic acid derivative
CN102123586A (en) * 2008-07-18 2011-07-13 法弗根股份有限公司 Methods of treating an overweight or obese subject

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SHENGTIAN CHEN等: "Structurally diverse secondary metabolites from a deep-sea-derived fungus Penicillium chrysogenum SCSIO 41001 and their biological evaluation", 《FITOTERAPIA》 *
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