CN103724290A - Cyclopeptide compound clavatustide A as well as producing strain, preparation method and application thereof - Google Patents
Cyclopeptide compound clavatustide A as well as producing strain, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a cyclopeptide compound clavatustide A as well as a producing strain, a preparation method and an application thereof. The structural formula of clavatustide A is represented as formula (I): the preparation method comprises steps as follows: activated aspergillus clavatus CCTCC No. M2013421 is inoculated into a fermentation culture solution for fermentation culture; after fermentation is finished, a mycelium and fermentation broth are obtained through separation; a mycelium extract or a fermentation broth extract is subjected to chromatographic separation and purification treatment, and the cyclopeptide compound is prepared; and the application relates to an application of the cyclopeptide compound in preparation of antineoplastic drugs or cancer prevention health food. The cyclopeptide compound has better antitumor activity in vitro, the IC50 value of the cyclopeptide compound to hepG2 cells is 15 mu g/mL, and the cyclopeptide compound has an excellent development prospect; and the preparation method is simple and convenient to operate and suitable for large-scale production.
Description
Technical field
The invention belongs to thalassiomycetes active substance field, be specifically related to a kind of cyclic peptide compounds clavatustide A and produce bacterium, preparation method and application.
Background technology
Marine microorganism is owing to living under pressure environment, and its secondary metabolic product often has the biological activity that land fungus twice meta-bolites does not possess, and becomes in recent years the focus of drug development.Marine microorganism not only struggle for existence pressure is very large; pH scope, temperature, pressure, dissolved oxygen, light, nutrition and the salinity etc. of its residing ocean environment are all different from terrestrial environment; this is the multifarious basis of marine microorganism secondary metabolic product (Debbab, A.; Aly, A.H.; Lin, W.H.; Proksch, P.Microb.Biotech.2010,3,544 – 563; Saleema, M.; Ali, M.S.; Hussain, S.; Jabbar, A.; Ashraf, M.; Lee, Y.S.Nat.Prod.Rep.2007,24,1142 – 1152; Wu, B.; Wu, X.; Sun, M.; Li, M.Mar.Drugs2013,11,2713-2721.).
Thalassiomycetes is as the important component part of marine microorganism, and the aspects such as, oil degradation synthetic at medicine, environment remediation have vital role.Thalassiomycetes had both had the typical protein modified performance of eukaryote, had again upper easy, the advantage fast of microorganism operation, as new eukaryotic expression system, had huge potential and wide application prospect.Along with going deep into that marine microorganism is studied, found increasing anti-tumor active substance from thalassiomycetes in recent years, thalassiomycetes becomes the another study hotspot after marine actinomycete.
(Xu is red for natural pond Tian Dun etc., Gu Qianqun, the refined .2001 of Cui Cheng. the progress [J] of studies of antitumor metabolites from marine microorganisms. Chinese Sea medicine .6:44-49.) penicillium being separated to from Enteromorpha marine alga Enteromorpha intestinalis body surface has been carried out to a series of research.This bacterial strain is placed in to the artificial seawater substratum containing 2% glucose, 1% peptone and 0.5% yeast extract paste; cultivate 3 weeks for 27 ℃; first separate and obtain 2 cell toxicant meta-bolites Communesins A and B, in the two structure, only acyl side-chain is deposited difference, but in activity, has differed nearly 10 times.
(Xu is red for natural pond Tian Dun etc., Gu Qianqun, the refined .2001 of Cui Cheng. the progress [J] of studies of antitumor metabolites from marine microorganisms. Chinese Sea medicine .6:44-49.) also from be located away from the GI aspergillus Aspergillus of ocean fish Pseudolabrus japonicus fummigatus mycelium, be separated to a series of cytotoxic compound Fumiquinazolines A~G, P385 cell is had to medium cytotoxicity.The Aspergillus versicolor that the people such as Fenieal separate from Caribbean Sea green alga Penicillus capitatus body surface obtains four sesquiterpene oil of mirbane ester cpds, wherein 9a, the IC of 14-dihydroxyl-6 p-nitrobenzoic acid cinnamic ester to 60 of NCI mankind's tumor cell groups
50mean value be 1.1mg/L, but to the selective toxicity of 5 kinds of kidney cancer cells (786-0, ACHN, CAK-1, TK-10, VO-31), average IC
50for 0.51mg/L.
Ocean hydrothermal solution mouth is most active environment (Thornburg, C.C. on the earth; Zabriskie, T.M.; McPhail, K.L.J.Nat.Prod.2010,73,489 – 499.), hydrothermal solution mouth crab (Xenograpsus testudinatus) just lives in extremely, toxicity, be covered with near the hydrothermal solution mouth of sulfide.This hydrothermal solution mouth crab is found (eng, M.-S. in Kueishan Island, Taiwan; Ng, N.K.; Ng, P.K.L.Nature2004,432,969.), up to now, nobody studies the meta-bolites of the epiphytic fungi of this hydrothermal solution mouth crab.
Summary of the invention
The invention provides a kind of cyclic peptide compounds clavatustide A, this compound extracts and obtains from hydrothermal solution mouth crab Xenograpsus testudinatus grows nonparasitically upon another plant excellent aspergillus Aspergillus clavatus C2WU fermenting culture, has stronger anti-tumor activity.
A kind of cyclic peptide compounds clavatustide A, structural formula is as shown in formula I:
The present invention also provides a kind of excellent aspergillus, Classification And Nomenclature is excellent aspergillus (Aspergillus clavatus), strain number is C2WU, this bacterial strain has been preserved in Chinese Typical Representative culture collection center (the China Center for Type Culture Collection that is positioned at Luo Jia Shan, Wuhan City Wuhan University on September 14th, 2013, CCTCC), preserving number is: CCTCC No.M2013421.
Described excellent aspergillus (Aspergillus clavatus) C2WU separates and obtains from hydrothermal solution mouth crab (Xenograpsus testudinatus), is the epiphytic fungi of hydrothermal solution mouth crab (Xenograpsus testudinatus).
The present invention also provides the preparation method of described cyclic peptide compounds clavatustide A, comprises the following steps:
(1) activated excellent aspergillus (Aspergillus clavatus) CCTCC No.M2013421 is inoculated in fermentation culture, carries out fermentation culture;
Due to excellent aspergillus (Aspergillus clavatus), C2WU is fungi, adopts conventional PDA liquid nutrient medium or malt extract medium to can be used for fermentation.But in order to simulate better the primary ocean environment of excellent aspergillus C2WU, enough nutritive ingredients are provided to microorganism growth metabolism, as preferably, in step (1), in 1L, the consisting of of described fermentation culture: starch 1-5g, wheat bran 10-20g, yeast extract paste 3-15g, KH
2pO
40.1-0.8g, MgSO
47H
2o0.1-0.8g, surplus is seawater.
Or, in 1L, the consisting of of described fermentation culture: potato 200-600g, peptone 2-10g, yeast extract paste 1-5g, glucose 5-20g, surplus is seawater; Nutrient solution initial pH value is 6.0~7.0.
Or, in 1L, the consisting of of described fermentation culture: sucrose 20-40g, yeast extract 8-12g, malt extract 18-22g, MgSO
47H
2o0.3-0.5g, KH
2pO
40.3-0.5g, surplus is seawater.
As further preferably, in step (1), in 1L, the consisting of of described fermentation culture: starch 1-5g, wheat bran 10-20g, yeast extract paste 3-15g, KH
2pO
40.1-0.8g, MgSO
47H
2o0.1-0.8g, surplus is seawater.
Or, in 1L, the consisting of of described fermentation culture: potato 200-600g, peptone 2-10g, yeast extract paste 1-5g, glucose 5-20g, surplus is seawater; Nutrient solution initial pH value is 6.0~7.0.
Or, in 1L, the consisting of of described fermentation culture: sucrose 20.0g, yeast extract 10.0g, malt extract 20g, MgSO
47H
2o0.4g, KH
2pO
40.4g, surplus is seawater.
It is 10 that described excellent aspergillus C2WU is made to concentration
7~10
9the spore suspension of cfu/mL, is inoculated in fermentation culture with 8~12% inoculum sizes, carries out fermentation culture.Preferably, the temperature of described fermentation culture is 20-30 ℃, and the time is 10-60 days.More preferably, the temperature of described fermentation culture is 22-26 ℃, and the time is 15-25 days.Most preferably, the temperature of described fermentation culture is 24 ℃, and the time is 20 days.Under this fermentation culture conditions, the output of described cyclic peptide compounds clavatustide A is the highest.
Fermentation culture adopts static cultivation mode, also can carry out shake-flask culture, while adopting shake-flask culture, and optionally corresponding shortening of fermentation time.
(2), after having fermented, separate and obtain mycelium and fermented liquid;
In the present invention, the mycelium obtaining from fermenting with in fermented liquid, all can extract, ly obtain described cyclic peptide compounds clavatustide A.Wherein, while utilizing mycelium to obtain clavatustide A, mycelium is placed in to organic solvent lixiviate, obtain vat liquor, again with distilled water to vat liquor suspendible, obtain aqueous suspension, then with ethyl acetate or chloroform, aqueous suspension is extracted, be extracted liquid A, extraction liquid A obtains mycelium extract after concentrated;
During lixiviate, the weightmeasurement ratio of mycelium and organic solvent is preferably 50~500g:1L, more preferably 100g:1L.Described organic solvent can be selected one or both in methyl alcohol, ethanol, ethyl acetate and acetone, is preferably methyl alcohol or acetone, and under methyl alcohol or acetone soak, mycelia physical efficiency, by abundant broken wall, makes the effective stripping of intracellular organic matter.
While utilizing fermented liquid to obtain clavatustide A, fermented liquid is extracted with ethyl acetate or chloroform, be extracted liquid B, extraction liquid B obtains fermentation broth extract after concentrated.
(3) mycelium extract or fermentation broth extract are carried out to chromatographic separation, purification process, make described cyclic peptide compounds clavatustide A;
As preferably, described chromatographic separation is purification on normal-phase silica gel column chromatography for separation, and eluent is petrol ether/ethyl acetate mixed solution.During chromatographic separation, successively take volume ratio as 9:1, the petrol ether/ethyl acetate mixed solution of 6:1,1:1,1:3,1:9 carries out wash-out, collected volume is than the cut eluting for the petrol ether/ethyl acetate mixed solution of 6:1.
In the target fraction obtaining through purification on normal-phase silica gel column chromatography for separation, described cyclic peptide compounds also contains a small amount of impurity, obtains the target compound that purity is higher if want, also needs this target fraction to be further purified processing.The method of described purification process is optional: recrystallization, reversed-phase silica gel column chromatography separate or high performance liquid chromatography separates.
Wherein, adopt reversed-phase silica gel column chromatography separation or high performance liquid chromatography to separate while carrying out purifying, preferred eluent is methanol/water mixed solution; More preferably, described methanol/water mixed solution by volume 9:1,5:1,1:1,1:3,1:9 carries out gradient elution, and collected volume is than the cut eluting for the methanol/water mixed solution of 1:3.
Thus, the present invention also provides described cyclic peptide compounds clavatustide A in the application of preparing in antitumor drug or tumor prevention protective foods.This antitumor drug, take cyclic peptide compounds of the present invention as main active ingredient, adds acceptable auxiliary material in pharmaceutics and makes, and can make preparation according to the formulation preparation method of recording in pharmaceutics.Described preparation can be injection liquid, drip liquid, powder injection, granule, tablet, electuary, powder, oral liquid, sugar coated tablet, film coated tablet, enteric coated tablet, suck agent, granule, pill, paste, sublimed preparation, sprays, pill, disintegrating agent, orally disintegrating tablet, micropill etc.This tumor prevention protective foods, take cyclic peptide compounds of the present invention as main active ingredient, adds acceptable protective foods auxiliary material and makes.
As preferably, described antitumor drug is medicines resistant to liver cancer, and described tumor prevention protective foods is prevention of hcc protective foods.Utilize this cyclic peptide compounds to carry out antitumor activity in vitro, show that this cyclic peptide compounds has good anti tumor activity in vitro, its IC to HepG2 cell
50value is 15 μ g/mL.
Compared with prior art, beneficial effect of the present invention is:
The present invention utilizes polarity difference extraction from the fermenting culture of crab epiphytic fungi rod aspergillus C2WU, the separation of cyclic peptide to obtain a kind of cyclic peptide compounds clavatustide A with novel structure, the method is easy and simple to handle, extraction yield is high, product purity is high, is applicable to large-scale production; And this compound has good anti tumor activity in vitro, its IC to HepG2 cell
50value is 15 μ g/mL, can be used for preparing medicines resistant to liver cancer or prevention of hcc protective foods, has good DEVELOPMENT PROSPECT.
Accompanying drawing explanation
Fig. 1 is cyclic peptide compounds clavatustide A's of the present invention
1h
1h COSY, the HMBC collection of illustrative plates of being correlated with;
Fig. 2 is the circular dichroism spectrogram of cyclic peptide compounds clavatustide A of the present invention; Wherein, upper figure is CD spectrogram, and the Y-axis mdeg of CD spectrogram represents milli degree, and X-axis is wavelength nm; Figure below is ultraviolet spectrogram, and the Y-axis of ultraviolet spectrogram represents absorbancy, and X-axis is wavelength nm;
Fig. 3 is cyclic peptide compounds clavatustide A's of the present invention
1h NMR nuclear magnetic spectrum;
Fig. 4 is cyclic peptide compounds clavatustide A's of the present invention
13c NMR nuclear magnetic spectrum;
Fig. 5 is the DEPT NMR nuclear magnetic spectrum of cyclic peptide compounds clavatustide A of the present invention;
Fig. 6 is the COSY NMR nuclear magnetic spectrum of cyclic peptide compounds clavatustide A of the present invention;
Fig. 7 is the HSQC NMR nuclear magnetic spectrum of cyclic peptide compounds clavatustide A of the present invention;
Fig. 8 is the HMBC NMR nuclear magnetic spectrum of cyclic peptide compounds clavatustide A of the present invention;
Fig. 9 is the infrared spectrogram of cyclic peptide compounds clavatustide A of the present invention.
Figure 10 is the structural formula of cyclic peptide compounds clavatustide A of the present invention.
Embodiment
Embodiment 1
(1) from hydrothermal solution mouth crab, separate fungi
Hydrothermal solution mouth crab Xenograpsus testudinatus receives in the Ye Ying of oceanography institute of Zhejiang University and teaches.First use aseptic seawater flushing hydrothermal solution mouth crab 3 times, remove non-attached microbial; By shell and the separate tissue of hydrothermal solution mouth crab, under aseptic condition, scrape lower housing powder again; Then in shell powder, add seawater to make serial gradient dilution (1:5,1:25,1:125,1:625), with vortex vibrator vibration 10min, obtain suspension; Centrifugal this suspension 20min under 5000r/min,, supernatant liquor inclines; A small amount of aseptic seawater resuspension for throw out, gets the resuspended liquid of 0.1mL and coats GYP substratum (1.0g glucose, 0.1g yeast extract, 0.5g peptone, 15g agar, 1L seawater) flat board; At 20 ℃ of room temperatures, cultivate after 10d, picking list bacterium colony moves at 4 ℃, inclined-plane and saves backup after line purifying.
(2) differentiate and separate fungi
Separate fungi while growing on PDA substratum, white mycelium, have every, colony edge has the thin layer aerial hyphae of wide 2-6mm, aerial hyphae is felt shape, is close to matrix growth; The spore head growing from the raw mycelia of base is abundant, and the top capsule of spore head is bar-shaped, and surface is robin's egg blue, is white during children; All there is transparence secretory product on the top of top capsule and aerial hyphae, and bacterium colony reverse side is tawny, has the radial texture of concentric(al) circles, and texture place color is darker.
Extract strain gene group DNA simultaneously, its ITS gene carry out sequencing of increasing, the base sequence of ITS gene is as shown in SEQ ID No.1.
According to the morphological specificity of bacterial strain and ITS the sequencing results, identify that this bacterial strain is excellent aspergillus Aspergillus clavatus, called after rod aspergillus C2WU.
(3) fermentation culture
Activated excellent aspergillus C2WU is made to concentration 10
8cfu/mL spore suspension, is inoculated in fermentation culture with 10% inoculum size, in 24 ℃ of static fermentations, cultivates 20 days.
Wherein, nutrient solution formula is: potato 400g, peptone 6g, yeast extract paste 2g, glucose 10g, seawater 1000mL; The initial pH value of fermentation culture is 6.5.
(4) cyclic peptide compounds preparation
After rod aspergillus C2WU fermentation culture, get 5L fermentation culture, centrifuging and taking precipitation obtains mycelium; Methyl alcohol lixiviate 1 week for mycelium, obtains vat liquor; Vat liquor with 1L distilled water suspendible, obtains aqueous suspension after concentrated; 6L ethyl acetate extraction for aqueous suspension, obtains acetic acid ethyl acetate extract, and acetic acid ethyl acetate extract obtains medicinal extract 10g after concentrated; Silica gel for medicinal extract (100 orders, 100g) is mixed to sample, carry out purification on normal-phase silica gel column chromatography for separation (200-300 order, 100g; Silicagel column size L50mm,
), with the petrol ether/ethyl acetate mixed solution of volume ratio 9:1,6:1,1:1,1:3,1:9, carry out gradient elution successively, each 300mL; TLC detects cut, and the cut of collecting wash-out ratio 6:1 merges, concentrates, then uses recrystallizing methanol, obtains target compound.
(1) with embodiment 1 (1) part.
(2) with embodiment 1 (2) part.
(3) fermentation culture
Activated excellent aspergillus C2WU is made to concentration 10
9cfu/mL spore suspension, is inoculated in fermentation culture with 8% inoculum size, in 22 ℃ of static fermentations, cultivates 25 days.
Wherein, the formula of fermentation culture is: starch 3g, wheat bran 14g, yeast extract paste 6g, KH
2pO
40.5g, MgSO
47H
2o0.4g, seawater 1000mL.
(4) cyclic peptide compounds preparation
After rod aspergillus C2WU fermentation culture, get 5L fermentation culture, centrifuging and taking supernatant obtains fermented liquid; After fermented liquid is concentrated, with 10g diatomite, mix sample, 1L ethyl acetate backflow, carries out purification on normal-phase silica gel column chromatography for separation (200-300 order, 1kg; Silicagel column size L50mm,
), with the petrol ether/ethyl acetate mixed solution of volume ratio 9:1,6:1,1:1,1:3,1:9, carry out gradient elution successively, each 300mL; TLC detects cut, and the cut of collecting wash-out ratio 6:1 merges; Target fraction high-efficient liquid phase chromatogram purification, with methanol-water (60-40) wash-out, detects wavelength 254nm, and the cut of collecting 78min merges, concentrates, then uses recrystallizing methanol, obtains target compound.
Embodiment 3
(1) with embodiment 1 (1) part.
(2) with embodiment 1 (2) part.
(3) fermentation culture
Activated excellent aspergillus C2WU is made to concentration 10
7cfu/mL spore suspension, is inoculated in fermentation culture with 12% inoculum size, in 25 ℃ of static fermentations, cultivates 20 days.
Wherein, nutrient solution formula is: sucrose 20.0g, yeast extract 10.0g, malt extract 20g, MgSO
47H
2o0.4g, KH
2pO
40.4g, seawater 1000mL.
(4) cyclic peptide compounds preparation
After rod aspergillus C2WU fermentation culture, fermentation culture is centrifugal, obtain respectively mycelium and fermented liquid.
Get mycelium and carry out vacuum-drying, dry weight obtains 253g, with methanol extraction (cold soaking) 3 times (3 × 1L), obtains methanol extract liquid, the concentrated medicinal extract 13g that obtains; 500mL distilled water suspendible for medicinal extract, obtains aqueous suspension; Aqueous suspension is extracted with ethyl acetate (3 × 3L) 3 times, is extracted liquid A; Fermented liquid (30L) is extracted with ethyl acetate (3 × 20L) 3 times, is extracted liquid B;
Extraction liquid A and extraction liquid B are merged, use purification on normal-phase silica gel column chromatography for separation, with the petrol ether/ethyl acetate mixed solution of volume ratio 9:1,5:1,1:1,1:3,1:9, carry out gradient elution successively, each 300mL; TLC detects cut, and the cut that to collect wash-out ratio be 1:3 carries out purifying with preparation HPLC: flow velocity 8mL/min, detects wavelength 254nm, with methanol-water (15-85) wash-out; Collect 17min cut, utilize Sephadex LH-20column to carry out purifying, then carry out purifying through preparation HPLC: flow velocity 8mL/min, detects wavelength 254nm, with methanol-water (15-85) wash-out; Collect 17min cut, obtain target compound.
Adopt HPLC to carry out Purity to the target compound making, purity is greater than 95% sample and uses mass spectrum and nuclear magnetic resonance technique to carry out Structural Identification, and nucleus magnetic resonance is measured with Bruker AVANCE DRX-500NMR Sectrometer, mark in TMS does; High resolution mass spectrum is measured with Agilent6210TOF LC/MS; Electrospray ionization mass spectrum ESI-MS Bruker Esquire3000
plusspectrometer measures.Infrared use NicoletAvatar-360FT-IR infrared spectrometer.
The one-dimensional NMR analytical results of target compound is in Table 1, and two-dimentional NMR analytical results is shown in Fig. 1, and circular dichroism spectrum analytical results is shown in Fig. 2, and nuclear magnetic spectrum is shown in Fig. 3-8, and infared spectrum is shown in Fig. 9.
NMR data (the CDCl of table 1 cyclic peptide compounds
3)
Position | δ C | δ H(J?in?Hz) |
D-phenyllactic?acid | ? | ? |
1 | 169.6,C | ? |
2 | 71.7,CH | 5.49,dd(8.3,6.3) |
3 | 37.6,CH 2 | 3.46,dd(13.7,8.3);3.25, |
4 | 135.9,C | ? |
5/9 | 129.7,CH | 7.34, |
6/8 | 128.7,CH | 7.37,overlap |
7 | 127.3,CH | 7.34,overlap |
anthranilic?acid?I | ? | ? |
10 | 167.1,C | ? |
11 | 127.8,C | ? |
12 | 129.7,CH | 7.63,d(7.6) |
13 | 126.6,CH | 7.30,overlap |
14 | 132.5,CH | 7.50,t(7.6) |
15 | 125.8,CH | 7.22,d(7.6) |
16 | 134.4,C | ? |
NH | ? | 8.07,s |
anthranilic?acid?II | ? | ? |
17 | 167.3,C | ? |
18 | 123.0,C | ? |
19 | 126.2,CH | 7.58,d(7.6) |
[0078]?
20 | 123.1,CH | 7.10,t(7.6) |
21 | 132.3,CH | 7.46,t(7.6) |
22 | 122.0,CH | 8.57,d(7.6) |
23 | 138.2,C | ? |
NH | ? | 10.23,s |
glycine | ? | ? |
24 | 168.1,C | ? |
25 | 51.7,CH 2 | 5.24,d(15.2);3.29,d(15.2) |
26 | 43.3,CH 2 | 3.95,m;3.27,m |
27 | 13.7,CH 3 | 1.02,t,(7.2) |
Through high resolution mass spectrum and electrospray ionization mass spectrum, measure, the molecular weight of target compound is 472.1872, the analytical results of associative list 1, Fig. 1-9, and known this material is cyclic peptide compounds, molecular formula is C
27h
26n
3o
5, structural formula is shown below, and called after clavatustide A determines that 2 absolute configurations are R type:
The anti-tumor activity analysis of embodiment 5 cyclic peptide compounds
HepG2 cell cultures is in containing the RP-MI1640 substratum of 10% calf serum, penicillin 100IU/mL and Streptomycin sulphate 100 g/mL, and every 3d changes liquid 1 time, and every 5d goes down to posterity 1 time.Cell is all placed in 37 ℃.
The HepG2 cell in vegetative period of taking the logarithm, after fully digesting containing 0.25% tryptic PBS liquid, is diluted to 5 × 10 with RPMI1640 substratum
4the single cell suspension of/mL, is inoculated in 96 porocyte culture plates, each concentration multiple cropping 3 holes, every hole 180 μ L; Put dosing after 37 ℃ of incubator incubation 12h, the test liquid of the every Kong Jiahan different concns of medicine group (0.5 μ g/100 μ L, 1 μ g/100 μ L, 2 μ g/100 μ L, 3 μ g/100 μ L, 3.5 μ g/100 μ L, 4 μ g/100 μ L, 5 μ g/100 μ L) clavatustide A, the parallel blank group of establishing, cultivates 48h altogether.
By formula, calculate inhibiting rate and the half-inhibition concentration (IC of compound clavatustide A to growth of tumour cell
50).From experimental result, the IC of this compound to HepG2 cell
50=15 μ g/mL, show that this compound has good antitumor action.
For understanding clavatustide A, suppress the mechanism of HepG2 cell, HepG2 cell is fixing at 4 ℃ with 70% cold ethanol, cell and 0.5mL DNA Prep LPR(Coulter DNA-Prep Reagents kit after fixing, Beckman Coulter, USA) in dark condition, mix 20 minutes, then with flow cytometer, analyze, and with the each phase cell mass of ModFit LT computed in software, calculation result is in Table 2.
Table 2 cell mass in each period composition
? | G1phase | S?phase | G2phase |
Clavatustide?A | 59.17% | 17.73% | 23.10% |
Blank?control | 41.03% | 19.49% | 39.48% |
From table 2, Clavatustide A blocked by the induction HepG2 cell G1 phase, and suppressed G1/S conversion, thereby realized the growth-inhibiting to HepG2 cell.
Get 0.5g cyclic peptide compounds Clavatustide A and mix with 10.5g PEG-4000, heating and melting, moves to after material in dripping pill drip irrigation, and liquid drops in 6~8 ℃ of whiterusss, and oil removing makes 300 of dripping pills.
Embodiment 7 is containing the preparation of cyclic peptide compounds lyophilized injectable powder
Get cyclic peptide compounds Clavatustide A0.5g, glucose 4.5g, Sulfothiorine 0.9g and distilled water 1000mL, after said components mixes, lyophilize, 400 of packing, obtain.
Claims (9)
1. a cyclic peptide compounds clavatustide A, is characterized in that, structural formula is as shown in formula I:
2. an excellent aspergillus, is characterized in that, called after rod aspergillus (Aspergillus clavatus) C2WU, and preserving number is CCTCC No.M2013421.
3. the preparation method of cyclic peptide compounds clavatustide A as claimed in claim 1, is characterized in that, comprises the following steps:
(1) activated excellent aspergillus (Aspergillus clavatus) CCTCC No.M2013421 is inoculated in fermentation culture, carries out fermentation culture;
(2), after having fermented, separate and obtain mycelium and fermented liquid;
Mycelium is placed in to organic solvent lixiviate, obtains vat liquor, then with distilled water to vat liquor suspendible, obtain aqueous suspension, then with ethyl acetate or chloroform, aqueous suspension is extracted, be extracted liquid A, extraction liquid A obtains mycelium extract after concentrated;
Or, fermented liquid is extracted with ethyl acetate or chloroform, be extracted liquid B, extraction liquid B obtains fermentation broth extract after concentrated;
(3) mycelium extract or fermentation broth extract are carried out to chromatographic separation, purification process, make cyclic peptide compounds clavatustide A claimed in claim 1.
4. preparation method as claimed in claim 3, is characterized in that, in step (1), the temperature of described fermentation culture is 20-30 ℃, and the time is 10-60 days.
5. preparation method as claimed in claim 4, is characterized in that, in step (1), described fermentation culture temperature be 22-26 ℃, the time is 15-25 days.
6. preparation method as claimed in claim 3, is characterized in that, in step (2), described organic solvent is one or both in methyl alcohol, ethanol, ethyl acetate and acetone.
7. preparation method as claimed in claim 3, is characterized in that, in step (3), described chromatographic separation is purification on normal-phase silica gel column chromatography for separation, and eluent is petrol ether/ethyl acetate mixed solution.
As claimed in claim 1 cyclic peptide compounds clavatustide A in the application of preparing in antitumor drug or tumor prevention protective foods.
9. application as claimed in claim 8, is characterized in that, described antitumor drug is medicines resistant to liver cancer, and described tumor prevention protective foods is prevention of hcc protective foods.
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