CN1243100C - Method for production of cytosporasp B and the use in preparation of anti-tumor and antifungal medicine - Google Patents

Abstract

The present invention relates to a Cytospora bacterin B compound from endogeny fungus-Dothiorella HTF3 in a mangrove plant, a preparation method thereof and an application in the preparation of anti-tumor drugs and anti-fungus drugs. The method comprises the steps that the seeds of the endogeny fungus are cultured and the endogeny fungus is fermented; the fermentation culture solution is filtered to remove fermentation solution so as to obtain a thallus; after the thallus is dried, the thallus is repeatedly soaked; acetic acid and ethyl ester are mixed through pressure reduction and concentration, chromatographic separation and gradient elution to collect eluent with the proportion of 1/1; when pressure is reduced, the eluent is concentrated to obtain the compound. The Cytospora bacterin B compound can be used for preparing the anti-tumor drugs and the anti-fungus drugs, has the advantages of high yield and 9 mg/g of thallus by dry weight, and can be used for industrial production.

Description

Cytosporasp B method for making and the application in preparing antitumor and antifungal drug

(1) technical field

The present invention relates to a kind of mangrove endogeny eumycete one Dothiorella ribis HTF that derives from ₃(Dothiorella sp.HTF ₃) the preparation method and the application in the antitumor and antifungal drug of preparation thereof of cytosporasp B compound.

(2) background technology

Mangrove forest is to be grown in the woody plant community, xylium that the geographic mud flat in estuary is coated, and it is special to play a part in balance of nature as the extra large Lu Bianyuan ecosystem of uniqueness.Mangrove plant is not only important economic plants, also is medicinal plant commonly used among the people, can be used to control blood urine as the bark extractive of mangrove acanthus (Acanthus ilicifolius); The bark of Common Ceriops (Ceriops tagal) is smashed to pieces can hemostasis, sore is disliked in defaecation and treatment, and the seed oil expression can antipruritic and treatment tinea, also can treat pernio etc.

Exist abundant endogenetic fungus in the mangrove, there is report to think that the medicinal ingredients of mangrove is all produced by its endogenetic fungus, few document (1, Brady, S., Wagenaar, M.W., Singh, M., et al.The cytosporonesisolated from an endophytic fungus.Org.Lett.2000,2 (25): 4043-4046; 2, Bandaranayake, W.M..Traditional and medicinal uses of mangroves.Mangroves and Salt Marshes, 1998,2:133-148; 3, Bandaranayake, W.M..Bioactivities, bioactive compounds and chemicalconstituents of mangrove plants.Wetland Ecology and Management, 2002,10:421-452; 4, Lin Y., Wu X., Deng z., et al.The metabolites of the mangrove fungus Verruculina enalia No.2606 from a salt lake in the Bahamas.Phytochemistry, 2002,59:469-471) the report endogenetic fungus that also proved mangrove can produce the compound with new texture and strong physiologically active really.But domestic and international at present work in this respect just just begins, and its research has high theoretical value and actual application value for this reason.

(3) summary of the invention

The object of the present invention is to provide a kind of separating and extracting method and its application in preparation antitumor drug and antifungal drug that derives from the compound of mangrove endogeny eumycete (cytosporone B).

Mangrove endogeny eumycete one Dothiorella ribis HTF3 (the Dothiorella sp.HTF that the present invention is used ₃) being preserved in Chinese typical culture collection center (CCTCC, Chinese Wuhan University are in the school), preserving number is CCTCC NO:M203067, preservation date is on August 29th, 2003.

The structural formula of the said cytosporasp B of the present invention (cytosporone B) compound is:

(3,5-dihydroxyl-methyln-hexyl ketone base)-Phenylacetic acid ethylester

The preparation method of said cytosporasp B compound is:

1) endogenetic fungus (Dothiorella sp.HTF ₃) seed culture:

Substratum is unit with g, peeling potatoes, chopping 20, and poach adds glucose 1～3 in filtrate filtered, agar 1.5～2.0, seawater is settled to 100ml, and the test tube slant is made in sterilization, and the picking bacterial classification inserts the inclined-plane, cultivates under the room temperature 5～15 days;

2) fermentation culture of endogenetic fungus:

Fermention medium is unit with g: peeling potatoes, chopping 20, and poach adds glucose 1～3 in filtrate filtered, and seawater is settled to 100ml, and sterilization chooses cultured bacterial strain on the inclined-plane into fermention medium, cultivates under the room temperature 5～15 days;

3) after being removed by filter fermented liquid, above-mentioned fermentation culture obtains thalline;

4) thalline oven dry, soak repeatedly with ethyl acetate, combined ethyl acetate, concentrating under reduced pressure, the paste of gained separates with silica gel column chromatography, and gradient elution is carried out to 0/100 in petrol ether/ethyl acetate=100/0, collects the two ratio and be 1/1 o'clock elutriant, concentrating under reduced pressure promptly obtains this compound, and content is the 9mg/g dry cell weight.

The experimental data of this compound of gained is:

Colorless oil; EMS:m/z (rel.int.): 323.3[M+H] ⁺(100), 345.3[M+Na] ⁺(11.4), 361.2[M+K] ⁺(4.2); ¹H NMR and ¹³C NMR (400MHz, CDCl ₃, ppm) see Table 1.

The said cytosporasp B compound of the present invention can be used for preparing antitumor drug, and its anti-tumor activity test experience is as follows:

1) tumor cell line

People B Lymphoma Raji Cells, human oral dermoid cancer KB cell, people's osteogenic sarcoma MG-63 cell, people's cancer A549 cell, human cervical carcinoma hela cell etc.

The NMR data of table 1. cytosporasp B

?Position	? 1H-NMR	Position	? 13C-NMR
				?2	?3.70(s)	1	?171.0
?4	?6.25(s)	2	?41.8
				?6	?6.25(s)	3	?136.0
?9		4	?112.0
				?10	?2.75(t)	5	?163.1
?11	?1.6	6	?103.2
				?12	?1.22	7	?161.0
?13	?1.19	8	?116.0
				?14	?1.20	9	?207
?15	?1.20	10	?43.7
				?16	?0.8	11	?25.0
?17	?4.2(q，7)	12	?29.4
				?18	?1.20(t，7)	13	?29.2
?OH-5		14	?31.8
				?OH-7		15	?22.8
		16	?14.2
						17	?61.0
		18	?14.1

2) material

A MTT (tetrazolium bromide):

Phosphate buffered saline buffer (PBS) dissolving MTT (Thiazoyl blue) with 0.01mol/L arrives final concentration 5mg/ml, and 0.22 μ m filtering with microporous membrane degerming is kept in Dark Place in 4 ℃ after the packing;

B SDS lysate:

100g sodium laurylsulfonate (SDS), 1N HCL10ml, heating for dissolving, distilled water is settled to 1000ml.

C cell culture medium (full training):

One bag of 10g dry powder RMPI 1640 (Gibco Co.Ltd.) cell culture medium is dissolved in the 1L distilled water; Add 2gNaHCO ₃Seal after stirring evenly dissolving, place 4 ℃ to spend the night, remove impurity with natural sedimentation; Add next day 10%-15% deactivation (56 ℃, calf serum 30min) and more than 1% anti-mother liquors; Behind the mixing with the membrane filtration degerming in 0.22 μ m aperture.

3) configuration of compound c ytosporone B

Get a certain amount of cytosporasp B, with dissolve with methanol and to adjust concentration be 5mg/ml, the membrane filtration degerming in 0.22 μ m aperture, 4 ℃ of preservations are standby.

4) cultivation of tumour cell

The a cell activation:

Get a clean beaker, the clean warm water of packing into, water temperature transfers to 37～40 ℃; Frozen pipe is taken out the rapid warm water that drops into thaw from liquid nitrogen, and the freeze-stored cell access is equipped with in the culturing bottle of cell culture medium in advance, at 37 ℃, 5%CO ₂, 100% humidity condition under cultivate, the observation of cell growing state is in time changed nutrient solution, is divided bottle.

The b cell counting:

Select logarithm to generate the phase cell, trysinization moves in the centrifuge tube, adds full the training to 10ml, gets one after another drop ofly to go in the tally one side groove, and inverted microscope is counting down.Adjust cell count to 1 * 10 ⁵/ ml.

C is active to be detected:

1. 96 orifice plates shine 1h under the UV-light in Bechtop;

2. in each hole, add cell suspension 80 μ l, 37 ℃, 5%CO ₂, 100% humidity condition under cultivate 24h;

3. add 20 μ l with training the solution that gradient dilution becomes a series of concentration entirely, continue to cultivate 48h;

4. every hole adds MTT solution 10 μ l, jolts gently to make the particle dissolving, places 3h for 37 ℃;

5. take out culture plate, every hole adds 10%SDS solution 100 μ l, and 37 ℃ of dissolvings are spent the night;

6. measure each hole light absorption value (reference wavelength is 655nm) with integrated enzyme reaction instrument 570nm, calculate inhibiting rate by following formula:

Inhibiting rate=(control group OD value-experimental group OD value)/control group OD value * 100%

7. be ordinate zou with the inhibiting rate, the logarithm of given the test agent concentration is X-coordinate mapping, and the concentration of obtaining inhibiting rate and be at 50% o'clock is IC ₅₀

The d test-results:

Cytosporone B is to the IC of people B Lymphoma Raji Cells, human oral dermoid cancer KB cell, people's osteogenic sarcoma MG-63 cell, people's lung cancer A549 cell, human cervical carcinoma hela cell ₅₀Be respectively 62.5ng/ml, 4 μ g/ml, 1 μ g/ml, 4 μ g/ml and 1 μ g/ml.

The said cytosporasp B compound of the present invention can be used for preparing antifungal drug, and its anti-mycotic activity test experience is as follows:

1) fungi indicator strain:

Saccharomyces albicans, black-koji mould, Trichoderma, sickle-like bacteria, Alternaria bacterium, white bread mould, cladosporium sp

2) configuration of compound c ytosporone B

Get a certain amount of cytosporone B, with dissolve with methanol and to adjust concentration be 1mg/ml, the membrane filtration degerming in 0.22 μ m aperture, 4 ℃ of preservations are standby.

3) the active detection: (mensuration of minimum inhibitory concentration MIC)

Each indicator that a will grow on PDA (potato-glucose-agar) slant medium with sterilized water washes, and making spore concentration is 10 ⁷The bacteria suspension of individual/ml, 4 ℃ of preservations are standby;

The sample solution of b cytosporone B is pressed double dilution method, is diluted to a series of concentration gradient with methyl alcohol, gets 20 μ l and joins in 96 orifice plates that shine 30min under the ultraviolet lamp of Bechtop.Blower fan air blast with Bechtop dries up methyl alcohol;

C fungi suspension is diluted to 10 with PD (potato-glucose) substratum ⁴The bacterium liquid of individual/μ l is got 100 μ l and is joined on above-mentioned 96 orifice plates, cultivates 48h for 25 ℃, naked-eye observation, and the minimum weaker concn that does not have fungi to generate is MIC.

The d experimental result:

Cytosporone B to the MIC of saccharomyces albicans, black-koji mould, Trichoderma, sickle-like bacteria, Alternaria bacterium, white bread mould, cladosporium sp is respectively: 0.156mg/ml, 0.125mg/ml, 0.0625mg/ml, 0.0625mg/ml, 0.07mg/ml, 0.125mg/ml and 1mg/ml.

In sum, the invention provides the preparation method that a class derives from the compound of mangrove endophytic fungus, the output height reaches the 9mg/g dry cell weight, can be used for industrial production; This compound all shows good inhibitory effect to multiple human body tumour cell, can be used for preparing the medicine of anti-this class tumour; This compound also has good inhibitory effect to human body cause illness fungi and phytopathogenic fungi, can be used for preparing this class antifungal drug.

(4) embodiment

Embodiment 1

Following examples provide the preparation method of said cytosporasp B compound.

1) seed culture of endogenetic fungus:

With 20g peeling potatoes, chopping, poach 30min adds glucose 1g in filtrate filtered, agar 1.6g, and 50% seawater is settled to 100ml, and the test tube slant is made in sterilization, and the picking bacterial classification inserts the inclined-plane, cultivates 10 days down for 25 ℃;

2) fermentation culture of endogenetic fungus:

With 20g peeling potatoes, chopping, poach 30min adds glucose 1.5g in filtrate filtered, and 50% seawater is settled to 100ml, and sterilization chooses cultured bacterial strain on the inclined-plane into fermention medium, cultivates 8 days down for 26 ℃;

3) after being removed by filter fermented liquid, above-mentioned fermentation culture obtains thalline;

4) thalline oven dry, soak repeatedly with ethyl acetate, combined ethyl acetate, concentrating under reduced pressure, the paste of gained separates with silica gel column chromatography, and gradient elution is carried out to 0/100 in petrol ether/ethyl acetate=100/0, collects the two ratio and be 1/1 o'clock elutriant, concentrating under reduced pressure promptly obtains this compound, and content is the 9mg/g dry cell weight.

Embodiment 2

With 20g peeling potatoes, chopping, poach 30min adds glucose 1.5g in filtrate filtered, agar 1.8g, and 50% seawater is settled to 100ml, and the test tube slant is made in sterilization, and the picking bacterial classification inserts the inclined-plane, cultivates 8 days down for 30 ℃; With 20g peeling potatoes, chopping, poach 30min adds glucose 2.5g in filtrate filtered again, and 50% seawater is settled to 100ml, and sterilization chooses cultured bacterial strain on the inclined-plane into fermention medium, cultivates 15 days down for 20 ℃; Obtain thalline after above-mentioned fermentation culture removed by filter fermented liquid; Soak repeatedly with ethyl acetate thalline oven dry back, combined ethyl acetate, concentrating under reduced pressure, the paste of gained separates with silica gel column chromatography, gradient elution is carried out to 0/100 in petrol ether/ethyl acetate=100/0, collect the two ratio and be 1/1 o'clock elutriant, concentrating under reduced pressure promptly obtains this compound, and content is the 9mg/g dry cell weight.

Embodiment 3

With 20g peeling potatoes, chopping, poach 30min adds glucose 2.0g in filtrate filtered, agar 1.5g, and 50% seawater is settled to 100ml, and the test tube slant is made in sterilization, and the picking bacterial classification inserts the inclined-plane, cultivates 15 days down for 28 ℃; With 20g peeling potatoes, chopping, poach 30min adds glucose 2g in filtrate filtered again, and 50% seawater is settled to 100ml, and sterilization chooses cultured bacterial strain on the inclined-plane into fermention medium, cultivates 10 days down for 25 ℃; Obtain thalline after above-mentioned fermentation culture removed by filter fermented liquid; Soak repeatedly with ethyl acetate thalline oven dry back, combined ethyl acetate, concentrating under reduced pressure, the paste of gained separates with silica gel column chromatography, gradient elution is carried out to 0/100 in petrol ether/ethyl acetate=100/0, collect the two ratio and be 1/1 o'clock elutriant, concentrating under reduced pressure promptly obtains this compound, and content is the 9mg/g dry cell weight.

Embodiment 4

With 20g peeling potatoes, chopping, poach 30min adds glucose 2.5g in filtrate filtered, agar 2.0g, and 50% seawater is settled to 100ml, and the test tube slant is made in sterilization, and the picking bacterial classification inserts the inclined-plane, cultivates 12 days down for 20 ℃; With 20g peeling potatoes, chopping, poach 30min adds glucose 1g in filtrate filtered again, and 50% seawater is settled to 100ml, and sterilization chooses cultured bacterial strain on the inclined-plane into fermention medium, cultivates 12 days down for 30 ℃; Obtain thalline after above-mentioned fermentation culture removed by filter fermentation liquid; Soak repeatedly with ethyl acetate thalline oven dry back, combined ethyl acetate, concentrating under reduced pressure, the paste of gained separates with silica gel column chromatography, gradient elution is carried out to 0/100 in petrol ether/ethyl acetate=100/0, collect the two ratio and be 1/1 o'clock elutriant, concentrating under reduced pressure promptly obtains this compound, and content is the 9mg/g dry cell weight.

Embodiment 5

With 20g peeling potatoes, chopping, poach 30min adds glucose 3.0g in filtrate filtered, agar 1.7g, and 50% seawater is settled to 100ml, and the test tube slant is made in sterilization, and the picking bacterial classification inserts the inclined-plane, cultivates 5 days down for 23 ℃; With 20g peeling potatoes, chopping, poach 30min adds glucose 3.0g in filtrate filtered again, and 50% seawater is settled to 100ml, and sterilization chooses cultured bacterial strain on the inclined-plane into fermention medium, cultivates 5 days down for 28 ℃; Obtain thalline after above-mentioned fermentation culture removed by filter fermented liquid; Soak repeatedly with ethyl acetate thalline oven dry back, combined ethyl acetate, concentrating under reduced pressure, the paste of gained separates with silica gel column chromatography, gradient elution is carried out to 0/100 in petrol ether/ethyl acetate=100/0, collect the two ratio and be 1/1 o'clock elutriant, concentrating under reduced pressure promptly obtains this compound, and content is the 9mg/g dry cell weight.

Claims (1)

1, the preparation method of cytosporasp B compound is characterized in that the steps include:

1) seed culture of endogenetic fungus-Dothiorella ribis CCTCC NO:M203067:

Peeling potatoes, chopping 20g, poach adds glucose 1～3g in filtrate filtered, agar 1.5～2.0g, seawater is settled to 100ml, and the test tube slant is made in sterilization, and the picking bacterial classification inserts the inclined-plane, cultivates under the room temperature 5～15 days;

2) fermentation culture of endogenetic fungus-Dothiorella ribis CCTCC NO:M203067:

Peeling potatoes, chopping 20g, poach adds glucose 1～3g in filtrate filtered, and seawater is settled to 100ml, and sterilization chooses cultured bacterial strain on the inclined-plane into fermention medium, cultivates under the room temperature 5～15 days;

3) after being removed by filter fermented liquid, above-mentioned fermentation culture obtains thalline; With

4) thalline oven dry, soak repeatedly with ethyl acetate, combined ethyl acetate, concentrating under reduced pressure, the paste of gained separates with silica gel column chromatography, and gradient elution is carried out to 0/100 in petrol ether/ethyl acetate=100/0, collects the two ratio and be 1/1 o'clock elutriant, concentrating under reduced pressure promptly obtains this compound, and content is the 9mg/g dry cell weight.