The become mildewed fermented liquid of bag hole bacterium of utilization separates the method for preparing the eburicoic acid compound
Technical field
The present invention relates to the become mildewed fermented liquid of bag hole bacterium of a kind of utilization and separate the method for preparing the eburicoic acid compound.
Background technology
According to document L.Francisco, Q.Jose, R.Augusto, et al., Lanostanoid triterpenes fromLaetiporus sulphureus and apoptosis induction on HL-60 human myeloid leukemiacells.J.Nat.Prod., 2004,67 (12), 2008. record, the compound eburicoic acid is induced people HL-60 bone marrow leukemia cells effect of apoptosis, its IC
50=23-27 μ M, 3 beta-hydroxy acetylize eburicoic acids show the strongest people HL-60 bone marrow leukemia cells effect of apoptosis (IC that induces
50=13-15 μ M).Its chemical structure is as follows:
In view of this compound and acetylate thereof are being induced the fine activity that shows aspect the people HL-60 bone marrow leukemia cells apoptosis, the work that eburicoic acid is developed to the bulk drug of treatment human leukemia medicine has the value of further further investigation.
The bag hole bacterium (Trametes versatillis.Berk) that becomes mildewed is polyporaceae (Polyporaceae) fungi, and sporophore is general medium big, keratin, and 3-7cm * 4-10cm, thick 2-3mm, grey is become mildewed on the surface.The bacterial context near-white, thick 2-3mm.The long 7mm of tube, wall is thin, and nozzle diameter 0.5-1.5mm is polygonal, microscler, grey.Be born on the rotten wood of needle or broad-leaf forest, mainly be distributed in ground such as Hebei, Anhui, Jiangsu, Jiangxi, Hainan, Guangdong, Yunnan.So far, become mildewed bag hole mushroom entity and fermented liquid chemical ingredients all do not appeared in the newspapers.We find that in bag hole bacterium process is become mildewed in research this bacterium is a bacterial classification that well produces eburicoic acid, we utilize conventional separation means that the fermented liquid of the bag hole bacterium that becomes mildewed has been carried out isolation identification, result of study shows that eburicoic acid is a main component, mainly from mycelium (its content account for mycelium dry weight 1%).Result of study shows that this bacterial classification produces mycelia many (every liter of fermented liquid can produce exsiccant mycelium 8 grams), fermentation time short (14 days).Therefore the optimization work that carries on culture medium prescription has very high researching value and using value.
Summary of the invention
The purpose of this invention is to provide the become mildewed fermented liquid of bag hole bacterium of a kind of utilization and separate the method for preparing the eburicoic acid compound.
For achieving the above object, the technical solution used in the present invention is: the become mildewed fermented liquid of bag hole bacterium of a kind of utilization separates the method for preparing the eburicoic acid compound, makes by following step operation: adopt plate to change the method for shaking bottle liquid culture, at first use glucose, peeled potatoes, MgSO
4, KH
2PO
4, VB
1Add water mixing stirring and make substratum; Get fermented liquid in cultivation and fermentation in the dark then, be divided into two parts of fermented liquid and mycelium; Extractive fermentation liquid obtains medicinal extract; The oven dry mycelium obtains medicinal extract with chloroform-methanol extraction; Merge two portions medicinal extract after TLC detects, last column chromatography for separation is with chloroform-methanol system gradient elution; Obtain containing one group of mixture of compound eburicoic acid in chloroform-methanol wash-out part through column chromatography repeatedly; This mixture adopts sherwood oil-acetone wash-out to separate, and obtains the pure product of compound eburicoic acid.
The inventive method can be made by following concrete steps operation: adopt plate to change the method for shaking bottle liquid culture, at first use glucose 15-25g, peeled potatoes 180-250g, MgSO
41-2g, KH
2PO
42.5-3.5g, VB
18-12mg adds water and mixes to 1000ml and make substratum; Then in temperature: 20-32 ℃, rotating speed: under the 150-200r/min condition in the dark after cultivation and fermentation 12-20 days altogether fermented liquid 8-11 liter, be divided into two parts of fermented liquid and mycelium; Fermented liquid obtains 1.8-2.4 gram medicinal extract with ethyl acetate extraction twice; Mycelium weight in wet base 400-450 gram, the heavy 70-85 gram in oven dry back; Mycelium after the oven dry is the chloroform-methanol extraction twice of 30-70: 70-30 with the volume ratio, obtains the medicinal extract of 5.5-6.5 gram altogether; Merge two portions medicinal extract after TLC detects, last silicagel column separates, and is the chloroform-methanol system gradient elution of 100-80: 0-20 with the volume ratio, and every 100ml is a stream part; Obtain containing one group of mixture of compound eburicoic acid in the chloroform-methanol wash-out part that with the volume ratio is 99-90: 1-10 through column chromatography repeatedly; It is sherwood oil-acetone wash-out separation of 75-65: 25-35 that this mixture adopts volume ratio, obtains the pure product of compound eburicoic acid.
Adopt technical solutions according to the invention can solve the raw material sources problem of eburicoic acid for the bulk drug that eburicoic acid is developed to treatment human leukemia medicine, this technology has the culture condition gentleness, the substratum preparation is simple, fermentation time short, output big, be easy to advantage such as scale operation.
Embodiment
Example 1
Adopt plate to change the method for shaking bottle liquid culture, at first use glucose 20g, peeled potatoes 200g, MgSO
41.5g, KH
2PO
43g, VB
110mg adds water and mixes to 1000ml and make substratum; Then in temperature: 25 ℃, rotating speed: under the 170r/min condition in cultivation and fermentation in the dark after 15 days altogether 10 liters of fermented liquids, be divided into two parts of fermented liquid and mycelium; Fermented liquid obtains 2 gram medicinal extract with ethyl acetate extraction twice; Mycelium weight in wet base 420 grams, the oven dry back weighs 80 grams; The exsiccant mycelium extracts twice with chloroform-methanol (volume ratio 40: 60), obtains the medicinal extract of 6 grams altogether; Merge two portions medicinal extract after TLC detects, last silicagel column separates, and is the chloroform-methanol system gradient elution of 100-80: 0-20 with the volume ratio, and every 100ml is a stream part; Obtain containing one group of mixture of compound eburicoic acid in chloroform-methanol (volume ratio 95: 5) wash-out part through column chromatography repeatedly; This mixture adopts sherwood oil-acetone (volume ratio 70: 30) wash-out to separate, and obtains the pure product of compound eburicoic acid.
Example 2
Adopt plate to change the method for shaking bottle liquid culture, at first use glucose 15g, peeled potatoes 180g, MgSO
41.0g, KH
2PO
42.5g, VB
18mg adds water and mixes to 1000ml and make substratum; Then in temperature: 20 ℃, rotating speed: under the 150r/min condition in cultivation and fermentation in the dark after 12 days altogether about 10 liters of fermented liquid, be divided into two parts of fermented liquid and mycelium; Fermented liquid obtains 1.8 gram medicinal extract with ethyl acetate extraction twice; Mycelium weight in wet base 400 grams, the oven dry back weighs 75 grams; The exsiccant mycelium extracts twice with chloroform-methanol (volume ratio 70: 30), obtains the medicinal extract of 5.5 grams altogether; Merge two portions medicinal extract after TLC detects, last silicagel column separates, and is the chloroform-methanol system gradient elution of 100-80: 0-20 with the volume ratio, and every 100ml is a stream part; Obtain containing one group of mixture of compound eburicoic acid in chloroform-methanol (volume ratio 90: 10) wash-out part through column chromatography repeatedly; This mixture adopts sherwood oil-acetone (volume ratio 75: 25) wash-out to separate, and obtains the pure product of compound eburicoic acid.
Example 3
Adopt plate to change the method for shaking bottle liquid culture, at first use glucose 25g, peeled potatoes 220g, MgSO
42.0g, KH
2PO
43.2g, VB
112mg adds water and mixes to 1000ml and make substratum; Then in temperature: 32 ℃, rotating speed: under the 200r/min condition in cultivation and fermentation in the dark after 18 days altogether 12 liters of fermented liquids, be divided into two parts of fermented liquid and mycelium; Fermented liquid obtains 2.5 gram medicinal extract with ethyl acetate extraction twice; Mycelium weight in wet base 450 grams, the oven dry back weighs 82 grams; The exsiccant mycelium extracts twice with chloroform-methanol (volume ratio 65: 35), obtains the medicinal extract of 6.5 grams altogether; Merge two portions medicinal extract after TLC detects, last silicagel column separates, and is the chloroform-methanol system gradient elution of 100-80: 0-20 with the volume ratio, and every 100ml is a stream part; Obtain containing one group of mixture of compound eburicoic acid in chloroform-methanol (volume ratio 92: 8) wash-out part through column chromatography repeatedly; This mixture adopts sherwood oil-acetone (volume ratio 65: 35) wash-out to separate, and obtains the pure product of compound eburicoic acid.
Respectively the pure product of gained compound eburicoic acid in the example 1,2,3 are carried out physics and chemistry and spectral data analysis, the result is as follows:
Eburicoic acid, C
31H
50O
3, colourless needle, m.p.:274-275 ℃, [α]
D 26=+39 ° of (pridine; C=1.0).EI-MS?m/z(%):470(14,[M]
+),455(30),437(100),419(48),55(88)。HR-ESI-MS:470.3712 (calculated value is 470.3759).
1H-NMR(C
5D
5N,400MHz):δ1.03(3H,d,J=7.0Hz,H-26),1.04(3H,d,J=7.0Hz,H-27),1.04(6H,br.s?H-18,H-26),1.08(3H,s,H-19),1.10(3H,s,H-30),1.25(3H,s,H-28),2.29(1H,m,H-25),2.66(1H,t,J=11.3Hz,H-20),3.45(1H,m,H-3),4.90(1H,s,H-31a),4.94(1H,s,H-31b)。
13C-NMR(C
5D
5N,100MHz):δ36.1(t,C-1),28.7(t,C-2),78.0(d,C-3),39.5(s,C-4),50.9(d,C-5),18.7(t,C-6),28.7(t,C-7),135.2(s,C-8),134.3(s,C-9),37.4(s,C-10),21.3(t,C-11),29.4(t,C-12),44.9(s,C-13),49.9(s,C-14),30.9(t,C-15),27.5(t,C-16),47.7(d,C-17),16.3(q,C-18),19.4(q,C-19),49.2(d,C-20),178.6(s,C-21),31.8(t,C-22),32.8(t,C-23),155.9(s,C-24),34.2(d,C-25),22.0(q,C-26),21.9(q,C-27),28.6(q,C-28),16.4(q,C-29),24.5(q,C-30),107.0(t,C-31)。Above data and document T.Tai, A.Akahori, T.Shingu, Triterpenes of Poria cocos.Phytochemistry, record value unanimity in 1993,32,1239..