CN103044515B - A kind of method extracting 17 (21)-hopenen-6,12 beta-diols from aschersonia aleyrodis - Google Patents

Abstract

A kind of open method extracting 17 (21)-hopenen-6,12 beta-diols from aschersonia aleyrodis of the present invention.Comprise the cultivation of meta-bolites, extraction, concentrate to obtain crude extract, crude extract crosses 200 ~ 300 order silicagel columns, with sherwood oil: ethyl acetate gradient, by sherwood oil: ethyl acetate volume ratio 20:1 ~ 15:1 elutriant merges concentrated, utilize 17 (21)-hopenen-6,12 beta-diols that recrystallization obtains in aschersonia aleyrodis.

Description

A method for extracting 17(21)-hoppene-6,12β-diol from Ascospora whitefly

technical field

The invention relates to a method for extracting 17(21)-hoppene-6,12β-diol from Secospora whitefly.

Background technique

Aschersonia aleyrodis belongs to Ascomycota, Copromycetes , Hypocreales, Ergotaceae, Ascheria genus, is an important entomogenic fungus, mainly parasitic on whitefly, its spores It has strong whitefly killing activity and has been made into a commercial insecticide. The metabolites also have activities such as killing whitefly and aphids, and have broad-spectrum antibacterial properties, and the metabolites are not affected by seasons, temperature, and rain. And so on, has a good application prospect.

With the gradual research on the bacterium, some compounds have been isolated from its metabolites and have certain activities, such as triterpenoids zeoxaline and dustanin have anti-tuberculosis activity, anthraquinones fine Penicillin and quinone acanthin have cytotoxic activity, 17(21)-hoppene-6,12β-diol is a five-membered ring triterpenoid with molecular formula C ₃₀ H ₅₀ O ₂ , has anti-plasmodium activity, IC ₅₀ is 15μM, the substance has potential medical value.

Contents of the invention

The object of the present invention is to disclose a method for extracting 17(21)-hopene-6,12β-diol {17(21)-hopene-6,12β-diol} from Ascospora whitefly, with To promote research and development in the field of medicine.

Technical scheme of the present invention:

The invention discloses a method for extracting 17(21)-hoppene-6,12β-diol from Ascospora whitefly, comprising the following steps:

1) After activation of Ascospora whitefly (Qiu Junzhi et al., Preliminary study on the infestation behavior of the entomopathogenic fungus Ascospora whitefly to whitefly whitefly. Acta Mycophyta Sinica, 2004, 23(1):115～121), taken from Bacterial blocks were inoculated into M102 medium, cultured continuously for 5-10 days under the conditions of 120-160r/min, 20-28°C, namely primary culture, and then transferred to M102 medium again according to 8%-10% inoculum amount. The secondary metabolites can be obtained after static culture at 20-28°C for 30-40 days.

2) Bacterial liquid and mycelium are filtered and separated, mycelium: methanol volume ratio = 1: 5~10, soaked for 24~48 hours, the soaking solution is concentrated to dryness by rotary evaporation, methanol is recovered, and water with one volume of mycelium is used Dissolve the spin-dried product, extract the water-soluble product and ethyl acetate at a volume ratio of 1:1~1:3 in a shaker flask, shake for 30~60 minutes and stand still for 30~60 minutes, then concentrate part of the ethyl acetate to obtain an extract.

3) Put the extract on a 200-300-mesh silica gel column, elute with a gradient of petroleum ether:ethyl acetate volume ratio of 70:1 to 1:1, combine the eluent with a volume ratio of 20:1 to 15:1, and evaporate to dry naturally , 17(21)-Hoppene-6,12β-diol was obtained by recrystallization.

The preparation method of the M102 medium is as follows, take 30 g of sucrose, 20 g of malt extract, 2.0 g of peptone, 1.0 g of yeast extract, 0.5 g of KCl, 0.5 g of MgSO ₄ , 0.5 g of KH ₂ PO ₄ , mix well and add Distilled water to 1L, divided into 250ml Erlenmeyer flasks, 100ml per bottle, sterilized at 121℃ for 20min under high pressure.

The structural formula of the 17(21)-hoppene-6,12β-diol is as follows (molecular formula C ₃₀ H ₅₀ O ₂ ):

Significant advantage of the present invention:

1) It is convenient to obtain materials

2) The process is simple and easy to operate

3) Low cost and high purity.

4) Promote the research and development of 17(21)-Hoppene-6,12β-diol in the field of medicine.

Description of drawings

Fig. 1 is the hydrogen spectrum of 17(21)-hopene-6,12β-diol;

Figure 2 is the carbon spectrum of 17(21)-hoppene-6,12β-diol.

Detailed ways

The preparation method of M102 medium in the following examples is as follows: take 30 g of sucrose, 20 g of malt extract, 2.0 g of peptone, 1.0 g of yeast extract, 0.5 g of KCl, 0.5 g of MgSO ₄ , 0.5 g of KH ₂ PO ₄ , and mix After homogeneity, add distilled water to make it up to 1L, divide into 250ml Erlenmeyer flasks, each bottle contains 100ml, and sterilize under high pressure at 121°C for 20min.

Example 1

1) After the activation of Ascospora whitefly, take the bacterial block and inoculate it into the M102 medium, and cultivate it continuously for 8 days under the conditions of 150r/min and 25°C, which is the primary culture, and transfer it to the M102 medium again according to the inoculation amount of 9%. Secondary metabolites were obtained by static culture at 25°C for 38 days.

2) Bacterial liquid and mycelium were separated by filtration, mycelium: methanol volume ratio = 1: 8 soaked for 36 hours, the soaking liquid was concentrated to dryness by rotary evaporation, methanol was recovered, and the spin-dried product was dissolved in water with one volume of mycelium. Extract the water-soluble matter and ethyl acetate in a shaker flask at a volume ratio of 1:2.5, shake for 50 minutes and rest for 50 minutes, and concentrate the ethyl acetate part to obtain the extract.

3) Put the extract on a 200-300-mesh silica gel column, elute with a gradient of petroleum ether:ethyl acetate volume ratio of 70:1 to 1:1, combine the eluent with a volume ratio of 20:1 to 15:1, and evaporate to dry naturally , 17(21)-Hoppene-6,12β-diol product was obtained by recrystallization.

Extraction rate% = weight of pure 17(21)-hopaene-6,12β-diol/total amount of extract x 100%=4.18%.

The hydrogen spectrum and carbon spectrum analysis of the above-mentioned recrystallized product are carried out. The hydrogen spectrum and carbon spectrum of the above-mentioned recrystallized 17(21)-Hoppene-6,12β-diol are shown in Fig. 1 and Fig. 2, which proves that the substance is 17(21)-Hoppene-6,12β-diol.

Example 2

1) After activation of Ascospora whitefly, take the bacterial block and inoculate it into the M102 medium, and cultivate it continuously for 5 days under the conditions of 120r/min and 20°C, which is the primary culture, and transfer it to the M102 medium again according to the inoculation amount of 8%. Secondary metabolites were obtained by static culture at 20°C for 30 days.

2) Bacterial liquid and mycelium are filtered and separated, mycelium: methanol volume ratio = 1:5 and soaked for 24 hours, the soaking solution is concentrated to dryness by rotary evaporation, methanol is recovered, and the spin-dried product is dissolved in water with one volume of mycelium. The water-soluble matter and ethyl acetate were extracted in a shaker flask at a volume ratio of 1:1, shaken for 30 minutes and stood still for 30 minutes, and the ethyl acetate was partially concentrated to obtain an extract.

3) Put the extract on a 200-300 mesh silica gel column, elute with a gradient of petroleum ether:ethyl acetate volume ratio of 70:1 to 1:1, combine the eluent with a volume ratio of 20:1 to 15:1, and evaporate to dryness naturally , 17(21)-Hoppene-6,12β-diol product was obtained by recrystallization.

Extraction rate% = weight of pure 17(21)-hopaene-6,12β-diol/total amount of extract × 100% = 3.32%.

The hydrogen spectrum and carbon spectrum analysis of the above-mentioned recrystallized product are carried out. The hydrogen spectrum and carbon spectrum of the above-mentioned recrystallized 17(21)-Hoppene-6,12β-diol are shown in Fig. 1 and Fig. 2, which proves that the substance is 17(21)-Hoppene-6,12β-diol.

Example 3

1) After activation of Ascospora whitefly, take the bacterial block and inoculate it into the M102 medium, and culture it continuously for 10 days under the conditions of 160r/min and 28°C, which is the primary culture, and transfer it to the M102 medium again according to 10% inoculum amount Secondary metabolites were obtained by static culture at 28°C for 40 days.

2) Bacterial liquid and mycelium were separated by filtration, mycelium: methanol volume ratio = 1:10 and soaked for 48 hours, the soaking liquid was concentrated to dryness by rotary evaporation, methanol was recovered, and the spin-dried product was dissolved in water with one volume of mycelium. The water-soluble matter and ethyl acetate were extracted in a shaker flask at a volume ratio of 1:3, shaken for 60 minutes and rested for 60 minutes, and the ethyl acetate was partially concentrated to obtain an extract.

3) Put the extract on a 200-300 mesh silica gel column, elute with a gradient of petroleum ether:ethyl acetate volume ratio of 70:1 to 1:1, combine the eluent with a volume ratio of 20:1 to 15:1, and evaporate to dryness naturally , 17(21)-Hoppene-6,12β-diol product was obtained by recrystallization.

Extraction rate% = weight of pure 17(21)-hopaene-6,12β-diol/total amount of extract × 100%=3.56% .

The hydrogen spectrum and carbon spectrum analysis of the above-mentioned recrystallized product are carried out. The hydrogen spectrum and carbon spectrum of the above-mentioned recrystallized 17(21)-Hoppene-6,12β-diol are shown in Fig. 1 and Fig. 2, which proves that the substance is 17(21)-Hoppene-6,12β-diol.

Claims (1)

1. one kind is extracted the method for 17 (21)-hopenen-6,12 beta-diols from aschersonia aleyrodis, it is characterized in that: the concrete steps of described method are as follows:

A: after aschersonia aleyrodis activation, getting bacterium block is inoculated in M102 substratum, cultured continuously i.e. elementary cultivation in 5 ~ 10 days under 120 ~ 160r/min, 20 ~ 28 DEG C of conditions, again be transferred in M102 substratum by 8% ~ 10% inoculum size, under 20 ~ 28 DEG C of conditions, static gas wave refrigerator 30 ~ 40 days, obtains secondary metabolite;

B: bacterium liquid and mycelium filtering separation, mycelium: methyl alcohol volume ratio=1:5 ~ 10 are soaked 24 ~ 48 hours, soak solution concentrated by rotary evaporation is to dry, reclaim methyl alcohol, thing is spin-dried for by the water dissolution of 1 times of mycelium volume, by the water solubles and ethyl acetate by volume 1:1 ~ 1:3 carry out shaking flask extraction, shake the static 30 ~ 60min of 30 ~ 60min, to obtain medicinal extract by concentrated for ethyl acetate portion;

C: 200 ~ 300 order silicagel columns on medicinal extract, with sherwood oil: ethyl acetate volume ratio 70:1 to 1:1 gradient elution, merges the elutriant of volume ratio 20:1 to 15:1, naturally volatilizes, utilize recrystallization to obtain 17 (21)-hopenen-6,12 beta-diols;

The structural formula of described 17 (21)-hopenen-6,12 beta-diols is as follows:

The preparation method of described M102 substratum is as follows: get sucrose 30g, malt extract 20 g, peptone 2.0 g, yeast extract 1.0 g, KCl 0.5 g, MgSO ₄0.5 g, KH ₂pO ₄0.5 g, adds single water that steams and is settled to 1L, be dispensed in 250ml triangular flask, every bottled 100ml, sterilizing 20min under 121 DEG C of high pressure after mixing.