CN103044510B - The isolation technique of ergosterol in Phellinus bacterium - Google Patents

Abstract

Do you the invention discloses a kind of Phellinus bacterium (phelliuns igniarius <i>Phellinus igniarius</i> (L ex Fr) Quel, phellinus linteus <i>phellinus linteus</i> (Berk et Curt) Teng Phellinus baumii, breathe out base of a fruit phellinus <i>Phellinus hartigii</i> (Allesch et Schnabl) Imaz) separation method of ergosterol in.First Phellinus bacterium crude extract is prepared; then carry out purification on normal-phase silica gel chromatography, adopt sherwood oil and acetone gradient elution, carry out TLC detection; reuse purification on normal-phase silica gel chromatography; then be methanol gel chromatography, after PLC detects, carry out reversed-phase silica gel chromatography; suitable merging elutriant; drying under reduced pressure, carries out methanol gel chromatography again, both obtains ergosterol.

Description

Separation technology of ergosterol from Phellinus fungus

technical field

The invention belongs to the field of bioengineering pharmacy.

Background technique

Phellinus (Phellinus), fruiting body sessile, cap flat hemispherical or horseshoe-shaped, 2-12*3-21 cm, 1.5-10 cm thick, woody, light liver brown to dark gray or black, often cracked when old, No cortex, with fine hairs at the beginning, then become glabrous, with concentric ring ribs, blunt edges, dark cinnamon to light brown, no hymenium on the lower side. Dark brown flesh, hard, woody. Tubes and flesh Nearly the same color, multi-layered, but the layers are not obvious, the old tube layer is filled with white hyphae. The tube mouth is rusty brown to caramel, round, 4-5 per mm. The spores are nearly spherical, smooth, colorless, 5- 6*3-4 microns. Setae are sharp at the top and swollen at the base, 10-25*5-7 microns. Mycelia are unbranched, without septa, 3-5 microns in diameter.

At present, there are many purification methods for fungal products, mainly including fractional precipitation, preparative high performance liquid chromatography, column chromatography, ultrafiltration, centrifugal precipitation chromatography, and several other methods. Among them, column chromatography is the most widely used method, which is divided into two categories: one is gel column chromatography with only molecular sieve effect, and commonly used gels include dextran gel and agarose gel. The second is ion exchange chromatography. However, it is mainly used to separate polysaccharides, hyaluronic acid, etc., and most of the products obtained after separation are mixtures. The present invention combines multiple chromatographic techniques and has high separation degree, and the pure product can be refined by applying the present invention.

Contents of the invention

The invention discloses a method for separating ergosterol from Phellinus fungus. First prepare the crude extract of Phellinus flavinus, then carry out normal phase silica gel chromatography, adopt petroleum ether and acetone gradient elution, carry out TLC detection, use normal phase silica gel chromatography again, then methanol gel chromatography, after PLC detection , carry out reverse-phase silica gel chromatography, combine the eluents appropriately, dry under reduced pressure, and carry out methanol gel chromatography again to obtain 5,7,22-triene-3-ergosterol.

Technical scheme of the present invention is as follows:

Phellinus crude extract, prepared by the following method:

(1) The fermentation medium formula is in grams/100 milliliters:

Corn starch 1-5% Glucose 1-5%

Peptone 0.1-0.5% Yeast Extract 0.1-0.5%

Magnesium sulfate 0.1-0.5% Potassium dihydrogen phosphate 0.01-0.05%

(2) Inoculate the Phellinus bacterium into the Erlenmeyer flask that liquid culture medium is housed in conventional method, with 20～35 ℃ of temperature, shake flask rotating speed is 80～280r/min, under the condition of pH3～8, vibration culture 7～ 15 days; when the pH value drops to 2.5-4 during cultivation, inoculate the seeds in the shaker flask into the culture medium of a 50L fermenter at a temperature of 20-35°C, a pressure of 0.1-0.2 kg/cm2 in the fermenter, and a pH of 3 ～8, under the conditions of ventilation volume 0.5～1.1vvm, stirring speed 100～280 rpm, culture for 7～15 days, the Phellinus crude extract can be prepared by using Phellinus mycelia fermentation whole liquid;

(3) Take the Phellinus mycelia fermentation whole liquid obtained in step (2), and concentrate it under reduced pressure in a conventional manner, so that its volume is concentrated to 1/2 to 1/5 of the original volume;

(4) Extract the concentrated fermented liquid of the above step (3) with ethanol that is 60 to 95% by volume, wherein the amount of ethanol added is 2 to 5 times the volume of the concentrated solution, so that the concentration of ethanol in the extract can be Reach 60-90%;

(5) Heat the extract obtained in step (4) at 50-70°C for 1-2 hours; separate by conventional methods, and remove impurities by secondary filtration to separate and obtain the ethanol extract; the above-mentioned ethanol extract Concentrate under reduced pressure in a conventional manner, so that its volume is concentrated to 1/5 to 1/10 of the original volume;

(6) Dry the concentrated solution obtained in the step (5) by a low-temperature freeze-drying method to obtain a crude Phellinus extract.

Method for isolating ergosterone:

Phellinus crude extract → normal phase silica gel chromatography → chloroform and methanol gradient elution → TLC detection → normal phase silica gel chromatography → methanol gel chromatography → TLC detection → reversed phase silica gel chromatography → TLC detection → methanol gel Chromatography → evaporate to dryness under reduced pressure → 5,7,22-triene-3-ergosterol.

The specific method is:

(1) preparing Phellinus crude extract;

(2) Mix the crude extract obtained in the above step (1) with normal-phase silica gel, mix and stir, and dry, perform silica gel normal-phase column chromatography, and elute with eluent for 2-7 times;

(3) Collect the last eluate in step (2), dry it under reduced pressure, mix the sample with an equal volume of silica gel, perform normal phase silica gel chromatography again, and elute with an eluent;

(4) Collect the eluate obtained in the above step (3), concentrate under reduced pressure, dissolve in methanol, perform methanol gel column chromatography, elute with eluent, use TLC to detect the collected eluate, and combine the eluents appropriately Deliquification, drying under reduced pressure;

(5) The product obtained in step (4) is subjected to reverse-phase silica gel chromatography, and the eluent is methanol and water;

(6) The product obtained in step (5) is subjected to TLC detection, and the eluent is appropriately combined, dried under pressure, and subjected to methanol gel chromatography again, and eluted with an eluent;

(7) Collect the eluate obtained in step (6), detect by TLC, and dry under pressure, which is ergosterol.

The present invention has the remarkable advantages of the separation technology of ergosterol in Phellinus bacterium: the method adopts a combination of multiple chromatography techniques, and can be refined to ergosterol with a clear structure and a purity greater than 95%. The technical route is mature and clear, efficient and precise.

(4) Description of drawings

Fig. 1 is the structural formula of 5,7,22-triene-3-ergosterol of the present invention;

Figure 2 is the one-dimensional NMR H spectrum of 5,7,22-triene-3-ergosterol.

(5) Specific implementation methods

Example 1:

Phellinus crude extract, prepared by the following method:

(1) The fermentation medium formula is in grams/100 milliliters:

Corn Starch 1% Glucose 1%

Peptone 0.1% Yeast Extract 0.1%

Magnesium sulfate 0.1% Potassium dihydrogen phosphate 0.01%

(2) Phellinus bacterium classification is inoculated in the Erlenmeyer flask that liquid culture medium is housed with 25 ℃ of temperature with 25 ℃ of temperature, and the rotating speed of shaking flask is 110r/min, under pH7 condition, vibrating culture 7 days; When the temperature drops to 3, inoculate the seeds in the shake flask into the culture solution of a 50L fermenter at a temperature of 25°C, a fermenter pressure of 0.1 kg/cm2, pH 3, an aeration rate of 0.5-1.1vvm, and a stirring speed of 100 rpm Conditions, cultivated for 7 days, can use Phellinus mycelia to ferment the whole solution to prepare Phellinus crude extract;

(3) Take the Phellinus mycelia fermentation whole liquid obtained in step (2), and concentrate it under reduced pressure in a conventional manner, so that its volume is concentrated to 1/3 of the original volume;

(4) extract the fermented liquid after the concentration of the above step (3) with 70% ethanol by volume percentage, wherein the amount of ethanol added is 5 times the volume of the concentrated solution, so that the concentration of ethanol in the extract can reach 55%;

(5) heat the extract obtained in step (4) at 70° C. for 1 hour; separate in a conventional manner, and remove impurities by secondary filtration to obtain an ethanol extract; reduce the above ethanol extract in a conventional manner Concentrate under pressure to make its volume concentrated to 1/5 of the original volume;

(6) Dry the concentrated solution obtained in the step (5) by a low-temperature freeze-drying method to obtain a crude Phellinus extract.

Weigh 300 g of the above crude extract and mix it with an equal volume of 100 mesh normal-phase silica gel, mix and stir, and perform silica gel normal-phase column chromatography. The chromatographic stationary phase is 200-300 mesh normal-phase silica gel, the column height is 1m, and the diameter is 20cm. The eluents are chloroform, chloroform:methanol=100:1, 50:1, 10:1, and 5:1 respectively. 3 column volumes. And the obtained eluents were named as Fr-1, Fr-2, Fr-3, Fr-4, Fr-5. . The Fr-5 equal volume silica gel sample was mixed, the silica gel used was 100 mesh normal phase silica gel, and five times the volume of silica gel was chromatographed, and the silica gel used was 200-300 mesh normal phase silica gel. The eluents were chloroform and methanol. The eluate was concentrated under reduced pressure, dissolved in methanol, and subjected to methanol gel column chromatography. Elute with an eluent, use TLC to detect the collected eluate, combine the eluate appropriately, and dry under reduced pressure. Then perform reverse phase silica gel chromatography, the eluent is methanol: water = 10%-80%. The eluents collected by TLC were properly combined, dried under reduced pressure, and methanol gel chromatography was performed again, and eluted with methanol, and the eluent was subjected to TLC detection. The developing solvent was chloroform:methanol=6:1-8:1, Evaporate to dryness under pressure, carry out 1D-HNMR nuclear magnetic resonance analysis, the frequency is 400MHZ, the analysis result is δ5.57(d, J=3.6Hz, 3H), 5.52–5.15(m, 10H), 5.13(s, 1H), 3.63(d,J=4.2Hz,4H),2.46(s,2H),2.28(s,2H),1.23–1.09(m,6H),1.09–0.65(m,69H),0.63(s,7H) .Proved to be 5,7,22-triene-3-ergosterol.

Example 2:

Phellinus crude extract, prepared by the following method:

(1) The fermentation medium formula is in grams/100 milliliters:

Corn Starch 3% Glucose 2%

Peptone 0.5% Yeast Extract 0.5%

Magnesium sulfate 0.5% Potassium dihydrogen phosphate 0.05%

(2) Phellinus bacterium classification is inoculated in the Erlenmeyer flask that liquid culture medium is housed with 30 ℃ of temperature with 30 ℃ of temperature, and the rotating speed of shaking flask is 180r/min, under the condition of pH6, vibration culture 15 days; When the temperature drops to 2.5, inoculate the seeds in the shake flask into the culture medium of a 50L fermenter at a temperature of 30°C, a fermenter pressure of 0.2 kg/cm2, a pH of 3, an aeration rate of 0.5-1.1vvm, and a stirring speed of 180 rpm Conditions, cultivated for 15 days, can use Phellinus mycelia to ferment the whole liquid to prepare Phellinus crude extract;

(3) Take the Phellinus mycelia fermentation whole liquid obtained in step (2), and concentrate it under reduced pressure in a conventional manner, so that its volume is concentrated to 1/5 of the original volume;

(4) extracting the fermented liquid after the concentration of the above step (3) with 90% ethanol by volume percentage, wherein the amount of ethanol added is 4 times the volume of the concentrated solution, so that the concentration of ethanol in the extract can reach 70%;

(5) heat the extract obtained in step (4) at 55° C. for 2.5 hours; separate in a conventional manner, and remove impurities by secondary filtration to obtain an ethanol extract; reduce the above ethanol extract in a conventional manner Concentrate under pressure to make its volume concentrated to 1/10 of the original volume;

(6) Dry the concentrated solution obtained in the step (5) by a low-temperature freeze-drying method to obtain a crude Phellinus extract.

Weigh 200 g of the above crude extract and mix it with an equal volume of 100 mesh normal-phase silica gel, mix and stir, and perform silica gel normal-phase column chromatography. The chromatographic stationary phase is 200-300 mesh normal phase silica gel, the column height is 0.8m, and the diameter is 15cm, and the column volumes are respectively eluted for 3 columns. And the obtained eluents were named as Fr-1, Fr-2, Fr-3, Fr-4, Fr-5. The Fr-5 equal volume silica gel sample was mixed, the silica gel used was 100 mesh normal phase silica gel, and five times the volume of silica gel was chromatographed, and the silica gel used was 200-300 mesh normal phase silica gel. The eluents were chloroform and methanol. The eluate was concentrated under reduced pressure, dissolved in methanol, subjected to methanol gel column chromatography, and eluted with eluent. The eluents collected by TLC were appropriately combined, evaporated to dryness under reduced pressure, dissolved in 10% methanol, and subjected to reversed-phase silica gel chromatography. The eluent was methanol and water, and methanol gel chromatography was carried out again, eluting with methanol. The eluate was detected by TLC, combined appropriately, dried under pressure, and analyzed by 1D-HNMR nuclear magnetic resonance, the frequency was 400MHZ, and the analysis result was δ5.57(d, J=3.6Hz, 3H), 5.52–5.15(m, 10H ),5.13(s,1H),3.63(d,J=4.2Hz,4H),2.46(s,2H),2.28(s,2H),1.23–1.09(m,6H),1.09–0.65(m, 69H), 0.63(s,7H). Proved to be 5,7,22-triene-3-ergosterol.

Claims (8)

1. the separation method of ergosterol in Phellinus fungus, its sequence of steps is as follows: (1) preparing Phellinus crude extract; (2) Mix and stir the ethanol precipitate obtained in the above step (1) with normal phase silica gel, and dry, carry out silica gel normal phase column chromatography, and elute with eluent for 2-7 times; Described eluent is chloroform and methanol; (3) Collect the last eluent in step (2), dry under reduced pressure, mix the sample with an equal volume of silica gel, perform normal phase silica gel chromatography again, and elute with an eluent; Described eluent is chloroform and methanol; (4) Collect the eluate obtained in the above step (3), concentrate under reduced pressure, dissolve in methanol, perform methanol gel column chromatography, elute with eluent, use TLC to detect the collected eluate, and combine the eluents appropriately Deliquification, drying under reduced pressure; (5) The product obtained in step (4) is subjected to reverse-phase silica gel chromatography, and the eluent is methanol and water; (6) The product obtained in step (5) is subjected to TLC detection, and the eluent is appropriately combined, dried under pressure, and subjected to methanol gel chromatography again, and eluted with an eluent; (7) Collect the eluent obtained in step (6), detect by TLC, and dry under pressure, which is ergosterol; The Phellinus crude extract is prepared by the following method: (1) The fermentation medium formula is in grams/100 milliliters: Corn starch 1-5%, glucose 1-5%, peptone 0.1-0.5%, yeast extract 0.1-0.5%, magnesium sulfate 0.1-0.5%, potassium dihydrogen phosphate 0.01-0.05%; (2) the method for fermenting and cultivating the crude extract of Phellinus Phellinus is to inoculate Phellinus bacterium classification in the Erlenmeyer flask that liquid culture medium is housed with conventional method, with 20～35 ℃ of temperature, shake flask rotating speed is 80～280r /min, under the condition of pH 3-8, shake culture for 7-15 days; when the pH value drops to 2.5-4 during the culture, inoculate the seeds in the shake flask into the culture solution of a 50L fermenter at a temperature of 20-35°C , Fermentation tank pressure 0.1-0.2 kg/cm2, pH 3-8, ventilation rate 0.5-1.1vvm, stirring speed 100-280 rpm, culture for 7-15 days, you can use Phellinus mycelium to ferment the whole Liquid preparation Phellinus crude extract; (3) Take the Phellinus mycelia fermentation whole liquid obtained in step (2), and concentrate it under reduced pressure in a conventional manner, so that its volume is concentrated to 1/2 to 1/5 of the original volume; (4) extract the fermented liquid after the concentration of the above step (3) with the ethanol of 60～95% by volume percentage, wherein, the amount of adding ethanol is 2～5 times of the concentrated liquid volume, can make the ethanol concentration in the extract liquid Reach 60-90%; (5) Heat the extract obtained in step (4) at 50-70°C for 1-2 hours; separate by conventional methods, and remove impurities by secondary filtration to separate and obtain the ethanol extract; the above-mentioned ethanol extract Concentrate under reduced pressure in a conventional manner, so that its volume is concentrated to 1/5 to 1/10 of the original volume; (6) drying the concentrated solution obtained in step (5) by a low-temperature freeze-drying method to obtain the crude extract of Phellinus Phellinus; Described ergosterol is 5,7,22-triene-3-ergosterol; The gel described in step (4) is SephadexLH-20, SephadexLH-25, and the eluent is methanol. 2. the separation method of ergosterol in Phellinus as claimed in claim 1, is characterized in that, the sample mixing silica gel described in step (2) is 100 mesh normal phase silica gel. 3. the separation method of ergosterol in Phellinus as claimed in claim 1, is characterized in that, the chromatographic silica gel described in step (2) is normal phase 200-300 order. 4. the separation method of ergosterol in Phellinus as claimed in claim 1, is characterized in that, the silica gel consumption described in step (3) is equivolume. 5. the separation method of ergosterol in Phellinus as claimed in claim 1, is characterized in that, the reverse phase material described in step (5) is C-18. 6. the method for separating ergosterol in Phellinus as claimed in claim 1, is characterized in that, the eluent described in step (5) is methyl alcohol: water=10%-80%. 7. the method for separating ergosterol in Phellinus as claimed in claim 1, is characterized in that, the eluent described in step (6) is methyl alcohol. 8. the separation method of ergosterol in Phellinus as claimed in claim 1, is characterized in that, the TLC developing agent described in step (7) is chloroform: methyl alcohol=6:1-8:1.