A method for extracting myristic acid from marine coccidioides, comprising: extracting with ethyl acetate; performing chromatography with a reversed-phase silica gel column; performing chromatography with a Sephadex LH-20 column; Chromatography was performed on a phase silica gel column; chromatography was performed on a normal phase silica gel column; myristic acid was extracted from marine coccidioides by the method of PTLC preparation.
Specifically include the following steps:
Step 1, preparing the ethyl acetate phase extract of the marine coccidioides: carrying out extraction treatment with ethyl acetate as the extractant, combining the extracts, and concentrating under reduced pressure to obtain the ethyl acetate phase extract of the marine coccidioides;
The marine coccidioides were grown to a stable stage, harvested, freeze-dried to obtain 19.3740 g of marine coccidioides algae powder, leached with 75% ethanol to obtain 5.1247 g of ethanol extract, added equal volumes of ethyl acetate and water, and extracted repeatedly , collecting the ethyl acetate phase, combining, concentrating to dryness under reduced pressure, and obtaining 4.2357 g of the ethyl acetate phase extract of the marine coccidioides;
Step 2: Preparation of primary crude myristic acid by reverse-phase silica gel column chromatography: The ethyl acetate phase extract of the obtained marine coccidioides was subjected to reverse-phase silica gel column chromatography, collected, detected, combined, and concentrated to dryness under reduced pressure. Obtain a crude product of myristic acid;
After dissolving 4.2357g of the marine coccidioides ethyl acetate phase extract with an appropriate amount of analytically pure methanol, carry out reverse-phase silica gel (180g) column chromatography; water successively, the mass fraction is 25% methanol solution, and the mass fraction is 50% Methanol solution with a mass fraction of 75% methanol solution, methanol elution, 1.5L for each gradient elution, flow rate control is 30mL/min, collect 1 tube per 200mL, rotary evaporation, preliminary analysis of effluent by TLC, combined palm The acid high-concentration flow is segmented, concentrated to dryness under reduced pressure, and the primary crude myristic acid is obtained;
Step 3, SephadexLH-20 Sephadex column chromatography to prepare the secondary crude myristic acid: the obtained marine coccidioides ethyl acetate phase extract was chromatographed on SephadexLH-20 Sephadex column, Collect, detect, merge, and concentrate to dryness under reduced pressure to obtain the secondary crude myristic acid;
Dissolve the primary myristic acid with an appropriate amount of methanol, put it on a Sephadex LH-20 Sephadex column, and use methanol as the eluent for elution. The flow rate is controlled at 8S/drop, and the fractions are collected by an automatic collector every 30 minutes. One tube, the effluent was initially analyzed by TLC, the fractions from tubes 21 to 25 were combined, concentrated to dryness under reduced pressure, and the secondary crude myristic acid was obtained;
Step 4: Preparing primary myristic acid by reverse-phase silica gel column chromatography: the obtained secondary crude myristic acid is chromatographed on a reverse-phase silica gel column, collected, detected, combined, and concentrated to dryness under reduced pressure to obtain myristic acid Sour primary product;
The secondary crude myristic acid was subjected to RPC18 reversed-phase silica gel column chromatography with mass fractions of 60%, 70%, 80%, 90%, and 100% acetone-water system gradients in sequence, each gradient eluted 200mL, and the flow rate was controlled Rotary evaporation at 10mL/min, the effluent was initially analyzed by TLC, the high-concentration stream of myristic acid was combined into sections, concentrated to dryness under reduced pressure, and the primary product of myristic acid was obtained;
Step 5, normal phase silica gel column chromatography to prepare the secondary product of myristic acid: the obtained primary product of myristic acid is chromatographed on a normal phase silica gel column, collected, detected, combined, and concentrated to dryness under reduced pressure to obtain myristic acid sour secondary product;
The primary product of myristic acid was subjected to normal-phase silica gel column chromatography, and the sample was mixed with silica gel powder. The silica gel was saturated with n-hexane and then loaded into the column. The volume ratio is 50:1, add 2 drops of glacial acetic acid, collect the fractions, combine tubes 63-67, concentrate to dryness under reduced pressure, and obtain the second refined product of myristic acid;
Step 6. Preparation of pure myristic acid by PTLC: perform PTLC on the obtained secondary refined myristic acid, sample spotting, development, scraper, sample recovery, and store at -20°C to obtain pure myristic acid;
Pass the secondary product of myristic acid through PTLC to finally obtain myristic acid: use a high-efficiency thin-layer chromatography plate, develop it in a developer with a volume ratio of petroleum ether: ethyl acetate = 15:1, and develop color at the front and rear ends , scraped the middle part with a razor blade, combined, dissolved in methanol, centrifuged at high speed, collected the supernatant, concentrated to dryness under reduced pressure, and finally obtained myristic acid.
