1) extraction: get 1kg of sorrel powder and extract twice, each time with 1L mass concentration be 70% ethanol aqueous solution, 45 ° C stirring and extracting 2h, filter, the filtrate of twice filtration is merged, under reduced pressure conditions Swirl to recover solvent and concentrate to 2L.
2) extraction: the concentrated solution obtained in step 1) was adjusted to pH 2 with 1 mol/L hydrochloric acid, extracted 3 times with 2L of water-saturated n-butanol, combined organic layers and concentrated to 400 mL;
3) Crystallization for the first time: the concentrated solution obtained in step 2) was added to 960mL of acetone and 240mL of methyl tert-butyl ether mixed solvent, heated to reflux under stirring, then cooled to 20 ° C to separate out white precipitate, filtered, 45 °C drying to obtain 49g of white crystals;
4) Crystallization for the second time: the 49g crystals obtained in step 3) were dissolved in 200mL of hot water at 100°C, stirred for 20min, then the solution was cooled to 0°C to separate out a white solid, suction filtered, and the solid was Dry under 45 ℃ of conditions, obtain β-escin 32g;
5) preparation of finished product of aescin: the 32g3-escin that obtained in step 4) was dissolved in 300mL of ethanol and 15mL of n-hexane mixed solvent, 4.8gL-proline was added to the above-mentioned system, and the system was heated to reflux for 30min. Be cooled to 50 DEG C, add 0.2g purity aescin B at this moment and reach 98% as crystal seed, continue stirring and be cooled to 20 DEG C, crystallize, until white crystal is no longer separated out, suction filtration, the solid is baked at 45 DEG C Dry to obtain 12.5 g of aescin B white crystals.
In the present embodiment, the purpose of step 1) is to extract and separate the total saponins of Aesculus chinensis from Salvia japonica, and this is the conventional technique of the extraction of total saponins from Salvia japonicas, and the extraction method can adopt the reflux, soaking, leakage etc. commonly used in this area method. Among them, the concentration of ethanol will have an important impact on the purity and yield of total escin, too low ethanol concentration will lead to insufficient product extraction, and too high ethanol concentration will lead to increased impurity content.
The purpose of the extraction in step 2) and the two crystallizations in steps 3) and 4) is to purify the extract to obtain high-purity β-escin. Among them, the purpose of adjusting the pH is to improve the stability and extraction efficiency of aescin, and if the pH is too high, the water solubility of aescin will increase, and the n-butanol extraction efficiency will decrease; if the pH is too low, the stability of aescin will be reduced. The ratio of acetone to methyl tert-butyl ether in step (3) can affect the precipitation rate and yield of aescin crystals.
The organic solvent in step 5) can also select any mixing of one or more of methanol, ethanol, isopropanol, acetone, tetrahydrofuran, methyl tert-butyl ether, n-hexane, n-heptane, etc. β-Aescin can be dissolved well, and at the same time, it can better precipitate aescin B13 in the induced recrystallization. The amount of L-proline directly affects the separation and precipitation of aescin B. In this example seven Leaf saponin B can not only selectively and smoothly precipitate crystals, but also the yield and purity are relatively high, and the crystal morphology is good.
Adopt high performance liquid phase method to detect the aescin B in the product, and the specific conditions are: the chromatographic column is a C18 column, and the particle diameter is 5um; : 65, mobile phase PH=2.,5; detection wavelength is 220nm; flow rate is 1.0ml/min, injection volume is 10μ1;
The content of aescin B in the product is 99.7%, and the yield (the total amount of aescin B in the product accounts for the percentage of the total amount of aescin B in the feeding salvia seeds) is 61%.