Han Fang is the root of Stephania tetrandra S. Mcore, a hurricane antipyretic analgesic drug whose active ingredient is alkaloid. Mainly Han Fang and Han Fang have been B. Clinically, in addition to the treatment of hypertension, neuropathic pain, and anti-ameba protozoa, the anti-alkaloid iodine methyl or bromomethyl compound is also used as a muscle relaxant. In addition, Han Fang has been in the form of a muscle relaxant. There are anti-cancer and dilated blood vessels in animal experiments.
Purpose requirement
Through the extraction, separation and identification of several alkaloids in Hanfang, the following knowledge and skills are required.
1. A general extraction method for alkaloids.
2. Separation by low pressure column chromatography, purification of monomers and identification by thin layer chromatography
Known structure and properties of alkaloids
The total alkaloid content in the roots of Hanfang has 1.5~2.3%, mainly Hanfang A, the content is about 1%, Hanfang has B, the content is about 0.5%; the ring vinegar base, the content is 0.2%; And several other trace alkaloids.
1. Han Fang has a (Tetrandrine, Han anti-alkali, powder anti-alkali)
Colorless needle crystal, insoluble in water and petroleum ether, soluble in organic solvents such as ethanol, acetone, ethyl acetate, ether and chloroform and dilute acid water, soluble in benzene, mp 216 ° C, double melting point, from acetone In the middle crystal, after melting at about 150 °C, it is heated and solidified, and remelted at 213 °C.
2. Han Fang has a single substance (Fangchinoline, also known as anti-nolinine, noodazole anti-alkali)
The dissolution behavior is similar to that of Hanfang. Because of a phenolic hydroxyl group, the polarity is slightly higher than that of Hanfang. The solubility in benzene is less than that of Hanfang and is higher than that of Hanfang. . Therefore, they can be separated from each other, and when they are reconstituted with different solvents, their crystal forms and melting points are different:
3. Ring phenololine (Cylanoline)
It is a water-soluble quaternary ammonium alkaloid, insoluble in polar solvent, chloride is colorless, octahedron-like knot, mp 214~6 °C, iodide is colorless silky crystal, mp185 °C; picrate is yellow Crystallization, mp 154~6 °C.
Extraction and separation of alkaloids
1. Extraction of total alkaloids and separation of lipophilic and hydrophilic alkaloids
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Note 1: Add the appropriate amount of acid water solution to the coarse powder of Hanfang, so as to wet the raw powder to a degree (about 150ml), mix well and let it sit for half an hour, evenly and densely into the infiltration cylinder, with a cone The bottom of the bottle or other flat-bottom tool is compressed for percolation at a flow rate of approximately 1.5 ml/min.
Note 2: The net sand must be washed and dried beforehand. The mixing volume should not exceed 120g, so that the Soxhlet extractor can not be installed at one time or installed too much. Can not extract alkaloids.
Note 3: The method of checking whether the alkaloids are exhausted is to take a few drops of the last ether extract, remove the ether, dissolve the residue with 5% HCl 0.5ml, add a drop of modified cesium iodide reagent, no precipitation Or when it is obviously turbid, it indicates that the alkaloid has been exhausted or substantially removed. On the contrary, it should continue to be extracted.
Note 4: First remove the filter paper tube inside the extractor. Then, the last ether extract in the extraction glass tube is poured out (other storage), and then the extraction glass tube is installed, heating is continued, and the diethyl ether in the flask is recovered in the glass cylinder until the volume of the ether extract in the flask is small, and stops. After recovery, the ether extract in the flask was decanted.
2. Separation of Han Fang and A by low pressure column chromatography
Low pressure column chromatography at low pressure (0.5~3kg/cm2, generally 0.3~1.2 kg/cm2) using thin layer chromatography with particle diameter between classical column chromatography (100~200μm) and HPLC (~37μm) A column chromatography column using silica gel (or alumina) H or G (50-75 μm) as a filler. The basic principle is the same as HPLC, and the separation effect is also between the classical column and HPLC. The column, the layer is tight and uniform, the distribution of the chromatographic band is neat and tidy, and the optimal separation solvent system for thin layer chromatography can be directly used for low pressure column chromatography. It is a good separation effect, simple equipment, convenient operation and fast. Methods. It is suitable for constant preparative separation from natural products.
(1) Packing column Decompression dry method, column specification: column length 30cm, inner diameter 2cm, a total of about 30g of silica gel (about 22cm high).
(2) Mixing sample and taking about 150mg of Chinese anti-alkali, adding a small amount of acetone to hot solution (just dissolve to degree) with a dropper added to 1.5g of silica gel, carefully mix well, steam dry on the water bath, grind fine, through a Carefully add the long-neck funnel to the top of the column and gently slam it. When the surface of the sample is flat and not mixed, cover the blank silica gel about 1~2cm high, and then cover a circular filter paper and press it tightly.
(3) Elution First check whether the valves from the air compressor to the column are normal, close each valve, and start the air compressor to the rated pressure (5.8kg/cm2) for use. Carefully add a small amount of eluent (cyclohexane-ethyl citrate-diethylamine/6:2:0.8) to the column wall of the column with a dropper. When the liquid level reaches a certain height, add the remaining elution again. Agent (a total of about 250ml), quickly put a glass standard plug joint on the top of the column, press it with iron clamp (the joint is opened when anti-pressurization), carefully open the air compressor valve, then open the needle valve and air filter Pressure device, (Note: the pressure is too large. The glass column will be fried, generally 2kg is safe, if necessary, wear a protective mask) Transfer the required pressure, 0.6~1.2kg/cm2, flow out after about 40 minutes, control the flow rate 1ml/ Points, every 10 minutes or so, a tube, 12 to 15 parts, the entire process of elution for about 3 hours.
(4) The respective fractions were transferred to a small glass evaporator, concentrated on a water bath, and examined by TLC, respectively. Adsorbent: silica gel G, developing solvent: cyclohexane-ethyl acetate-diethylamine/6:3: 1, modified cesium iodide reagent spray color development, Han Fang has A, B as the standard control, combined with the same components, respectively, obtained crude methyl A and B, recrystallized with acetone, mp.
Identification method
1. Derivative preparation
Take 0.2g of Chinese anti-A, dissolve in 2ml (acetone, add a bitter acid saturated aqueous solution until the yellow precipitate is no longer folded, collect the precipitate by suction filtration, wash with a small amount of water ether, recrystallize with ethanol, get Han Anti-A bitteric acid salt, mp 235~242 ° C (dc).
2. Organic amine base TLC
Adsorbent: Thin layer chromatography silica gel G from Qingdao Ocean Chemical Plant, made with 0.3% CMC-Na aqueous solution, and activated at 110 ° C for 1 hour.
Sample: Han Fang has been isolated, 1, B, 2, total alkali 3
Developer: Cyclohexane - EtoAc-NHEt2 (6:2:1)
Color developer: modified cesium iodide reagent (expanded with a hair dryer and then sprayed with a coloring agent to avoid interference with diethylamine)
Phenomenon: A color is light brown after color development, and fades in about 2 hours, while B is brown, and it will not fade for a long time, which can help identify it.
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