Extraction, isolation and characterization of the alkaloids of Hantahexaenia japonica

Summary

Han Fangji is the root of Stephania tetrandra S. Mcore (防己科千金藤属物), an expectorant, antipyretic and analgesic drug whose active ingredients are alkaloids. The main ones are hanfengzhi methylin and hanfengzhi ethylin. In addition to its clinical use in the treatment of hypertension, neuropathic pain, anti-amoeba protozoa, the iodomethyl, or bromomethyl compounds of the alkaloids of Stephania tetrandra S. Mcore are used as muscle relaxants, and Stephania tetrandra S. Mcore has anticarcinogenic and vasodilatory effects in animal experiments.

Operation method

chromatography

Principle

The total alkaloid content in the root of Hambusa is 1.5~2.3%, mainly Hambusin A, the content is about 1%, Hambusin Ethyl, the content is about 0.5%; Verticillin Phenolic Alkaloid, the content is 0.2%; and several other kinds of trace alkaloids.1. Hambusin A (Tetrandrine, Hambusin, Powdered Hambusin Alkaloid) is colorless pin crystals, insoluble in water and petroleum ether, easily soluble in ethanol, acetone, Ethyl acetate, ethyl ether and trichloromethane and other organic solvents and dilute acid water, soluble in benzene, mp 216 ℃, there is a double melting point phenomenon, from the acetone in the crystallization of the melting around 150 ℃ and then solidified after heating to 213 ℃ to re-melting. 2. HANPHONTZE BETAINE (Fangchinoline, anti-fenoline alkaloids, northeastern powder preventive alkaloids) solubility behavior is similar to that of HANPHONTZE METHYLENE, due to the phenolic hydroxyl group, so the polarity is higher than that of HANPHONTZE METHYLENE. Because of the phenolic hydroxyl group, the polarity is slightly higher than that of hanfengine, and the solubility in benzene is smaller than that of hanfengine, while in ethanol it is larger than that of hanfengine. Here can be separated from each other, with different solvents recrystallization, its crystal shape and solubility are different: 3. Cylanoline (Cylanoline) for water-soluble quaternary alkaloids, insoluble in polar solvents, chloride for colorless, octahedral shaped knots Chang, mp 214~6 ℃, iodide for the colorless sericeous crystals, mp185 ℃; picrate for yellow crystals, mp154~6 ℃.

Materials and Instruments

Crude powder of HANPROFEN
Acetone Ethanol Water Petroleum ether Ethyl acetate Silica gel Aluminum oxide Bismuth potassium iodide reagent Acetone
Round-bottomed flasks Chromatography columns Long-necked funnels Filter paper sheets Air pumps Dropper tubes Iron clamps Glass stopper fittings Glass evaporators

Move

I. Extraction and separation experiments
1. Reflux extraction
100 g of crude powder in 1000 ml round-bottomed flask, add 500 ml of 95% ethanol, reflux 1 h, filter, the same method of extraction once, combined with the ethanol extract, concentrated to dryness, to get the extract.
2. Low-pressure column chromatography
Low-pressure column chromatography at low pressure (0.5~3 ^kg/cm2, generally 0.3~1.2 ^kg/cm2 ) using the particle diameter between the classical column chromatography (100~200 μm) and HPLC (~ 37 μm) thin layer chromatography with silica gel (or alumina) H or G (50~75 μm) as a filler of a kind of column chromatography column, the basic principle of the same with HPLC, the separation of Its basic principle is the same as that of HPLC, and the separation effect is also between classical column and HPLC. The column is loaded by dry method with reduced pressure, the layer is compact and uniform, the distribution of chromatographic bands is concentrated and neat, meanwhile, the optimal separating solvent system of the thin-layer chromatography can be directly used in the low-pressure column chromatography, which is a method with better separation effect, simple equipment, convenient operation and rapid method, and it is suitable for the constant amount of the natural products' preparative separation.
(1) Column loading
Decompression dry loading method, chromatography column specifications: column length 25 cm, inner diameter 1.8 cm, a total of about 25 g of silica gel (height of about 20 cm).
(2) Mixing and spiking
Take about 150 mg of Hambucine, add a small amount of acetone hot soluble (just dissolved for the degree) with a dropper to 1.5 g silica gel, carefully mixed, water bath evaporation, crushed fine, through a long-necked funnel carefully added to the top of the column, gently vertical tonk, to be the surface of the sample is flat and not mixing, and then covered with a blank silica gel about 1 ~ 2 cm high, then add a round piece of filter paper, pressed tightly.
(3) Elution
Turn on the air pump and wait for use. Use a dropper to add a small amount of eluent (petroleum ether: ethyl acetate 3:1) carefully along the wall of the chromatographic column, when the liquid level reaches a certain height, and then once again add the rest of the eluent (a total of about 250 ml), quickly on the top of the column mounted on a glass standard mouth plug connector, press with an iron clip (to prevent the connector from rushing away when pressurized), connected to the air pump connector, control the flow rate of 1 ml/min, every 10 minutes or so, a tube, receiving 12-15 copies, the whole process of elution is about 2 hours.
(4) Inspection
Each stream of copies were moved into a small glass evaporator, concentrated on a water bath, respectively, through the TLC check, adsorbent: silica gel G, unfolding agent: petroleum ether: ethyl acetate 3:1, modified bismuth iodide potassium reagent spray color, with hanpaoxin acephate, acephate as a standard for control, combined with the same components, respectively, to obtain the acephate, acephate crude, recrystallized with acetone, determination of mp.
The following experiments can be chosen after class
3. Extraction of total alkaloids and separation of lipophilic and hydrophilic alkaloids


Note 1: Add the crude powder of hanfengshi with appropriate amount of acid-water solution to the extent that it can wet the powder of the raw herb (about 150 ml), mix well, leave it for half an hour, and fill it evenly and densely into the percolating cylinder, press it tightly with the bottom of a conical flask or other flat-bottomed tool for percolation, at a flow rate of about 1.5 ml/minute.
Note 2: The sand must be washed and dried beforehand, and it is better not to mix more than 120 g, so as to avoid the Soxhlet extractor can not be loaded at one time or loaded too much. The alkaloids cannot be extracted.
Note 3: Check whether the alkaloid extraction method, is to take the last ether extract about a few drops, evaporate the ether, residue plus 5% HCl 0.5 ml dissolved, plus a drop of modified bismuth potassium iodide reagent, no precipitate folding or obvious turbidity, indicating that the alkaloid has been extracted, or basically extracted. On the contrary, extraction should be continued.
Note 4: First, remove the filter paper cartridge from the extractor. Then extract the last ether extract in the glass cylinder dumped (another storage), and then the extraction of the glass cylinder installed, continue to heat, recycling ether in the flask in the glass cylinder, to the ether extract in the flask volume is small, stop recycling, will be dumped out of the flask in the ether extract.
Identification methods
1. TLC of organic amine base
Adsorbent: thin-layer chromatography silica gel G, plate with 0.3% CMC-Na aqueous solution, 110 ℃ activation for 1 hour. Samples: Separated hanpenguai methyl ①, ethyl ②, total base ③ Expanding agent: petroleum ether- EtoAc-NHEt2 (6:2:1) Colorant: modified bismuth potassium iodide reagent (blow dry with a hairdryer after unfolding before spraying the colorant to avoid interference by diethylamine) Phenomenon: aether is light brown after color development and fades in about 2 hours, while ethylamine is brown and does not fade after a long period of time, which can help in its identification.

The following experiments can be optional after class
2. Derivative preparation

Take hanfeng has 0.2 g of methylin, dissolved in 2 ml (acetone, dropwise addition of a saturated aqueous solution of picric acid until no longer folded out of the yellow precipitate, filtration to collect the precipitate, in turn to a small amount of water ether washed, recrystallized from ethanol, to get the hanfeng has methylin picrate, mp 235~242 ℃.

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