The extraction and isolation of the free hydroxyanthraquinone components in Tiger Balm can be applied to (1) the preparation of drugs and (2) the study of pharmacological effects.
Operation method
extraction
Principle
Hydroxyquinones and stilbenes are soluble in ethanol, so ethanol can be used to take them out. Hydroxyanthraquinones are soluble in weak polar solvents such as ether, and resveratrol glycosides have a small solubility in ether, using the difference in their solubility in ether to separate the hydroxyanthraquinones from resveratrol glycosides, and then utilizing the structural differences of the hydroxyanthraquinones to express different acidity, and separating them by PH gradient extraction method.
Materials and Instruments
Tiger Balm Powder Move I. Extraction Preparation of total ethanol extract: Take 200g of Tiger Balm crude powder, reflux in 1000ml round-bottomed flask, the first time add 500ml ethanol reflux for one hour, the second time add 300ml ethanol reflux for 30 minutes, the third time add 250ml ethanol reflux for 30 minutes, combined three times of ethanol extracts, placed, if there is precipitation can be filtered once, the filtrate is decompressed and recycled ethanol to dryness to get a paste. Separation 1. Separation of lipophilic and hydrophilic components: Add 10ml of water and 100ml of ether to the above paste and place it after full shaking, pour the ether solution into a 500ml triangular flask (the water layer can not be poured out), then add 50ml of ether to the flask and shake, place it, pour out the ether solution, operate the same method for six times, and merge the ether solution that is lipophilic component - total free anthraquinone, and the residuals extracted from ether contain water-soluble components. 2. Free anthraquinone separation: (1) Strong acidic components of the sub-high: the ether solution containing the above total free anthraquinone placed in 100 ml dispensing funnel, add 5% sodium bicarbonate aqueous solution of 40 ml extraction (measured 5% sodium bicarbonate PH value), placed so that the full stratification, such as ether volatilization in the process of extraction can be supplemented, divided out of alkaline aqueous solution with the same method of extraction for two times, the combined alkaline water extract, under stirring and slowly add 6N hydrochloric acid drop to adjust to pH2 attention to observation Color changes, a little time to precipitate, filtration, water to wash the precipitate neutral, the precipitate will be placed on the surface of the dryer, the strongly acidic components. (2) medium acidic components - the separation of rhodopsin: sodium bicarbonate extracted ether solution with 5% sodium carbonate solution (measure the pH of 5% sodium carbonate aqueous solution) extraction 5 - 9 times, each time 40 ml, until the extract is shallow. Combine the sodium carbonate extracts, carefully add hydrochloric acid to adjust the pH 3, leave, extract and filter, wash the precipitate in water to neutral, drain, dry and weigh. Recrystallize with methanol a chloroform or benzene a chloroform (1:1) to obtain a mixture of rhubarb phenol and rhubarbine 6-methyl ether. (3) neutral components - sterols separation: sodium hydroxide extracted ether solution, washed with water to neutral, anhydrous sodium sulfate dehydration, recovery of ether residue, hot soluble in methanol for the second time (10 ml, 5 ml) filtration and combined with methanol solution, concentration, placed on the crystallization, filtration of the precipitate and a small amount of petroleum ether washed, and then recrystallized with methanol, β-sitosterol, mp136 --137 ℃, take a little crystallization, do acetic anhydride - concentrated sulfuric acid reaction, observe the phenomenon. Identification 1. Chemical identification: take a little rhubarb, rhubarb phenol, etc., dissolve with ether, do the following reaction: A: alkali test: take 1ml of test solution, add several drops of 20% NaOH, observe the color. B. Magnesium acetate reaction: take 1ml of test sample, add several drops of magnesium acetate reagent, observe the present bag. 2.Thin layer identification: Adsorbent: silica gel - CMC Expanding agent: C6H6-EtoAc (3:2 or 97:9). Color developer: (1) ammonia vapor fumigation. (2) 5% KOH spray. Caveat Rhubarb phenol and rhubarb 6-methyl ether are difficult to separate from each other, in this experiment thin layer conditions for the same spot, available chromatography with calcium hydrogen phosphate for column chromatography, eluted with petroleum ether, the lower layer of the yellow band is eluted to methanol recrystallization can be obtained rhubarb phenol, the upper layer of the yellow band is eluted to methanol recrystallization can be obtained rhubarb 6-methyl ether. Common Problems The structure and properties of the main components in Tiger Balm: 1. Emodin (Emodin): orange-yellow long needle crystals (acetone in the crystallization of orange, methanol in the crystallization of yellow), mp256-257 ℃. Several insoluble in water, for the following solvents: carbon tetrachloride 0.01%, chloroform 0.0718%, carbon disulfide 0.009%, ether 0.14%, benzene 0.0415. Soluble in ethanol, soluble in ammonia, sodium carbonate and sodium hydroxide aqueous solution. 2. Rheumol (Chrysophanol): golden yellow hexagonal flaky crystals (acetone crystals) or needle-like crystals (ethanol crystals). mp 196 ℃, can sublimate. Soluble in ether, chloroform, benzene, glacial acetic acid, ethanol, slightly soluble in methanol, insoluble in petroleum ether, insoluble in water, sodium bicarbonate and sodium carbonate aqueous solution. Soluble in water, sodium bicarbonate and sodium carbonate aqueous solution and hot sodium carbonate aqueous solution. 3. Emodin 6-methyl ether (Emodinmonomethl ether): golden yellow needle crystal, mp207 ℃, can sublimate. Soluble in sodium hydroxide aqueous solution, solubility similar to rhubarb phenol. 4. rhubarb 6-methyl ether 6-D-glucoside (Anthraglycosibe A): yellow needle crystal (dilute methanol in the crystallization) mp230-232 ℃. 5. Rhododendron 3-D-glucoside (Anthraglycoside B): light yellow needle crystal (dilute ethanol in the crystallization of 1 molecule of water mp190-191 ℃. 6. Resveratrol (Resveratrol): colorless needle crystals, mp256-257 ° C, 216 ° C sublimation, soluble in ether, chloroform, methanol, ethanol, acetone, etc.. 7.resveratrol glucoside (polydotinpeceid): colorless granular crystals, double melting point of 130-140 ° C, 225-226 ° C, soluble in methanol, ethanol, acetone, hot water, soluble in ethyl acetate, soluble in sodium carbonate and sodium hydroxide in aqueous solution, slightly soluble in cold water, insoluble in ether. 8.β-sitosterol 9. tannin: a condensed tannin, soluble in alcohol and water, insoluble in benzene, ethyl ether, chloroform and so on. In addition. Tiger Balm leaves and stems contain a small amount of hydroxyanthraquinone compounds, organic acids, quercetin, isoquercetin, tiger balm flavonoid glycosides, flatulence glycosides, chrysoeriol glycosides and rue glycosides, Vtio and so on. For more product details, please visit Aladdin Scientific website.
Ethanol Ethyl ether Distilled water Sodium bicarbonate Sodium carbonate Hydrochloric acid Sodium carbonate Magnesium acetate reagent Sodium hydroxide KOH spray Ammonia vapor
Round-bottomed flasks Triangular flasks Dispensing funnels pH meters Glass rods Ovens Filter extraction flasks Electronic balances Silica gel-CMC