In this article we will discuss about the isolation, identification and estimation of pseudoaconitine.
Isolation of Pseudoaconitine:
Procedure:
1. The roots of A. balfourii are air-dried, finely powdered and subjected to percolation with 95% ethanol.
2. The ethanolic extract is concentrated to a small volume under reduced pressure, and is treated with 0.5% HCl.
3. The acid solution contains impurities, like resin and oils which are removed by agitating with solvent ether.
4. The solution is made alkaline and extracted with several portions of solvent ether.
5. Ether extracts are combined together and evaporated. Pseudoaconitine crystallises out.
6. Crude pseudoaconitine is purified by converting it into its hydrobromide and re- crystallising it with ethanol.
Identification of Pseudoaconitine:
1. Dilute solution of aconitine + few drops of acetic acid + 2-3 drops of 0.2 N potassium permagnate → Rosettes of red prismatic crystals.
2. A drop of 2% solution of aconitine nitrate in 10% acetic acid is mixed on a slide with one drop of saturated solution of NaCl or KBr. The characteristic microcrystalline salt of the alkaloid is obtained.
3. Very dilute solution of aconitine + ammonium reineckate in slightly acid solution → ppt (observe under microscope) → Rosettes of fine crystals
Estimation of Pseudoaconitine:
Method I ― By HPLC:
The method comprises extraction of aconite powder first with ammonical ether and then with methanol (x 3 each); chromatography of the resultant extract over neutral alumina and elution with ethyl acetate-methanol (7:3); prior to HPLC. The method is found to be reproducible and suitable for routine analysis of aconite and its pharmaceutical preparations, quantitatively
Procedure:
1. Drug Materials:
Aconitine is purchased from Sigma Pharmaceuticals. The raw aconite roots and processed aconites (prepared from raw aconite roots by autoclaving at 120°C for 40 minutes) are finely powdered and dried in a desiccator.
2. Reagents:
Acetonitrile (HPLC grade), tetrahydrofuran, sodium hydrogen phosphate, phosphoric acid, sodium hexane sulphonate, alkaline alumina, neutral alumina.
3. Instrumental Conditions:
The analysis can be carried out on an instrument coupled to a stainless steel column (i.d.:30 cm x 4 mm), packed with ODS chemically bonded silica gel. The detector is a UV 8 variable wavelength monitor with a flow cell of volume 8 µl.
4. Extraction Steps:
i. Ammoniacal Chloroform/Ammoniacal Ether/Ammoniacal Methanol Method:
The sample (5 g) is stirred with one of the above-named organic solvents (60 ml), containing 5% aqueous NH3 (5 ml) at room temperature for 30 minutes. The liquid phase is filtered and the residue is further extracted 2 or 4 times in the same manner with the same solvent (60 ml each). The combined solution is evaporated to dryness at a temperature not exceeding 40°C.
ii. Methanol or Hydrochloric Acid Method:
The sample (5 g) is extracted 3 times with either methanol or 1 N HCl (60 ml each) at room temperature for 24 hours each. The liquid phase is filtered, combined and evaporated under diminished pressure at a temperature below 40°C.
iii. Ammoniacal Ether-Methanol Method:
The sample (5 g) is extracted 3 times with ammoniacal ether (60 ml each) for 30 minutes each, and then with methanol (100 ml each), once for 16 hours and twice for 3 hours each. The organic phase is combined and evaporated to dryness under reduced pressure at a temperature not exceeding 40°C.
5. Purification Steps:
i. Chloroform or Dichloromethane Partition Method:
The methanol extract prepared as above (575 mg), equivalent to 5 gm of crude drug, is suspended in CHCl3 or CH2Cl2 (20 ml) and extracted 3 times with 5 % HCl (15 ml each). The combined acidic layer is made alkaline (pH 10) with 25% aqueous NH3 and extracted 4 times with CHCl3 or CH2 Cl2 (20 ml each). The organic phase is washed once with water and evaporated to dryness.
ii. Celite or Alumina Chromatography Method:
The methanol extract equivalent to 5 gm of crude drug is mixed with celite (twice its weight), placed on the top of a column of celite or alumina (10 g) and eluted successively with benzene (100 ml), AcOEt-MeOH (7:3, 200 ml) and MeOH (100 ml). Each eluate is evaporated to dryness.
Method II ― By GLC/Selected Ion Monitoring (SIM):
SIM was used by Kitae et al. (1996) to determine aconitum alkaloids in the tubers of Aconitum japonicum.
Procedure:
1. Materials:
Aconitum tubers are purchased from a reliable source and authentified by a taxonomist.
2. Reference Alkaloids:
Standard samples of hypaconitine, mesaconitine, jesaconitine and aconitine are purchased from a commercial source.
3. Reagents:
BSTFA [N, O-bis (trimethylsilyl) trifluoroacetamide].
4. Extraction:
After the aconitum tubers are weighed and finely powdered, alkaloids are extracted using 40 ml of methanol/2.0 gm of powder for 1 hr. The extract is filtered and the residue is further extracted in the same manner. The combined solution after removing un-dissolved matter is evaporated to dryness. 30 ml of water is poured into the residue which is then acidified to pH 3 by the addition of 10% aqueous tartaric acid. The acidified solution is washed twice with 60 ml of diethyl ether.
After washing, the solution is made alkaline with 4% of aqueous NaOH and extracted twice with CHCl3 (60 ml each). The organic phases are combined and evaporated to dryness. Before converting to TMS (trimethylsilyl) derivatives, the extract is diluted with CHCl3 for analysis. Diluted sample extracts are then evaporated to dryness and converted into TMS derivatives by overnight treatment with 50 µl of BSTFA and used for GC/SIM analysis.
5. Apparatus:
All analyses are carried out on a Jeol model DX-303 system interfaced to a MS-GCG06 gas chromatograph. The column is a 15 m x 0.25 mm I.D. fused silica capillary cross-linked column with methylsilicone. Temperature of the column oven is started at 250°C and increased to 320°C at 16°C/minute. The carrier gas is helium with a linear velocity of about 25 cm/s. The temperatures of the injection port and transfer lines are kept at 320°C and 250°C, respectively, and that of the ion source at 250°C.
The ionisation, the trap current, and the accelerating voltage are 70 eV, 300 and 3 kV respectively. For GC/SIM analysis, the ions at m/z = 596 for hypaconitine, at m/z – 684 for mesaconitine, at m/z = 698 for aconitine, and at m/z = 728 for jesaconitine, are monitored with a dynamic resolution of 1,000 respectively.
No comments yet.