In this article we will discuss about the isolation, identification and estimation of nux-vomica alkaloids.
Nux-vomica consists of the dried, ripe seeds of Strychnos nux-vomica, belonging to the family loganiaceae. It contains indole type of alkaloids, namely strychinine (not less than 1.2 %) and brucine.
Strychinine is practically insoluble in solvent ether and is very little slightly in acetone and light petroleum. The base dissolves readily in chloroform (1 in 6) and in a mixture of equal parts of ether and chloroform. It is soluble in benzene (1 in 160) and in amyl alcohol (1 in 180). The base melts at 286-288°C.
Brucine is easily soluble in ethanol, chloroform and acetone. It is very slightly soluble in solvent ether. It is practically insoluble in dilute solutions of NaOH and KOH and is sparingly soluble in ammonia solutions. It melts at 178°C.
Isolation of Nux-Vomica Alkaloids:
Identification of Nux-Vomica Alkaloids:
1. Extract of the drug + 2 drops of sulphovanadic acid → purple-red colour (presence of stryhinine).
4. Mandelin’s Test (for Strychinine):
Sample solution + Mandelin’s reagent (H2SO4 + ammonium vanadate) → Violet-blue colour → cherry-red → orange colour.
5. Malaquin’s Test:
Malaquin described the following test for strychnine. It is very sensitive and can detect strychnine in a dilution of 1 in 100,000.
Estimation of Nux-Vomica Alkaloids:
Method I ― Estimation by Colorimetry (Strychinine):
The colorimetric method was used by Allen and Allport for the estimation of strychinine in Nux-vomica seed. It is based on the colour formation with Malaquin’s reagent.
Procedure:
1. 1 gm of finely powdered seed is accurately weighed and placed in a 100 ml conical flask; 3 ml of absolute alcohol is added and the mixture is heated on a boiling water bath, until most of the alcohol has evaporated.
2. 10 ml of tetrachloroethylene and 0.6 gm of piperazine are added; it is subjected to reflux condensation for 15 minutes. The contents are transferred to a small percolator packed well; the percolation is continued at a speed of 1 drop/second, until the extraction is completed (solvent which is heated extra is used for the percolation).
3. Sufficient 0.1 N H2SO4 is added to bring the volume of the percolate to 90 ml. The liquids are allowed to separate in a separating funnel.
4. After the immiscible liquids have separated, about 10 ml of the upper layer is pipetted out and filtered through a dry filter paper.
5. Exactly 5 ml of the filtrate is transferred to a conical centrifuge tube of 15 ml capacity. 1.5 ml of 9% w/v aqueous solution of oxalic acid and 1 ml of 5% w/v solution of potassium ferrocyanide in 0.2% w/v aqueous solution of sodium carbonate are added. The tube is allowed to stand at room temperature for 15 minutes and is then immersed in a freezing mixture. The tube is agitated until the contents are frozen, removed and gently warmed, until melting takes place.
6. Approximately 0.1 gm of acid washed Keiselguhr is added and the precipitate is centrifuged down. The clear supernatant is poured off and about 2 ml of a 0.1 % w/v aqueous solution of H2SO4 containing 1% of oxalic acid is added.
7. The precipitate is washed by stirring with a glass rod. The washing is repeated once more and the clear supernatant decanted, as completely as possible.
8. 1 ml of conc. HCl is added to the precipitate, the mixture is heated over a flame and 10 ml of 10% w/v of HCl is added. The mixture is transferred to a 100 ml volumetric flask.
The centrifuge tube is rinsed well with dilute HCl and the volumetric flask filled to the mark with the washings.
9. A portion of this solution is filtered and 5 ml of the filtrate is transferred to a test tube containing 0.2 gm of zinc amalgam. The tube is immersed in a boiling water bath for 7 minutes and cooled under a tap. 0.05 ml of freshly prepared solution (0.1%) of sodium nitrite is added. The absorbance is measured at 440 nm.
10. The calibration curve is prepared by using solutions of pure strychnine ferrocyanide.
Method II ― Estimation by Colorimetry (Brucine):
1. 0.1 gm of the alkaloidal sample (containing both brucine and strychinine) is dissolved in 20 ml of 1% H2SO4 with gentle heating.
2. The solution is transferred to a 100 ml graduated cylinder, rinsed and made up to 30 ml with 1% H2SO4.
3. If the alkaloids are present as salts, they must be converted to free bases by treating with alkali and extracting with chloroform. After evaporation of the chloroform, 0.1 gm of the dry residue is treated as above.
4. Different aliquots of standard brucine solutions are prepared from the stock solution.
5. 10 ml of the mixture (1:1) of nitric acid and 20% H2SO4 is added to the above test and standard samples. The solution is stirred and after one minute, 2 ml of a saturated aqueous solution of potassium chlorate is added to the sample and standard.
6. The solution is well stirred and, if necessary, diluted to 50 or 100 ml, according to colour intensity.
7. The absorbance is measured in a suitable absorption meter and the amount of brucine is calculated from a calibration curve.
Method III ― By HPLC:
A reversed-phase high-performance liquid chromatographic procedure can be used for the quantitative analysis of strychinine and brucine in S. nux-vomica and S. ignatii.
Procedure:
1. Chromatograph:
LKB gradient pump 2249 equipped with a diode-array detector, Hewlett-Packard model 1040 M, series 2, operating at 260 nm.
2. Column:
Lichrospher 60 RP8 Select B Merck (250 x 4 mm; with a particle size of 5 µm) as the stationary phase.
3. Standard Alkaloid Solution:
Approximately 10 mg each of strychnine and brucine alkaloids are precisely weighed and dissolved in 100 ml of ethanol. Injection volume – 10 µl.
4. Plant Material:
Strychnos nux-vomica seeds and Strychnos ignatti seeds are collected from a reliable source and authenticated by the taxonomist.
5. Extraction of the Alkaloids:
The dried powdered plant material (500 mg) is macerated with a mixture of diethyl ether (1.5 ml), ammonia (0.25 ml) and ethanol (0.5 ml) for 2 hr and then extracted with 50 ml of CHCl3. The residue after evaporating the solvent is dissolved in ethanol and the final volume is brought up to 50 ml with ethanol. Solutions are passed through a 0.45 µm filtration disc HVLP. Injection volume – 10 µl.
6. Mobile Phase:
Solvent A ― MeCN. Solvent B ― aq solution of sodium salt of heptanesulfonic acid (1 gm in 420 ml) and fitted at pH 3.2 with phosphoric acid (0.5%). Elution is done with 25% of solvent A and 75% of solvent B. Flow rate ― 1.0 ml/minute.
7. Linear relationships are obtained by plotting the peak area against the amount of each alkaloid injected (µg in 10 µl injection volume) with UV detection at 260 nm, at least up to 2.2 µg of strychnine and 2.3 µg of brucine.
No comments yet.