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SREEVIDYA & MEHROTRA: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 6, 2003
DIETARY SUPPLEMENTS
Spectrophotometric Method for Estimation of Alkaloids Precipitable with Dragendorff’s Reagent in Plant Materials NARASIMHAN SREEVIDYA and SHANTA MEHROTRA1 National Botanical Research Institute, 2, Rana Pratap Marg, Lucknow, India 226 001
A rapid, easy, and simple spectrophotometric method was developed for the estimation of total alkaloids precipitated by Dragendorff’s reagent (DR) in plant materials. It is based on the formation of yellow bismuth complex in nitric acid medium with thiourea. The yellow-colored complex formed obeys Lambert-Beer’s law in the concentration range of 0.06–50 mg/mL with lmax at 435 nm. Using this method, the alkaloidal percentage of certain alkaloids (ajamalicine, papaverine, cinchonine, piperine, berberine) and some plant materials containing alkaloids (Berberis aristata, Solanum nigrum, and Piper longum) were determined. The method was compared with other methods. It can be used for routine analysis of commercial samples by industries dealing with herbal drugs for standardization of plant materials containing alkaloids and for alkaloid-containing pharmaceutical products.
A
lkaloids are responsible for the therapeutic effect of many plant materials such as Rauwolfia serpentina, Strychnos nux-vomica, and cinchona bark. Some alkaloids, such as aristolochic acids, are toxic. Many references are available for the toxicity of aristolochic acids, and plants bearing them are banned for use in medications used by human beings (1). No single chemical component is responsible for the medicinal properties of plant-based drugs, and their synergic action or bioenhancement is due to the presence of other chemical substances in the plant material. Therefore, the determination of the total amount of different classes of components is essential for the standardization of the plants. As alkaloids have therapeutic efficacy and bioenhancing properties, the estimation of total alkaloids in plants bearing alkaloids and formulations that contain them as therapeutic agents becomes essential. Even though there are many methods for determining individual alkaloidal content in formulations and extracts, there are relatively few methods for determination of total alkaloidal content. Titrimetric methods using different types of dyes as indicators and colorimetric methods using cobalt
thiocyanates (2) and reinecke salts (3) are available. Chromatographic methods such as paper (4), high-performance thin-layer chromatography (5), and liquid chromatography (6) are also available for individual alkaloids, depending on the type. However, there are certain demerits in the aforesaid methods. With a titrimetric method, there are chances of manual errors depending on the skill of the person performing the procedure; hence the values for the samples may vary significantly, and an instrument method would be more reliable for validation purposes and in accordance with GLP norms. Modern chromatographic methods are not suitable for herbal materials where the therapeutic activity derives from a complex mixture of many closely related compounds or class of compounds, which could still be quantified by spectrophotometric methods or other traditional methods. Generally, reinecke salts and cobalt thiocyante salts are not used for routine alkaloid detection. When compared with the above 2 reagents, Dragendorff’s reagent (DR) is very common and is used for routine detection of alkaloids. In his review on the biological and phytochemical screening of plants, Fransworth (7) reported that in at least 19 surveys for alkaloids, DR was used. Many other workers have used DR for screening of alkaloids (8–10). Alkaloids were quantified with DR titrimetrically by estimation of liberated EDTA with 0.01M ZnSO4 with Na3BO3 buffer (11) and iodometrically (12). A spectrophotometric method of quantification method based on DR is more convenient for a routine analysis of the total alkaloidal content. Here we propose a simple, rapid, efficient spectrophotometric method, which can be used for routine estimation of alkaloids. The amount of bismuth present is estimated after precipitating the alkaloids with DR. DR cannot precipitate purine alkaloids (13); however, very few plants contain such alkaloids. Experimental Apparatus (a) Spectrophotometer.—UV 1601 UV-Vis spectrophotometer (Shimadzu, Tokyo, Japan) with 1 cm UV quartz cell. (b) Analytical balance.—AB54–S (Metler Toledo, Greifensee, Switzerland). Reagents
Received February 8, 2003. Accepted by JS May 20, 2003. 1 Author to whom correspondence should be addressed; e-mail:
[email protected].
(a) Bismuth nitrate pentahydrate.—Qualigens (Mumbai, India); standard solution; purity 99.8%.
SREEVIDYA & MEHROTRA: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 6, 2003 1125
50 mL 2% methanolic acetic acid for 1.5 h, and the extract was diluted to 100 mL with 2% methanolic acetic acid. (h) Extraction of Piper longum.—10 g coarsely powdered plant material (fruits) was Soxhlet-extracted with 100 mL ethanol for 2 h, and the extract was concentrated to 10 mL. To the concentrated extract, 10 mL 10% alcoholic KOH was added and further diluted to 100 mL with ethanol. Procedure for Calibration Curve
Figure 1. Absorption spectrum of bismuth nitrate–thiourea complex.
(b) Thiourea.—SD Fine (Mumbai, India). (c) Disodium sulfide.—Qualigens. (d) Ajamalicine.—Sisco (Sisco Research Laboratories, Bombay, India); purity 98%. (e) Papaverine.—Lancaster (Morecambe, UK); purity 98%. (f) Cinchonine.—Lancaster; purity 98%. (g) Piperine.—Lancaster; purity 98%. (h) Berberine.—Lancaster; purity 98%. All other chemicals used were of analytical grade. We collected genuine plant materials of Solanum nigrum and Piper longum from the botanic gardens, National Botanical Research Institute, Lucknow, India, and Berberis aristata from Dehradun, India. Solutions (a) DR.—Prepared by mixing (1) solution of 0.8 g bismuth nitrate pentahydrate in 40 mL distilled water and 10 mL glacial acetic acid, and (2) solution of 8.0 g potassium iodide in 20 mL distilled water. (b) Standard bismuth nitrate solution.—Bismuth nitrate stock solution was made by dissolving 10 mg Bi(NO3)3·5H2O in 5 mL concentrated nitric acid and diluting to 100 mL with distilled water. (c) Thiourea, 3%.—Prepared by dissolving 3 g in 100 mL distilled water. (d) Disodium sulfide, 1%.—Prepared by dissolving 1 g in 100 mL distilled water. (e) Stock solutions of alkaloid.—10 mg of each pure alkaloid was dissolved in 10 mL solvent, e.g., 10 mg berberine was dissolved in warm distilled water and diluted to 10 mL with warm distilled water. Similarly, stock solutions of piperine in 95% alcohol and papaverine, cinchonine, and ajamalicine were prepared in 95% alcohol containing 2% acetic acid. (f) Extraction of Berberis aristata.—10 g coarsely powdered plant material (root) was extracted with 25 mL 2% aqueous acetic acid at room temperature for 10 min. The procedure was repeated 3 times. The extracts were mixed and diluted to 100 mL with 2% aqueous acetic acid. (g) Extraction of Solanum nigrum.—10 g coarsely powdered plant material (whole plant) was Soxhlet-extracted with
The calibration curve was obtained with Bismuth nitrate pentahydrate stock solution. Series dilutions of the stock solution were made by pipetting out 1, 2, 3, 4, 5, 6, 7, 8, and 9 mL stock solution into separate 10 mL standard flasks and diluting to volume with distilled water. A 1 mL amount of this solution was taken, and 5 mL thiourea solution was added to it. The absorbance value of the yellow solution was measured at 435 nm against colorless reagent blanks. Figure 1 shows the spectrum of the yellow bismuth thiourea solution. Procedure for Assay of Alkaloids and Plant Extracts A 5 mL amount of the extract/solution was taken and the pH was maintained at 2–2.5 with dilute HCl. A 2 mL amount of DR was added to it, and the precipitate formed was centrifuged. The centrifugate was checked for complete precipitation by adding DR. After centrifugation, the centrifugate was decanted completely and meticulously. The precipitate was further washed with alcohol. The filtrate was discarded and the residue was then treated with 2 mL disodium sulfide solution. The brownish black precipitate formed was then centrifuged. Completion of precipitation was checked by adding 2 drops of disodium sulfide. The residue was dissolved in 2 mL concentrated nitric acid, with warming if necessary. This solution was diluted to 10 mL in a standard flask with distilled water; 1 mL was then pipetted out, and 5 mL thiourea solution was added to it. The absorbance was measured at 435 nm
Table 1. Optical characteristics and precision for bismuth–thiourea complex Parameters
Characteristics
Color
Yellow
lmax, nm
343 and 435
Stability
Stable
Beer’s law range, mg/mL
0.05–50
Molar absorptivity, L/mol
3.039 ´ 103
Limit of detection, mg/mL
0.02
Regression equation
y = mx + c
Regression coefficient (R2)
0.9994
Slope (m)
0.0662
Intercept (c)
0.0144 a
Standard error
0.0051
Coefficient of varianceb
0.1565
a,b
n = 3 replicates.
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Table 2. Determination of concentrations of different alkaloids for recovery and accuracya Alkaloids
Actual concn, Observed concn, mg/mL mg/mL
Recovery, %
Ajamalicine
0.100
0.109
109.00 ± 0.82
Papaverine
0.104
0.105
100.96 ± 0.86
Cinchonine
0.104
0.102
98.07 ± 0.57
Piperine
0.133
0.128
96.24 ± 0.62
Berberine
0.205
0.201
98.04 ± 0.45
a
Average of 3 determinations ± RSD, %.
against the blank containing nitric acid and thiourea. The amount of bismuth present in the solution was calculated by multiplying the absorbance values with the factor, taking suitable dilution factor into consideration. The factor is obtained from the standard curve, which is a constant for different concentrations. Factor = concentration/absorbance A comparison of total alkaloids in all the plant materials taken was made by using the Method II given in the CCRUM publication, Physicochemical Standards of Unani Formulations (14). According to this method, the weighed plant material (15 g) is extracted with 200 mL chloroform–ether–alcohol (90%; 23 + 8 + 2.5) for 10 min. To this, 6 mL dilute ammonia is added, and the flask is shaken for 1 h and allowed to stand for 8 h with occasional shaking. To this, 10 mL water is added, and the mixture is shaken vigorously. When the drug has settled, 100 mL of the solution is drawn out and filtered into a separator and washed with a few milliliters of a mixture of solvent ether and chloroform, and shaken with 0.5N sulfuric acid for complete extraction of alkaloids. The combined acid extract is filtered and made alkaline with dilute ammonia solution. The alkaloids are liberated with chloroform and washed with chloroform. The chloroform washes and the extract are poured in a tared conical flask. The chloroform is distilled off and the solvent is removed completely in a vacuum desiccator. A 5 mL amount of alcohol is added to the residue, and the solvent is again removed. The evaporation with alcohol is repeated, and the residue is dried to constant weight in vacuum desiccator and weighed as total alkaloids.
Results and Discussion The alkaloids are precipitated as (BiI3)(Alk·HI) (15) by DR (KBiI4). Bismuth forms a yellow bismuth complex {Bi[CS(NH2)3]}(NO3)3 in nitric acid medium with thiourea. Maintaining the pH is very important for precipitation by DR. If the medium is more acidic, the precipitate formed dissolves in excessive acid. The bismuth from the alkaloidal complex is completely released by disodium sulfide (12). If the concentration of bismuth is high, precipitation occurs after addition of thiourea solution. Also, if the concentration of thiourea is high, precipitation occurs. Therefore, dilute solutions and 3% solution of thiourea was used. The reagents used are stable, and the complex formed is also stable for more than 24 h. The calibration graph between concentration and absorbance is linear with R2 value 0.9994 and obeys Lambert-Beer’s law in the concentration range of 0.06–50 mg/mL (Table 1). Because the complex formed is 1:1, the amount of bismuth corresponds to the amount of alkaloids present; hence the method of preparation of DR does not affect the result. The amount of alkaloid present is calculated by multiplying absorbance with factor. The recovery was appreciable, indicating the accuracy of this experimental procedure (Table 2). Differences of estimated concentrations to actual concentrations varied from –1.5 to +10%. The differences may be due to the presence of some traces of unreacted reagent in the positive side, and differences in the negative side may be due to the impurity of the alkaloids, the purity of which was only 98%. When this method was applied to plant materials, the same type of accuracy was observed, which was determined by addition of different amounts of berberine solution to Berberis aristata alkaloidal extract (Table 3). A comparison with the Physicochemical Standards of Unani Formulations method (14) was made to check whether the results obtained by this method were comparable with the method developed using DR (Table 4). In conclusion, this method will determine the amount of alkaloids precipitable by DR and can be applied to single alkaloid or to alkaloid-bearing plants. This method can be used for routine analysis of commercial samples by industries dealing with herbal drugs for standardization of plant materials containing alkaloids and alkaloid-containing pharmaceutical products. This method is not applicable to plant materials containing purine alkaloids because DR cannot precipitate purine alkaloids. However, few plants contain such alkaloids.
Table 3. Recovery studies in Berberis aristataa Amount of alkaloid in Berberis aristata, mg/10 g material 56.693
Added amount of berberine, mg/mL
Observed amount of alkaloid, mg/10 g material
Recovery, %
1.000
57.543
99.73 ± 0.62
56.693
5.000
61.050
98.95 ± 0.71
56.693
10.000
65.945
98.87 ± 0.56
a
Average of 3 determinations ± RSD, %.
SREEVIDYA & MEHROTRA: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 6, 2003 1127 Table 4. Determination of total alkaloids in different plant materials by the proposed method using DR and Method II given in PMUa
(2) Deltombe, J., Leboutte, G., & Rosier, N. (1962) J. Pharm. Belg. 17, 236–238 (3) Weyers, J., & Skora, M. (1962) Diss. Pharm. 14, 201–205
Amount of total alkaloid by the proposed method using DR, mg/10 g material
Amount of alkaloid by Method II given in PMU, mg/10 g material
Berberis aristata
56.693 ± 0.67
58.059 ± 0.55
Piper longum
19.540 ± 0.78
18.245 ± 0.48
(7) Fransworth, N.R. (1966) J. Pharm. Sci. 55, 247
Solanum nigrum
43.612 ± 0.56
43.578 ± 0.75
(8) Kiang, A.K., & Douglas, B. (1957) in Proc. Third Congress, Pan Indian Ocean Science Association, Section G, Tananarive, India, pp 19–24
Plant
a
Average of 3 determinations ± RSD, %; PMU = Physicochemical Standards of Unani Formulations (14).
Acknowledgments We thank P. Pushpangadan (National Botanical Research Institute) for providing encouragement and facilities. N. Sreevidya is grateful to the Council of Scientific and Industrial Research (New Delhi, India) for financial support and to Subha Rastogi and R.P. Rastogi for helpful discussions (and to M. Vijayakumar for his assistance throughout the work). References (1) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans (2002) Vol. 82, IARC Press, Lyon, France, pp 79, 83–84
(4) Narain, S.T. (1967) Pharm. Zentralle Dtschl. 106, 443–447 (5) Wang, M., & Zhu, M. (1984) Yaowu Fenzi Zazhi 4, 12–15 (6) Cui, J., Zhang, G., & Wang, M. (1985) Yaoxue Xuebao 20, 59–66
(9) Webb, L.J. (1949) in Bull. 241, Commonwealth Scientific and Industrial Research Organisation, Melbourne, Australia, 99 pp (10) Abish, E., & Reichstein, T. (1960) Hel. Chem. Acta 43, 1844–1861 (11) Surovi, Z., Dugandzic, M., & Blagojevic, Z. (1963) Arch. Farm 13, 299–304 (12) Petkov, P.A. (1965) Sb. Tr. Visshiya Med. Inst. IP. Pavlov, Plovdiv 17, 23–31 (13) Evans, W.C. (2002) Trease and Evans Pharmacognosy, 15th Ed., W.B. Saunders, Edinburgh, UK, p. 336 (14) Physicochemical Standards of Unani Formulations (1986) Part 1, Central Council for Research in Unani Medicine, New Delhi, India, pp 229–230 (15) Francois, M., & Blanc, L.G. (1922) Comt. Rend. 175, 273–274
