1124 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 al-
kaloids (ajamalicine, papaverine, cinchonine,
piperine, berberine) and some plant materials con-
taining 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 sam-
ples by industries dealing with herbal drugs for
standardization of plant materials containing alka-
loids 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 alka-
loids, 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 hu-
man 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 com-
ponents is essential for the standardization of the plants. As al-
kaloids have therapeutic efficacy and bioenhancing proper-
ties, the estimation of total alkaloids in plants bearing alka-
loids and formulations that contain them as therapeutic agents
becomes essential.
Even though there are many methods for determining indi-
vidual 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
Received February 8, 2003. Accepted by JS May 20, 2003.
1
Author to whom correspondence should be addressed; e-mail:
pharmacognosy1@rediffmail.com.
thiocyanates (2) and reinecke salts (3) are available. Chro-
matographic methods such as paper (4), high-performance
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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 man-
ual errors depending on the skill of the person performing the
procedure; hence the values for the samples may vary signifi-
cantly, and an instrument method would be more reliable for
validation purposes and in accordance with GLP norms. Mod-
ern chromatographic methods are not suitable for herbal mate-
rials where the therapeutic activity derives from a complex
mixture of many closely related compounds or class of com-
pounds, which could still be quantified by spectrophotometric
methods or other traditional methods. Generally, reinecke
salts and cobalt thiocyante salts are not used for routine alka-
loid 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) re-
ported that in at least 19 surveys for alkaloids, DR was used.
Many other workers have used DR for screening of alka-
loids (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 spectrophotomet-
ric method, which can be used for routine estimation of alka-
loids. The amount of bismuth present is estimated after precipi-
tating 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
(a) Bismuth nitrate pentahydrate.—Qualigens (Mumbai,
India); standard solution; purity 99.8%.
 SREEVIDYA & MEHROTRA: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 6, 2003 1125
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 bis-
muth 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 alka-
loid 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 pow-
dered plant material (root) was extracted with 25 mL 2%
aqueous acetic acid at room temperature for 10 min. The pro-
cedure was repeated 3 times. The extracts were mixed and di-
luted to 100 mL with 2% aqueous acetic acid.
(g) Extraction of Solanum nigrum.—10 g coarsely pow-
dered plant material (whole plant) was Soxhlet-extracted with
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 eth-
anol 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
The calibration curve was obtained with Bismuth nitrate
pentahydrate stock solution. Series dilutions of the stock solu-
tion 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
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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 centri-
fuged. The centrifugate was checked for complete precipita-
tion 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 solu-
tion. The brownish black precipitate formed was then centri-
fuged. 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 solu-
tion was diluted to 10 mL in a standard flask with distilled wa-
ter; 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.
1126 SREEVIDYA & MEHROTRA: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 6, 2003

Table 2. Determination of concentrations of different Results and Discussion

alkaloids for recovery and accuracya

Actual concn, Observed concn, The alkaloids are precipitated as (BiI3)(Alk·HI) (15) by DR
Alkaloids mg/mL mg/mL Recovery, % (KBiI4). Bismuth forms a yellow bismuth complex
Ajamalicine 0.100 0.109 109.00 ± 0.82 {Bi[CS(NH2)3]}(NO3)3 in nitric acid medium with thiourea.
Maintaining the pH is very important for precipitation by DR.
Papaverine 0.104 0.105 100.96 ± 0.86
If the medium is more acidic, the precipitate formed dissolves
Cinchonine 0.104 0.102 98.07 ± 0.57 in excessive acid. The bismuth from the alkaloidal complex is
Piperine 0.133 0.128 96.24 ± 0.62 completely released by disodium sulfide (12). If the concen-
Berberine 0.205 0.201 98.04 ± 0.45 tration of bismuth is high, precipitation occurs after addition
of thiourea solution. Also, if the concentration of thiourea is
a
Average of 3 determinations ± RSD, %. high, precipitation occurs. Therefore, dilute solutions and 3%
solution of thiourea was used. The reagents used are stable, and

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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 suit-
able dilution factor into consideration. The factor is obtained
from the standard curve, which is a constant for different con-
centrations.
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 Formula-
tions (14). According to this method, the weighed plant mate-
rial (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 set-
tled, 100 mL of the solution is drawn out and filtered into a
separator and washed with a few milliliters of a mixture of sol-
vent ether and chloroform, and shaken with 0.5N sulfuric acid
for complete extraction of alkaloids. The combined acid ex-
tract is filtered and made alkaline with dilute ammonia solu-
tion. 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 sol-
vent is again removed. The evaporation with alcohol is re-
peated, and the residue is dried to constant weight in vacuum
desiccator and weighed as total alkaloids.
the complex formed is also stable for more than 24 h. The cali-
bration graph between concentration and absorbance is linear
with R2 value 0.9994 and obeys Lambert-Beer’s law in the con-
centration range of 0.06–50 mg/mL (Table 1). Because the com-
plex 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 esti-
mated 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 dif-
ferences 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 al-
kaloids precipitable by DR and can be applied to single alka-
loid 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 con-
taining alkaloids and alkaloid-containing pharmaceutical
products. This method is not applicable to plant materials con-
taining 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, Added amount of Observed amount of alkaloid,

mg/10 g material berberine, mg/mL mg/10 g material Recovery, %

56.693 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 (2) Deltombe, J., Leboutte, G., & Rosier, N. (1962) J. Pharm.
plant materials by the proposed method using DR and Belg. 17, 236–238
Method II given in PMUa
(3) Weyers, J., & Skora, M. (1962) Diss. Pharm. 14, 201–205
Amount of total
alkaloid by the Amount of alkaloid by (4) Narain, S.T. (1967) Pharm. Zentralle Dtschl. 106, 443–447
proposed method Method II given in (5) Wang, M., & Zhu, M. (1984) Yaowu Fenzi Zazhi 4, 12–15
using DR, mg/10 g PMU, mg/10 g
Plant material material (6) Cui, J., Zhang, G., & Wang, M. (1985) Yaoxue Xuebao 20,
59–66
Berberis aristata 56.693 ± 0.67 58.059 ± 0.55
(7) Fransworth, N.R. (1966) J. Pharm. Sci. 55, 247
Piper longum 19.540 ± 0.78 18.245 ± 0.48
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,
a
Average of 3 determinations ± RSD, %; PMU = Physicochemical Tananarive, India, pp 19–24
Standards of Unani Formulations (14).

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(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,
Acknowledgments 1844–1861

We thank P. Pushpangadan (National Botanical Research
Institute) for providing encouragement and facilities. N.
Sreevidya is grateful to the Council of Scientific and Indus-
trial 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
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