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Summary. A new, simple, and reproducible HPTLC–densitometric method has been es-
tablished and validated for estimation of trigonelline in the leaves of Abrus precatorius L.
and its herbal formulation Chatak® using ICH guidelines. Accelerated solvent extraction
(ASE) as an alternative to conventional techniques was also explored for rapid extrac-
tion. The methanol extracts of leaves, its formulation, and standard solution of trigonel-
line were applied on silica gel F254 HPTLC plates and developed in a twin chamber using
the mobile phase toluene–ethyl acetate–formic acid–methanol (2:3:1.8:3.8, v/v/v/v). The
plates were scanned at 268 nm (λmax of trigonelline) using a Camag TLC scanner 3 with
the CATS 4 software. A linear relationship was obtained between the response (peak
area) and the amount of trigonelline in the range 40–200 ng per spot; the correlation coef-
ficient was 0.9957. The ASE method gives higher extraction efficiency in less time com-
pared to Soxhlet extraction. The HPTLC–densitometric method showed good linearity,
recovery, and high precision. It is useful to analyze trigonelline in A. precatorius leaves
and routine quality control of its marketed formulation.
Key Words: trigonelline, Abrus precatorius L., accelerated solvent extraction, HPTLC–
densitometry, ICH guidelines
Introduction
Abrus precatorius L. (Fabaceae) is an important medicinal plant commonly
known as “Indian liquorice” and found throughout India. In Indian tradi-
tional systems of medicine, leaves of A. precatorius are used for the treat-
ment of gonorrhea, jaundice, biliousness, hemoglobinuric bile, leucoderma,
itching, skin diseases, etc. [1–3]. Pharmacological activities such as anti-
inflammatory [4], antimicrobial [5], neuromuscular [6], and toxicity studies
[7] have been reported for its leaves. It is also a major ingredient of many
marketed polyhedral digestive mixtures such as Chatak®.
Leaves are the foremost source of an alkaloid trigonelline [8] having
diverse pharmacological activities such as hypoglycemic, hypocholes-
terolemic, antiseptic, antimigraine, antitumor, mutagenic, and osmoregula-
tory [9]. Hence, the active principal trigonelline has been used as a marker
0231–2522 © 2012 Akadémiai Kiadó, Budapest
264 B.B. Kumbhalkar et al.
Experimental
Plant Material
The leaves of A. precatorius were collected during the winter of 2009 from
Pune, India. The plant sample was identified, authenticated, and deposited
in the crude drug repository of Agharkar Research Institute, Pune (Voucher
specimen number L-049). The sample was powdered, passed through an 80-
mesh sieve, and stored in an airtight container at 25°C for further studies.
Chemicals
Standard trigonelline (CAS 535-83-1; purity ≥98.0%) was purchased from
Fluka-Aldrich Chemical (Steinheim, Germany). Silica gel F254 HPTLC plates
were purchased from Merck (Darmstadt, Germany). Chatak®, a digestive
mixture (Aishwarya Products, Pune, India), was procured from the local
market. Other analytical-grade solvents and reagents were obtained from
SD Fine Chemicals (Mumbai, India).
Soxhlet Extraction
Accurately weighted (2.5 g) powdered leaves and the Chatak® sample were
extracted exhaustively with methanol (50 mL) using a Soxhlet apparatus for
6–10 h. The optimum yield was obtained in methanol with an extraction
time of 8 h. The extracts were concentrated under reduced temperature and
Development of an HPTLC–Densitometric Method 265
pressure using a rotary evaporator. The yields of the methanol extract of the
leaves and Chatak® samples were 0.9475 and 0.67 g, respectively.
Powdered leaves and the Chatak® sample (2.5 g) were extracted with
methanol and placed in the stainless steel cell of a Dionex ASE™ 100 system
(Sunnyvale, CA, USA). The extraction was performed at 100 bar and at
three temperatures, 60, 80, and 100°C, for 15 min (1 cycle). The optimum
yield was obtained at 80°C. In the experiment at 80°C, five replicate cycles
were performed. The extracts were concentrated under reduced tempera-
ture and pressure using a rotary evaporator. The yields of the methanol ex-
tract of the leaves and the Chatak® sample were 0.969 and 0.703 g, respec-
tively.
Chromatography
was repeated thrice to plot a graph of the response (peak area) versus the
amount of trigonelline.
Quantification of Trigonelline
Suitably diluted solutions of test samples (10 μL) were applied in triplicate
on an HPTLC plate along with the standard. The plate was developed un-
der predetermined conditions as described above and scanned at 268 nm
(λmax of trigonelline). The peak areas were recorded, and the trigonelline
content in the samples was calculated using the calibration plot.
Table II. Results from study of intraday and interday precision for trigonelline
80 1.09 0.94
120 0.89 0.83
160 1.18 1.23
aMean ± SD (n = 3)
270 B.B. Kumbhalkar et al.
Conclusion
A new, simple, accurate, precise, reproducible, and sensitive HPTLC–
densitometric method has been established for the determination of
trigonelline in leaves of A. precatorius and its commercial product Chatak®.
Comparison of the extraction yields of the trigonelline reveals that ASE is a
simple and efficient method which is helpful for standardization and rou-
tine quality control of raw materials and herbal products containing A. pre-
catorius leaves as ingredients. It provides significant advantages in terms of
greater specificity and rapid analysis.
Acknowledgments
The authors are grateful to the Director and the In-charge, Botany Group,
Agharkar Research Institute, Pune, India, for providing the facilities and en-
couragement throughout the work.
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Accepted by MWH