Transcript Document

Simultaneous quantification of
bergenin, catechin, and gallic acid
from
Bergenia ciliata and Bergenia ligulata
by using thin-layer chromatography
张
慧
2012213034
Journal of Food Composition and Analysis 21 (2008) 496– 500
Contents
1. Introduction
2. Materials and methods
3. Results and discussion
4. Conclusion
1. Introduction
Bergenia ciliata Sternb. and Bergenia ligulata Wall.
Bergenia genus belongs to the family Saxifragaceae.
It is native to central Asia and found in Afghanistan,
China and in Himalaya.
Indian Systems of Medicine
fevers, diarrhea, and pulmonary affections
phenolic compounds
bactericidal activity,
inhibition of HIV replication,
free radical scavenging activity,
cytotoxic activity,
gastric protective action
anti-inflammatory activity.
1. Introduction
The methanol extract of B. ciliata exhibited significant
anti-inflammatory potential by inhibition of 31.4±1.09%
in granuloma weight.
 B. ligulata extract had no effect in preventing stone
formation but was significantly beneficial in dissolving
preformed stones.
Alcohol extract of B. ligulata exhibits significant
anti-inflammatory, analgesic, and diuretic properties.
1. Introduction
quantification and standardization
Quantitative analysis aims to to separate and identify the
marker compounds from herbs or herbal preparations and
then uses them as indicators or standards to assess quality.
the therapeutic importance of B. ciliata and B. ligulata
A simple TLC densitometric method is developed.
2. Materials and methods
2.1. Plant material
B. ciliata and B. ligulata
2.2. Chemicals
All the chemicals used in the experiments were of analytical grade.
2.3. TLC conditions
 Spotting device: Linomat V Automatic sample spotter; CAMAG
 Densitometer:
TLC Scanner 3 is controlled by WinCats software; CAMAG;
 HPTLC plates:
20×10 cm2, 0.2mm thickness precoated with silica gel 60 F254;
 Solvent system: toluene:ethyl acetate:formic acid (4:6:1, v/v).
2. Materials and methods
2.4. Sample preparation
Dried powder of rhizomes, petiole, and leaf (2.5 g) were
extracted exhaustively with methanol (350 mL) separately
under reflux for 1 h on water bath.
2.5. Preparation of standard solution
The stock solutions of bergenin, catechin and gallic acid
(160 mg/mL) each were prepared in methanol.
Standard solutions were prepared by dilution of the
stock solution.
2. Materials and methods
2.6. Calibration curves
Standard solution (10 mL) of bergenin (16–80 ng/mL), catechin
(16–48 ng/mL), and gallic acid (16–56 ng/ml) were applied in
triplicate on precoated silica gel 60 F254 HPTLC plates (E. Merck)
of uniform thickness of 0.2mm.
The plates were developed in a solvent system of toluene:ethyl
acetate:formic acid (4:6:1, v/v) in CAMAG twin trough chamber up
to a distance of 8 cm.
 After development, the plate was dried in air and scanned at
280nm using absorbance reflectance mode by CAMAG Scanner 3
and WINCATS software for bergenin, catechin, and gallic acid.
The peak areas were recorded.
Respective calibration curves were prepared by
plotting peak area vs. concentration of bergenin,catechin, and
gallic acid applied.
2. Materials and methods
2.7. Simultaneous quantification of bergenin, catechin, and
gallic acid in different parts of B. ciliata and B. ligulata
 Suitably diluted sample solutions (10 ml) were applied in
triplicate on a precoated HPTLC plate with the
CAMAG Linomat V Automatic Sample Spotter.
 The plate was developed and scanned at 280 nm.
The peak areas and absorption spectra were recorded.
 The amount of bergenin, catechin,and gallic acid in the
sample was calculated using the respective calibration
curves.
To check the identity of the bands UV absorption
spectrum of each standard was overlayed with the corresponding
band in the sample track.
Overlaying the absorption spectra at start, middle and
end position of the band checked the purity of
the bands in the sample extract.
2.8. Method validation
The method was validated for precision, repeatability, and
accuracy.
3. Results and discussion
Mobile phase composition：
toluene:ethylacetate:formic acid (4:6:1, v/v).
3. Results and discussion
3.1. Linearity and Limits of detection (LOD) and limits
of quantification (LOQ)
The regression equations and correlation coefficients for the
references were :
Bergenin [y=5.552x+554.045] (R2 =0.9994)
Catechin [y =11.293x-142.462] (R2 =0.9994)
Gallic acid [y=10.375x+203.625] (R2 = 0.9996)
3.2. Instrumental precision and intra-day and
inter-day precision
The results were expressed as % relative standard deviation
(%R.S.D.) and standard error (S.E.) that indicated high
precision.
3.3. Recovery
3.4. Quantitative determination
It was found that rhizome, petiole, and leaf samples
of B. ciliata and B. ligulata show a similar but unique TLC
chromatogram.
The assay is proved to be accurate, reproducible, and
sensitive.
4. Conclusion
1.The rhizomes were found to contain higher
concentration of bergenin, catechin, and gallic acid than
other parts of the plants.
2. The method was found to be simple, precise, specific,
sensitive, accurate and can be used for their
quantification in the plant materials.
3. It can be also used in routine quality control of herbal
materials as well as formulations containing any or all of
these compounds.
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