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张 慧 2012213034

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  1. Simultaneous quantification of bergenin, catechin, and gallic acid fromBergenia ciliata and Bergenia ligulata by using thin-layer chromatography 张 慧 2012213034 Journal of Food Composition and Analysis 21 (2008) 496– 500

  2. Contents 1. Introduction 2. Materials and methods 3. Results and discussion 4. Conclusion

  3. 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.

  4. 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.

  5. 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.

  6. 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).

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 3. Results and discussion Mobile phase composition: toluene:ethylacetate:formic acid (4:6:1, v/v).

  12. 3. Results and discussion

  13. 3.1. Linearity and Limits of detection (LOD) and limits of quantification (LOQ)

  14. 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)

  15. 3.2. Instrumental precision and intra-day and inter-day precision

  16. The results were expressed as % relative standard deviation (%R.S.D.) and standard error (S.E.) that indicated high precision.

  17. 3.3. Recovery

  18. 3.4. Quantitative determination

  19. 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.

  20. 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.

  21. Thank you!