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Development of TLC fingerprint for quality assessment of Siriraj Kaewbenjakun recipe

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Nuntika Huntonpong , Phornnapa Chareonkij , Pravit Akarasereenont , Sirikul Chotewuttakorn ,
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Wanlapha Ananta , Prasopporn Punpeng , Tawee Laohapand .
1 Department of Pharmacology, Center of Applied Thai Traditional Medicine, Faculty of Medicine,
2 Siriraj Hospital, Mahidol University, Bangkok 10700. Thailand.
Objective: To develop fingerprint and parameters for quality assessment of Siriraj Kaewbenjakun recipe
using thin layer chromatography (TLC) technique.

Material and Methods: Water extract and 80% ethanolic extract of 4 batches of Siriraj Kaewbenjakun

solution (100 mg/ml) were separately loaded on TLC silica gel 60 F plate. Various mobile phases were
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optimized to establish TLC fingerprint of phenolic components. The chromatogram after being sprayed
with fast blue salt Reagent (FBS) were detected under UV , UV and visible light. Only clear and sharp
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bands by FBS were selected to analyze relative retention factor (rR). Consistency among batches was
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validated by number of band and %CV of rR . Synthetic steroid contamination in the recipe was detected
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by developing TLC plate with steroid mobile phase, then sprayed with Tetrazolium Blue Chloride Reagent
(TBC).

Results: One of six mobile systems for phenolic separation was selected to establish chromatogram

fingerprint in this study. By FBS spraying reagent, 2 bands were detected in water extract while 3 bands
were detected in 80% ethanolic extract. Using gallic and caffeic acid as phenolic markers, each rR of 4
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batches was calculated and summarized in the table below. For TBC spraying reagent, no band was

detected which indicated that there was no synthetic steroid contamination in any batches of Siriraj
Kaewbenjakun recipe.

Table rR of 4 batches of Siriraj Kaewbenjakun recipe water and 80% ethanolic extract detected by FBS spray.
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Extraction rR to gallic = R of substance/R of gallic acid rR to caffeic= R of substance/R of caffeic acid
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Batch1 Batch2 Batch3 Batch4 mean %cv Batch1 Batch2 Batch3 Batch4 mean %cv
Water b 1 1.79 1.79 1.75 1.75 1.77 1.30 1.02 1.02 1.00 1.00 1.01 1.14
b 2 2.88 2.92 2.92 3.00 2.95 1.71 1.64 1.67 1.67 1.67 1.66 0.90
EtOH b 1 ND 1.11 1.11 1.11 1.11 0.00 ND 0.59 0.59 0.59 0.59 0.00
b 2 ND 1.78 1.78 1.78 1.78 0.00 ND 0.94 0.94 0.94 0.94 0.00
b 3 ND 3.33 3.33 3.28 3.31 0.88 ND 1.76 1.76 1.73 1.75 0.99
Conclusion: Chromatogram fingerprint of phenolic components of Siriraj Kaewbenjakun was developed.

Number of bands and rR of the different batches were similar to each other. %CV of rR was less than 2.
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It, therefore, may suggest that these 2 parameters may be suitable for consistency assessment of the batch

preparations. Moreover, there was no synthetic steroid contamination in the 4 batches of the recipe assayed.