Determination of dehydroandrographolide in Andrographis paniculata by thin-layer scanning method - Master's thesis - Dissertation
Determination of Andrographis paniculata by dual-wavelength thin-layer scanning
Dehydroandrographolide is a key compound found in Andrographis paniculata, and the method described here offers an accurate and reliable way to determine its content. The procedure involves thin-layer chromatography followed by dual-wavelength scanning, which allows for precise quantification even when compounds have overlapping spots.
The sample recovery rate achieved in this study was as high as 99.9%, indicating that the method is highly effective and suitable for routine analysis. This technique is particularly useful in quality control settings where consistency and accuracy are essential.
Key words: thin layer scanning method, Andrographolide dehydration, Andrographolide content
Andrographis paniculata (Burn.f.) Nees is a well-known medicinal herb included in the 1995 edition of the Chinese Pharmacopoeia. It is valued for its ability to clear heat, detoxify, cool the blood, and reduce swelling. Research has identified several compounds in this plant, including andrographolide, neo-andrographolide, deoxy-andrographolide, and dehydrated andrographolide. These compounds have been isolated from the plant, with various analytical methods used to determine their concentrations.
Traditional methods such as alkaline hydrolysis, colorimetric analysis, and thin-layer scanning have been employed to measure these compounds. However, due to the similarity in retention times and UV absorption spectra, some compounds are difficult to distinguish. In this experiment, the dual-wavelength thin-layer scanning technique was successfully applied to overcome this challenge, enabling the accurate determination of dehydroandrographolide.
1 Experimental Part
1.1 Instruments and Reagents
The experiments were conducted using a Japan Island Law CS-930 double long thin-layer scanner, a CAMAG NANOMAT III thin-layer spotting instrument, and a CAMAG double groove expansion cylinder (10 cm × 20 cm). Silica gel G plates were obtained from Qingdao Marine Chemical Plant and used for thin-layer chromatography. All reagents were of high purity.
1.2 Thin-Layer Scanning Method for Determination of Dehydrated Andrographolide in Andrographis paniculata
1.2.1 Thin Layer Chromatography Conditions
A silica gel GF' plate (10 cm × 20 cm) was used, activated at 110°C for one hour. The mobile phase consisted of acetonide acetone (15:7.5). After development, the spot corresponding to deoxy-andrographolide showed no purple-red color under UV light (254 nm), while the dehydrated compound produced a distinct purple-red spot after spraying with a 2% 3,5-dinitrobenzoic acid solution and potassium hydroxide.
1.2.2 Scanning Conditions
Dual-wavelength reflection scanning was performed at 263 nm and 371 nm. The absorption spectrum of the reference substance was analyzed between 200–370 nm.
1.3 Standard Curve Preparation
A standard curve was constructed by preparing different concentrations of the reference solution. The regression equation was Y = 0.054 + 2.337X, with a correlation coefficient of r = 0.988. The linear range was 0.45–4.50 µg.
1.4 Spotting Precision Test
Six samples were prepared and scanned. The relative standard deviation (RSD) was 2.79%, indicating good precision.
1.5 Recovery Test
The average recovery was 99.9% (n=5, RSD=2.70%), confirming the reliability of the method.
1.6 Sample Determination
A total of 14 samples were analyzed, including leaves and stems. The results showed that the leaves had higher levels of dehydroandrographolide compared to the stems.
2 Summary
2.1 Extraction Methods
Three extraction techniques—ethanol, heat reflux, and cold soaking—were tested. The most efficient and practical method was found to be ethanol extraction combined with ultrasonic treatment. The room temperature during thin-layer development was maintained at around 26°C for optimal results.
2.2 Content Variation
The study revealed that the concentration of dehydroandrographolide varies significantly between leaves and stems. Based on these findings, the Chinese Pharmacopoeia (1995 edition) recommends that the leaves should contain at least 35% of the active compound to ensure medicinal quality.
This method provides a simple, accurate, and reproducible approach for the quality control of Andrographis paniculata, making it a valuable tool in pharmaceutical and herbal medicine research.
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