Determination of dehydroandrographolide in Andrographis paniculata by thin-layer scanning method - Master's thesis - Dissertation

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Determination of Andrographis paniculata by Dual-Wavelength Thin-Layer Scanning

Dehydroandrographolide content is simple to determine, with a sample recovery rate of 99.9%.

Key words: thin layer scanning method, dehydroandrographolide, andrographolide content

Andrographis paniculata (Burn.f.) Nees is included in the 1995 edition of the Chinese Pharmacopoeia, known for its heat-clearing, detoxifying, blood-cooling, and swelling-reducing effects. Several compounds have been isolated from this plant, including andrographolide, neo-andrographolide, deoxy-andrographolide, and dehydrated andrographolide. Current analytical methods include alkaline hydrolysis, colorimetry, and thin-layer scanning, primarily measuring the first three internal esters or total ester content. However, due to overlapping spots on the TLC plate, separation can be challenging. This study uses dual-wavelength thin-layer scanning to distinguish dehydrated andrographolide based on differences in ultraviolet absorption wavelengths.

1. Experimental Part

1.1 Instruments and Reagents

The equipment used includes the Japan Island Law CS-930 dual-wavelength thin-layer scanner, CAMAG NANOMAT III spotter, and a CAMAG double groove chamber (10 cm × 20 cm). The silica gel G plates were produced by Qingdao Marine Chemical Plant. 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

Silica gel GF₂₅₄ plates (10 cm × 20 cm) were used, activated at 110°C for 1 hour. The developing solvent was acetonitrile–methanol (15:7.5). After development, deoxy-andrographolide showed no fluorescence under UV light (254 nm), but formed a dark purple-red spot after spraying with 2% 3,5-dinitrobenzoic acid in hexanol and potassium hydroxide solution. Dehydrated andrographolide also showed a similar response.

1.2.2 Scanning Conditions

A dual-wavelength reflection method was applied with λ_max at 263 nm and λ_ref at 371 nm. The absorption spectrum of the reference substance was recorded between 200–370 nm.

1.3 Standard Curve Preparation

After vacuum drying for 24 hours, 5 mg of dehydrated andrographolide was dissolved in 5 ml hexanol. Solutions of 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 μL were spotted, developed, and scanned. A linear regression equation was obtained: Y = 0.054 + 2.337X, with r = 0.988, within a concentration range of 0.45–4.50 μg/spot.

1.4 Spotting Stability Test

The integral value of the spot remained stable for up to 2.5 hours, indicating good stability of the method.

1.5 Spotting Precision Test

Six replicate spots showed an RSD of 2.79%, confirming the method’s precision.

1.6 Recovery Test

0.25 g of powdered Andrographis paniculata was spiked with a known concentration of reference solution. The average recovery was 99.9% (n=5, RSD=2.70%).

1.7 Sample Analysis

0.5 g of coarse powder was extracted with ethanol, sonicated, filtered, and concentrated. The test solution was compared with the reference solution using TLC and scanning. A total of 14 samples from different sources were analyzed, with separate measurements for leaves and stems. Results are summarized in Table 1.

2. Conclusion

2.1 Extraction Methods

Three extraction methods—ethanol, heat reflux, cold soaking, and ultrasonic extraction—were tested. The most efficient and practical method was found to be ultrasonic extraction. The room temperature during development should be maintained around 26°C for consistent results.

2.2 Analytical Findings

The content of dehydroandrographolide varied between leaves and stems, with leaves generally containing higher levels. Based on these findings, the Chinese Pharmacopoeia (1995 edition) requires that the leaf content must not be less than 35%, ensuring the quality and efficacy of the medicinal material.