Determination of Quercetin in Extracts and Herbal Products of Phyllanthus niruri by TLC Densitometry Method

Phyllanthus niruri has many pharmacological activities such as antioxidant, anti-inflammatory and anticancer. Phyllanthus niruri has been widely developed from extracts into herbal products. The flavonoid quercetin became an analytical marker compound to ensure the quality of P. niruri extracts in the Indonesian herbal pharmacopeia II. This study aimed to determine the levels of quercetin in extract and herbal products using the TLC Densitometry method. TLC Densitometry method using chloroform: ethyl acetate: formic acid (5:4:1) as the mobile phase and silica GF254 as the stationary phase. The Rf value of the quercetin compound obtained is 0.50. The Rs quercetin is 1.56 in the extract and 2.55 in the product. The maximum wavelength for quercetin is 366 nm. The results of quercetin levels were 3.579% in the extract and 0.0412% in herbal products. The TLC densitometry method can analyze quercetin in extracts and herbal products.

Quercetin can be used as analytical marker compound to ensure quality Phyllanthus niruri extract. Phyllanthus niruri herb extract must contains a total flavonoid not less than 3.20% as quercetin (Indonesian Herbal Pharmacopoeia, 2017). Analytical marker compound was compound in plant that dominant in plant, easy analysis or have important activities (Ihsan et al., 2020). Some previous analytical method to analysis quercetin were spectrophotometry UV-Vis (Soylak et al., 2020), TLC (Ihsan et al., 2019), HPLC (Dinakaran et al., 2018), LC-MS (Kumar et al., 2015), HPTLC (Amir et al., 2013). TLC densitometry method was a simple, selective and inexpensive method for routine analysis in quality assurance of traditional medicines (Hafid et al., 2015). The purpose of this study was to determine the levels of quercetin in extract and herbal product using the TLC Densitometry method.
Extraction method 300 g of Phyllanthus niruri L. was dissolved in 1000 ml ethanol 96%. Then stirred 30 minutes. This solution was macerated for 24 hours (This Procedure was repeated 3 times). The collected filtrate was evaporated with a rotary evaporator until it became an extract.

Preparation of standard solution
Stock solution was prepared 200 ppm. Then diluted to five concentration.

Preparation of test solution
100 mg extract was dissolved with ethanol 96% in 25 ml volumetric flask. This solution was filtered before 3 µl applied in TLC plate. 1000 mg was dissolved with ethanol 96% in 10 ml volumetric flask. This solution was filtered before 8 µl applied in TLC plate.

Optimization of mobile phase
Three composition of mobile phase was evaluated for selection the best separation of quercetin in sample. The composition of mobile phase was shown in Table 1.

Qualitative analysis TLC method
Retardation factor (Rf) and Resolution (Rs) were used to evaluated separation. Spot quercetin standard, extract sample and herbal product were evaluated by scanning at 200-400 nm.

Quantitative analysis TLC method
The curve calibration was used to calculate concentration quercetin in sample. Five concentration of quercetin standard was spotted 2 µl and was scanned at 366 nm.

Result and Discussion
The yield of the Phyllanthus niruri extraction method in this study was 36%. Extraction used ethanol 96% because quercetin quite soluble in alcohol. Extraction of secondary metabolites was influenced by the type of solvent, the solvent to solid ratio, the extraction duration, and the amount of maceration. Mobile phase optimization aimed to obtain the best mobile phase composition that can separate quercetin from another component in sample. The composition and results of Retardation factor (Rf) and Resolution (Rs) were shown in Table 1. The Composition III (Chloroform: ethyl acetate: formic acid = 5:4 :1) was selected because show the best quercetin resolution in extract and herbal product. The Rf value of standard quercetin was 0.5. This Rf also meet the requirement between 0.2-0.8. The separation of quercetin in extract and herbal product of Phyllanthus niruri by composition III of mobile phase was shown in Figure 1.   The result of the determination of the quercetin content in extract was 3.5%. The concentration quercetin in the extract meet the Indonesian herbal pharmacopoeia II. Phyllanthus niruri L. herb extract contains a total flavonoid not less than 3.20% calculated as quercetin. This extraction method can be applied to production extract from Phyllanthus niruri. The result of the determination of the quercetin content in extract was 0.041%. Each herbal product had certain specifications. This is based on consideration of herbal product indication.

Conclusion
The thin layer chromatography densitometry method can be used for analysis of quercetin in extract and herbal product of Phyllanthus niruri.