Phyllanthus species (Family Euphorbiaceae) has been used in traditional medicine of several countries as a cure for numerous diseases, including jaundice and hepatitis. This study is an attempt to evaluate the immunomodulatory activity of various fractions, column eluents of ethyl acetate fraction, and their polyphenols. Phyllanthus maderaspatensis were standardized using high-performance liquid chromatography to identify and quantify polyphenols, and purification of polyphenols was carried out using vacuum liquid chromatography. Subsequently, we tested various fractions, column eluents of ethyl acetate fraction, and polyphenols in vitro to assess their impact on nitric oxide (NO) production in LPS-stimulated mouse macrophage RAW 264.7 cells. The ethyl acetate fraction (100 μg mL⁻¹) had a more significant stimulatory effect on LPS-stimulated NO production by the RAW 264.7 cells. We found that the ethyl acetate fraction contains a high amount of catechin, quercetin, ellagic acid kaempferol, and rutin, which are responsible for immunomodulation. The ethyl acetate fraction at concentrations of 25 and 50 μg mL⁻¹ had a significant inhibitory effect and 100 μg mL⁻¹ had a more significant stimulatory effect when compared with the LPS control. The percentage of inhibition by LPS control ranged from zero percentage, kaempferol ranged from 45.4% at 50 μg mL⁻¹ to 41.88% at 100 μg mL⁻¹, catechin ranged from 50% at 50 μg mL⁻¹ to 35.28% at 100 μg mL⁻¹, rutin ranged from 36.2% at 50 μg mL⁻¹ to 47.44% at 100 μg mL⁻¹, gallic acid ranged from 28.4% at 50 μg mL⁻¹ to 50.9% at 100 μg mL⁻¹, ellagic acid ranged from 45.12% at 50 μg mL⁻¹ to 38.64% at 100 μg mL⁻¹, and purified quercetin ranged from 26.2% at 50 μg mL⁻¹to 45.48% at 100 μg mL⁻¹. As NO plays an important role in the immune function, polyphenols’ treatment could modulate several aspects of host defense mechanisms owing to the stimulation of the inducible nitric oxide synthase.

Quantifying and optimizing the polyphenol content of Phyllanthus maderaspatensis was accomplished using a single-solvent HPTLC system. Analyzing hydroalcoholic extracts for kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid, we simultaneously quantified and optimized their concentration. In the experiment, the methanol to water ratio (%), temperature (°C), and time of extraction (min) were all optimized using a Box-Behnken statistical design. Kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid were among the dependent variables analyzed. In the HPTLC separation, silica gel 60F254 plates were used, and toluene, ethyl acetate, and formic acid (5:4:1) made up the mobile phase. For kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid, densitometric measurements were carried out using the absorbance mode at 254 nm. Hydroalcoholic extract of P. maderaspatensis contains rutin (0.344), catechin (2.62), gallic acid (0.93), ellagic acid (0.172), quercetin (0.0108) and kaempferol (0.06). Further, it may be affected by more than one factor at a time, resulting in a varying degree of reaction. A negative correlation was found between X1 (extraction time (min)) and X2 (temperature), as well as X1 and X3 (solvent ratios). Taking these characteristics into consideration, the method outlined here is a validated HPTLC method for measuring kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid.