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TLC-Based Metabolite Profiling and Bioactivity-Based Scientific Validation for Use of Water Extracts in AYUSH Formulations.

ABSTRACT: We aimed to develop a chromatographic method for scientific validation of water extract of some important Indian traditional plants used in AYUSH-based formulation as immunomodulator and to evaluate their bioactive potential. Fruits of Phyllanthus emblica L. and Piper nigrum L., stem of Tinospora cordifolia (Willd.) Miers, rhizome of Curcuma longa L., leaves of Ocimum sanctum L. and Achillea millefolium L., roots of Withania somnifera L., and stem bark of Azadirachta indica A. Juss. were coarsely powdered and extracted in three different solvents (water, ethanol, and hydroethanol). The antioxidant potential was determined through 1, 1-diphenyl-2-picrylhydrazyl and ferric reducing capacity methods. Thin-layer chromatography (TLC) was carried out for the comparative metabolite profiling of the extracts using toluene, ethyl acetate, and formic acid (5 : 4 : 1, v/v/v) as a solvent system. In vitro immunomodulatory activity of the extracts has been tested on splenocyte proliferation and pinocytic assay. Hydroethanolic extract (HEE) of most of the plant materials has the highest phenolic and flavonoid contents, followed by water extract (WE) and ethanolic extract (EE), whereas the water extracts of most of the plant material showed better antioxidant activity. Almost all extract exhibited splenocyte proliferation and pinocytic activity in a dose-dependent manner. But water extract showed significantly higher splenocyte proliferation and pinocytic activity as compared to the other two extracts. TLC analysis resulted in detection of totally 63 and 56 metabolites at 254 nm and 366 nm, respectively. Through principal component analysis (PCA), it was observed that metabolite pattern of different extracts from same plant materials may be different or similar. This preliminary result can be used for quality evaluation and to develop a synergy-based polyherbal combination of water extracts of selected plant materials.

SUBMITTER: Zahiruddin S

PROVIDER: S-EPMC8741349 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundIn molecular imaging with dynamic PET, the binding and dissociation of a targeted tracer is characterized by kinetics modeling which requires the arterial concentration of the tracer to be measured accurately. Once in the body the radiolabeled parent tracer may be subjected to hydrolysis, demethylation/dealkylation and other biochemical processes, resulting in the production and accumulation of different metabolites in blood which can be labeled with the same PET radionuclide as the parent. Since these radio-metabolites cannot be distinguished by PET scanning from the parent tracer, their contribution to the arterial concentration curve has to be removed for the accurate estimation of kinetic parameters from kinetic analysis of dynamic PET. High-performance liquid chromatography has been used to separate and measure radio-metabolites in blood plasma; however, the method is labor intensive and remains a challenge to implement for each individual patient. The purpose of this study is to develop an alternate technique based on thin layer chromatography (TLC) and a sensitive commercial autoradiography system (Beaver, Ai4R, Nantes, France) to measure radio-metabolites in blood plasma of two targeted tracers-[18F]FAZA and [18F]FEPPA, for imaging hypoxia and inflammation, respectively.ResultsRadioactivity as low as 17 Bq in 2 µL of pig's plasma can be detected on the TLC plate using autoradiography. Peaks corresponding to the parent tracer and radio-metabolites could be distinguished in the line profile through each sample (n = 8) in the autoradiographic image. Significant intersubject and intra-subject variability in radio-metabolites production could be observed with both tracers. For [18F]FEPPA, 50% of plasma activity was from radio-metabolites as early as 5-min post injection, while for [18F]FAZA, significant metabolites did not appear until 50-min post. Simulation study investigating the effect of radio-metabolite in the estimation of kinetic parameters indicated that 32-400% parameter error can result without radio-metabolites correction.ConclusionTLC coupled with autoradiography is a good alternative to high-performance liquid chromatography for radio-metabolite correction. The advantages of requiring only small blood samples (~ 100 μL) and of analyzing multiple samples simultaneously, make the method suitable for individual dynamic PET studies.

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TLC-Based Metabolite Profiling and Bioactivity-Based Scientific Validation for Use of Water Extracts in AYUSH Formulations.

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