New Report of Triterpenoid Betulinic Acid along with Oleanolic Acid fromAchyranthes asperaby Reversed-Phase-Ultra Flow Liquid Chromatographic Analysis and Confirmation Using High-Performance Thin-Layer Chromatographic and Fourier Transform-Infrared Spectroscopic Techniques

Triterpenoid betulinic acid (BA) was detected, quantified, and reported for the first time from leaf extract of Achyranthes aspera along with much known oleanolic acid (OA). Extraction was achieved using ultrasonic exposure, and reversed-phase.ultra flow liquid chromatographic (RP.UFLC) technique was employed during investigation. RP.UFLC separation was achieved on a Hibar 250–4.6 mm, 5 μ, Lichrospher 100, C18e column using methanol and water (90:10) as mobile phase with pH adjusted to 5.0 using glacial acetic acid (GAA) in an isocratic mode. The content of BA (0.25 mg g−1 fresh weight [FW]) was ∼75% higher than OA (0.06 mg g−1 FW). These results suggest BA to be the major triterpenoid compared to OA in the leaf of A. aspera. High-performance thin-layer chromatography (HPTLC) separation of the two triterpenic acids (oleanolic and betulinic acid) was also achieved on silica gel G 60 F254, 50 ×~ 100 mm glass TLC plates, using benzene, ethyl acetate, and formic acid as solvent system in a ratio of 67.9:22.7:9.4. Anisaldehyde reagent was used for detection. The method was used for the screening of oleanolic and betulinic acids from A. aspera leaf extract. Similarly, Fourier transform-infrared (FT-IR) spectroscopic analysis was done in the mid IR region of 400–4000 cm−1 with 64 scan speed using OMNIC 8.1 (ver. 8.1.210) software. The results were also supported by HPTLC and FT-IR data.