ABR Vol 14 [5] September 2023 319 | P age © 2023 Author Advances in Bioresearch Adv. Biores., Vol 14 (5) September 2023: 319-326 ©2023 Society of Education, India Print ISSN 0976-4585; Online ISSN 2277-1573 Journal’s URL:http://www.soeagra.com/abr.html CODEN: ABRDC3 DOI: 10.15515/abr.0976-4585.14.5.319326 ORIGINAL ARTICLE HPTLC Method Development and Validation for Quantification of Biomarker β-Sitosterol and Betulinic Acid in the Leaves, Stem and Roots of Spermadictyon suaveolens Roxb. Divya Lobo and Aparna Saraf* The Institute of Science, 15, Madame Cama Road, Fort, Mumbai-400032, India HOD, Department of Botany, The Institute of Science, Homi Bha Bha State University, 15, Madame Cama Road, Mumbai 400032, India. E-mail: draparnasaraf@yahoo.co.in ABSTRACT A simple method has been established for the, β-Sitosterol and Betulinic Acid biomarker in the Leaves, Stem and Roots of Spermadictyon suaveolens Roxb belonging to the Rubiaceae family. Chromatography was performed on Silica gel 60 F254 precoated plates using mobile phase solvents of n-Hexane: Ethyl Acetate 8:2 (v/v) for both the biomarkers, thereafter derivatized with Anisaldehyde Sulphuric Acid (ASA) and then scanned, quantified at 540 nm. A clear band for β-Sitosterol at RF 0.21 + 0.03 and for Betulinic Acid at RF 0.15 + 0.03 was observed. The intra-day and inter-day precisions with mean % RSD value (n=6) was found to be 1.902% and 1.186% and for Betulinic Acid was 2.980% and 2.117% which proves that the method created is in fact specific and reproducible. The Quantity of β-Sitosterol found in leaf, stem and root were 0.1518μg, 0.1582μg and 0.1014μg and for Betulinic Acid were, 0.0043μg, 0.0592μg and 0.1012μg of plant part respectively. The Recovery has been seen to be >95% for β-Sitosterol and >80% for Betulinic Acid in the root of the plant. Thus, quality assurance of crude drugs as well as the determination of dosage may be decided by referencing to this method. Keywords: Spermadictyon suaveolens Roxb., β-Sitosterol, Betulinic Acid, HPTLC, Quantification, Validation. Received 11.05.2023 Revised 21.06.2023 Accepted 25.09.2023 How to cite this article: Divya Lobo And Aparna Saraf. HPTLC Method Development and Validation for Quantification of Biomarker β-Sitosterol and Betulinic Acid in the Leaves, Stem and Roots of Spermadictyon suaveolens Roxb.. Adv. Biores. Vol 14 [5] September. 2023. 319-326. INTRODUCTION Many Phyto-constituents from plants act as marker compound to determine the authenticity of the part of the plant or plant itself. Well established and validated protocol may be used for the profiling of the phytochemical and quantification of standard compounds in medicinal plants. Spermadictyon suaveolens Roxb with synonym of Hamiltonia suaveolens, Forest Champa, is a woody shrub, 2-3 meters in height, from the family Rubiaceae mostly found in the Indo-Pak subcontinent [1]. Among the plethora of medicinal properties, its used for, particular usage in, viral infections, diabetes and wound healing are of significance [2]. Leaves and bark have shown anti-oxidant and anti-microbial properties [3] while the stem has been used by traditional practitioners to cure Herpes zoster virus known as ‘Nagin’[4]. Roots are applied to relieve joint pain [5] as well as in rheumatoid arthritis and bloody dysentery in animals [2]. β-Sitosterol and Betulinic Acid found in the leaves, stem and root of Spermadictyon suaveolens Roxb can be further utilized for authentication of the plant and its parts, as a biomarker. β-Sitosterol is a phytosterol which has numerous medicinal properties [6]. Found in most plants like nuts, legumes, olive oil etc. shows diverse nutraceutical benefits like anticancer, anti- inflammatory, anti-diabetic, antioxidant, wound healing, lipid lowering etc.[7]–[10]. Betulinic Acid is a Pentacyclic Triterpenoid that has proved to show anti-retroviral, anti-diabetic, anti- inflammatory, anti-malarial as well as anti-cancer and anti-obesity properties [11] . Found in dried fruits like raisins and trees like birch eucalyptus and plane trees [12]. Advances in Bioresearch