EVALUATING AND QUANTIFYING THE BIOACTIVE POTENTIAL OF HYGROPHILA SPINOSA
DOI:
https://doi.org/10.22159/ajpcr.2025v18i5.54283Keywords:
Phytochemical, Validation, High-performance thin-layer chromatographic, Quercetin, Lupeol, Hygrophila spinosaAbstract
Objectives: The quantification of Quercetin (QR) and Lupeol (LU) in the seed extract of Hygrophila spinosa has been accomplished using a highly efficient, rapid, and precise high-performance thin-layer chromatographic (HPTLC) method.
Methods: This advanced technique employed a carefully optimized mobile phase consisting of toluene, ethyl acetate, methanol, and formic acid in a precise ratio of 5:4:2:0.5 (v/v/v/v) to meticulously elute the QR and LU markers from the extract on silica gel 60 F254 HPTLC plates, measuring 10×10 cm. Detection was conducted at a wavelength of 236 nm, ensuring accurate identification of the compounds.
Results: Results revealed that the hydroalcoholic extract of H. spinosa contains substantial quantities of QR and LU, quantified at 17.0 mg/100 g and 17.4 mg/100 g, respectively. The robustness of the method has been thoroughly validated, demonstrating exceptional linearity, accuracy, precision, and specificity, as well as acceptable limits for both the limit of detection (LOD) and the limit of quantification. The calibration curve displayed remarkable linearity between 100 and 600 ng/band for both QR and LU, with a limit of quantitation of 4.97 ng/band for QR and 3.49 ng/band for LU, and LODs of 1.64 ng/band for QR and 1.15 ng/band for LU.
Conclusion: This innovative method represents a significant advancement in the quantification of valuable bioactive compounds. It can be confidently applied to measure QR and LU levels in H. spinosa seed extracts and related formulations, thereby contributing to the advancement of research and applications in phytochemistry and natural product development.
Downloads
References
Rakshit G, Singh V, Vichitra A, Rajpal R, Chandra P, Choudhury S. A multi-centric double blind homoeopathic pathogenetic trial of Hygrophila spinosa. Indian J Res Homoeopathy. 2014;8(1):9-18. doi: 10.4103/0974-7168.129672
Noor N, Satapathy KB. Phytodiversity of Dhauligiri hill and its adjoining area, Odisha, India: A floristic approach. Plant Arch. 2020 Apr 1;20(1):2093-102.
Kumar CK, Sree MS, Joshna A, Lakshmi SM, Kumar DS. A review on south Indian edible leafy vegetables. J Glob Trends Pharm Sci. 2013 Oct;4(4):1248-56.
Chauhan NS, Dixit VK. Asteracantha longifolia [L.] Nees, acanthaceae: Chemistry, traditional, medicinal uses, and its pharmacological activities-a review. Rev Bras Farmacogn. 2010;20(5):812-7. doi: 10.1590/S0102-695X2010005000022
Pal A, Paul AK. Bacterial endophytes of the medicinal herb Hygrophila spinosa T. Anders and their antimicrobial activity. Br J Pharm Res. 2014;3(4):795-806. doi: 10.9734/BJPR/2014/3698
Verma D, Singh S, Arya R, Rajan S, Arya BS, Khurana A, et al. Morpho-anatomical observations on homoeopathic plant drug Hygrophila spinosa T. Anderson. Pharmacogn J. 2019;11(2):286-91. doi: 10.5530/ pj.2019.11.44
Misra TN, Singh RS, Pandey HS, Singh BK, Pandey RP. Constituents of Asteracantha longifolia. Fitoterapia. 2001 Feb 1;72(2):194-6. doi: 10.1016/s0367-326x(00)00269-0, PMID 11223236
Ali A, Tripathi SK. A pentacyclic triterpenoid from Asteracantha longifolia ness. Asian J Chem. 2007 Jul 1;19(5):3765-9.
Shahid, M., M. M. Khan, A. Hameed, M. Ashraf, and A. Jamil. “Antioxidant enzymes and inorganic elements in seeds and leaves of four potential medicinal plants from Pakistan.” (2012): 281-91.
Patra A, Jha S, Murthy PN. Pharmacognostical standardization of leaves of Hygrophila spinosa T. Anders. Pharmacogn J. 2009;1(2):82-7. doi: 10.4103/0973-7847.70912
Godbole NN, Gunde BG, Srivastava PD. An investigation of oil from seed of Hygrophila spinosa. Oil Soap. 1941 Oct;18(10):206-7. doi: 10.1007/BF02544184
Perumal Samy R, Manikandan J, Al Qahtani M. Evaluation of aromatic plants and compounds used to fight multidrug resistant infections. Evid Based Complement Alternat Med. 2013;2013:525613. doi: 10.1155/2013/525613, PMID 24223059
Lakhanpal P, Rai DK. Quercetin: A versatile flavonoid. Internet J Med Update. 2007 Jul 1;2(2):22-37. doi: 10.4314/ijmu.v2i2.39851
Costa LG, Garrick JM, Roquè PJ, Pellacani C. Mechanisms of neuroprotection by quercetin: Counteracting oxidative stress and more. Oxid Med Cell Longev. 2016;2016(1):2986796. doi: 10.1155/2016/2986796, PMID 26904161
Saleem M, Maddodi N, Abu Zaid M, Khan N, Bin Hafeez B, Asim M, et al. Lupeol inhibits growth of highly aggressive human metastatic melanoma cells in vitro and in vivo by inducing apoptosis. Clin Cancer Res. 2008 Apr 1;14(7):2119-27. doi: 10.1158/1078-0432.CCR-07-4413
Shen X, Si Y, Wang Z, Wang J, Guo Y, Zhang X. Quercetin inhibits the growth of human gastric cancer stem cells by inducing mitochondrial-dependent apoptosis through the inhibition of PI3K/Akt signaling. Int J Mol Med. 2016 Aug 1;38(2):619-26. doi: 10.3892/ijmm.2016.2625, PMID 27278820
Chen S, Jiang H, Wu X, Fang J. Therapeutic effects of quercetin on inflammation, obesity, and type 2 diabetes. Mediators Inflamm. 2016;2016(1):9340637. doi: 10.1155/2016/9340637, PMID 28003714
Marchionatti AM, Pacciaroni A, Tolosa De Talamoni NG. Effects of quercetin and menadione on intestinal calcium absorption and the underlying mechanisms. Comp Biochem Physiol A Mol Integr Physiol. 2013 Jan 1;164(1):215-20. doi: 10.1016/j.cbpa.2012.09.007, PMID 23000882
Townsend EA, Emala CW Sr. Quercetin acutely relaxes airway smooth muscle and potentiates β-agonist-induced relaxation via dual phosphodiesterase inhibition of PLCβ and PDE4. Am J Physiol Lung Cell Mol Physiol. 2013 Sep 1;305(5):L396-403. doi: 10.1152/ ajplung.00125.2013, PMID 23873842
Oliveira-Junior MS, Pereira EP, De Amorim VC, Reis LT, Do Nascimento RP, Da Silva VD, et al. Lupeol inhibits LPS-induced neuroinflammation in cerebellar cultures and induces neuroprotection associated to the modulation of astrocyte response and expression of neurotrophic and inflammatory factors. Int Immunopharmacol. 2019 May 1;70:302-12. doi: 10.1016/j.intimp.2019.02.055, PMID 30852286
Sudhahar V, Kumar SA, Sudharsan PT, Varalakshmi P. Protective effect of lupeol and its ester on cardiac abnormalities in experimental hypercholesterolemia. Vascul Pharmacol. 2007 Jun 1;46(6):412-8. doi: 10.1016/j.vph.2006.12.005, PMID 17336164
Lee C, Lee JW, Seo JY, Hwang SW, Im JP, Kim JS. Lupeol inhibits LPS-induced NF-kappa B signaling in intestinal epithelial cells and macrophages, and attenuates acute and chronic murine colitis. Life Sci. 2016 Feb 1;146:100-8. doi: 10.1016/j.lfs.2016.01.001, PMID 26767626
Thirumalaisamy R, Ameen F, Subramanian A, Selvankumar T, Alwakeel SS, Govarthanan M. In-vitro and in-silico anti-inflammatory activity of lupeol isolated from Crateva adansonii and its hidden molecular mechanism. Int J Pept Res Ther. 2020 Dec;26(4):2179-89. doi: 10.1007/s10989-019-10006-5
Soni LK, Dobhal MP, Arya D, Bhagour K, Parasher P, Gupta RS. In vitro and in vivo antidiabetic activity of isolated fraction of Prosopis cineraria against streptozotocin-induced experimental diabetes: A mechanistic study. Biomed Pharmacother. 2018 Dec 1;108:1015-21. doi: 10.1016/j. biopha.2018.09.099, PMID 30372801
Esposito F, Mandrone M, Del Vecchio C, Carli I, Distinto S, Corona A, et al. Multi-target activity of Hemidesmus indicus decoction against innovative HIV-1 drug targets and characterization of lupeol mode of action. Pathog Dis. 2017 Aug;75(6):ftx065. doi: 10.1093/femspd/ ftx065, PMID 28637198
Shailajan S, Joshi H. Optimized separation and quantification of pharmacologically active markers quercetin, kaempferol, β-sitosterol and lupeol from Cuscuta reflexa Roxb. J Pharm Res. 2011 Jun;4:1851-3.
Upadhye M, Deokate U, Pujari R, Phanse M. Antidiabetic potential of Ficus glomerata roots with a special emphasis on estimation of bioactive compounds by a novel validated HPTLC technique. Indian J Pharm Educ Res. 2022 Apr 1;56(2):470-8. doi: 10.5530/ijper.56.2.68
Gurupriya S, Cathrine L, Pratheema P. HPTLC method for the determination of lupeol from Andrographis echioides leaves. Int J Pharm Pharm Sci. 2018;10(5):102-7. doi: 10.22159/ijpps.2018v10i5.24748
Dwivedi J, Gupta A, Verma S, Paliwal S, Rawat AK. Validated simultaneous high-performance thin-layer chromatographic analysis of ursolic acid, β-sitosterol, lupeol and quercetin in the methanolic fraction of Ichnocarpus frutescens. J Planar Chromatogr Mod TLC. 2019 Apr;32(2):103-8. doi: 10.1556/1006.2019.32.2.4
Kokate CK, Khandelwal GS. Practical Pharmacognosy. 4th ed. Pune: Nirali Prakashsa; 2019. p. 8.2-8.22.
Konuskan DB, Kamiloglu O, Demirkeser O. Fatty acid composition, total phenolic content and antioxidant activity of grape seed oils obtained by cold-pressed and solvent extraction. Indian J Pharm Educ Res. 2019 Jan 1;53(1):144-50. doi: 10.5530/ijper.53.1.19
Saykova I, Tylkowski B, Popovici C, Peev G. Extraction of phenolic and flavonoid compounds from solid wastes of grape seed oil production by cold pressing. J Chem Technol Metall. 2018 Mar 1;53(2):177-90.
ICH, Q2 (R1). Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonization Geneva; 2005. p. 1-13.
Hussain MS, Fareed S, Ali M. Simultaneous HPTLC-UV530 nm analysis and validation of bioactive lupeol and stigmasterol in Hygrophila auriculata (K. Schum) Heine. Asian Pac J Trop Biomed. 2012 Feb 1;2(2):S612-7. doi: 10.1016/S2221-1691(12)60283-4
Hussain MS, Fareed S, Ali M. Hyphenated chromatographic analysis of bioactive gallic acid and quercetin in Hygrophila auriculata (K. Schum) Heine growing wildly in marshy places in India by validated HPTLC method. Asian Pac J Trop Biomed. 2012 Feb 1;2(2):S477-83. doi: 10.1016/S2221-1691(12)60257-3
Ghule B, Agrawal P, Lal P, Kothari D, Kotagale N. Separation and quantification of lupeol in Hygrophila schulli by high-performance thin-layer chromatography. J Planar Chromatogr. 2021 Feb;34(1):79-87. doi: 10.1007/s00764-021-00079-8
Published
How to Cite
Issue
Section
Copyright (c) 2025 DR. LATA P. KOTHAPALLI, MS. GAIKWAD SMRUTI SHRIDHAR, DR. ASHA B. THOMAS

This work is licensed under a Creative Commons Attribution 4.0 International License.
The publication is licensed under CC By and is open access. Copyright is with author and allowed to retain publishing rights without restrictions.