Author(s):
Venkata Suresh Ponnuru, Prerna Mehta
Email(s):
Email ID Not Available
DOI:
10.52711/2231-5713.2025.00048 
Address:
Venkata Suresh Ponnuru1, Prerna Mehta2
1Department of Pharmaceutical Analysis, Chalapathi Institute of Pharmaceutical Sciences, Lam, Guntur, Andhra Pradesh, India.
2Department of Biotechnology, GD Rungta College of Science and Technology Bhilai - 490024, Chhattisgarh, India.
*Corresponding Author
Published In:
Volume - 15,
Issue - 4,
Year - 2025
ABSTRACT:
Enicostemma littorale blume (E. littorale) has traditionally been used to treat various health conditions, including diabetes. Swertisin (SWT), a confirmed phytoconstituent of E. littorale, is responsible for its antidiabetic properties. Although the SWT was previously extracted using conventional methods, the yield was low. To address this issue, researchers have developed improved extraction techniques for SWT. This new approach involved initial ethanol extraction and a sequential extraction process. After obtaining the resultant ethanol extract, experimental design procedures were used for maximum recovery. Using the Box-Behnken design, systematic optimization of the extraction efficiency was accomplished. It was found that n-butanol: Methanol (4.62:5.28, v/v), sonication time (30 min), and solvent volume (15 mL) from E. littorale showed a maximum recovery of 3.36 mg/g, and statistical analysis revealed that the optimized extraction conditions were significantly different (p<0.001) from the low, medium, and high-level extraction parameters. The developed method is effective in extracting SWT, and this approach can be utilized to enhance the extraction of phytochemicals from raw extracts.
Cite this article:
Venkata Suresh Ponnuru, Prerna Mehta. Systematic Optimization of Swertisin-Rich Extract from Enicostemma littorale using Box-Behnken Design and Quantification by LC-MS/MS: A Smarter Approach to Extraction Efficiency. Asian Journal of Pharmacy and Technology. 2025; 15(4):323-0. doi: 10.52711/2231-5713.2025.00048
Cite(Electronic):
Venkata Suresh Ponnuru, Prerna Mehta. Systematic Optimization of Swertisin-Rich Extract from Enicostemma littorale using Box-Behnken Design and Quantification by LC-MS/MS: A Smarter Approach to Extraction Efficiency. Asian Journal of Pharmacy and Technology. 2025; 15(4):323-0. doi: 10.52711/2231-5713.2025.00048 Available on: https://ajptonline.com/AbstractView.aspx?PID=2025-15-4-1
REFERENCES:
1. Murali B, Upadhyaya UM, Goyal RK. Effect of chronic treatment with Enicostemma littorale in non-insulin-dependent diabetic (NIDDM) rats. Journal of Ethnopharmacology. 2002; 81(2); 199-204. https://doi.org/10.1016/S0378-8741(02)00077-6
2. Vijayvargia R, Kumar M, Gupta S. Hypoglycemic effect of aqueous extract of Enicostemma littorale Blume (chhota chirayata) on alloxan induced diabetes mellitus in rats. Indian Journal of Experimental Biology. 2000; 38(8); 781-4.
3. Maroo J, Ghosh A, Mathur R, Vasu VT, Gupta S. Antidiabetic efficacy of Enicostemma littorale methanol extract in alloxan-induced diabetic rats. Pharmaceutical Biology. 2003; 41(5); 388–391. https://doi.org/10.1076/phbi.41.5.388.15943
4. Vasu VT, Ashwinikumar C, Maroo J, Gupta S, Gupta S. Antidiabetic effect of Enicostemma littorale Blume aqueous extract in newly diagnosed non-insulin-dependent diabetes mellitus patients (NIDDM): a preliminary investigation. Oriental Pharmacy and Experimental Medicine. 2003; 3; 84–89. https://doi.org/10.3742/OPEM.2003.3.2.084
5. Srinivasan M, Padmanabhan M, Prince PS. Effect of aqueous Enicostemma littorale Blume extract on key carbohydrate metabolic enzymes, lipid peroxides and antioxidants in alloxan‐induced diabetic rats. Journal of Pharmacy and Pharmacology. 2005; 57(4); 497-503. https://doi.org/10.1211/0022357055722
6. Upadhyay UM, Goyal RK. Efficacy of Enicostemma littorale in Type 2 diabetic patients. Phytother Res. 2004 Mar; 18(3); 233-5. https://doi.org/10.1002/ptr.1434
7. Nampalliwar, A. R., & Godatwar, P. Randomised Controlled Trial on the efficacy of Mamajjaka (Enicostemma littorale Linn.) in the Management of Madhumeha (Diabetes Mellitus). International Journal of Ayurvedic Medicine. 2024; 15(1); 148–153. https://doi.org/10.47552/ijam.v15i1.4276
8. Zanwar, S. B., Patel, K. V., & Mandhane, S. N. In Vitro and In Vivo Anticancer Activity of E. littorale Extract on Hepatocellular Carcinoma. Journal of Natural Remedies. 2024; 24(4); 869–876. https://doi.org/10.18311/jnr/2024/36504
9. Wang X, Wang T. Swertiamarin exerts anticancer effects on human cervical cancer cells via induction of apoptosis, inhibition of cell migration and targeting of MEK-ERK pathway. Trop J Pharm Res. 2021; 20(1); 75-81. https://doi.org/10.4314/tjpr.v20i1.12
10. Roy S, Niranjan C, Jyothi T, Shankrayya M, Vishawanath K, Prabhu K, et al. Antiulcer and anti-inflammatory activity of aerial parts Enicostemma littorale blume. J Young Pharm. 2010; 2(4); 369–73. https://doi.org/10.4103/0975-1483.71629
11. Vaidya H, Rajani M, Sudarsanam V, Padh H, Goyal R. Antihyperlipidaemic activity of swertiamarin, a secoiridoid glycoside in poloxamer-407-induced hyperlipidaemic rats. Journal of Natural Medicines. 2009; 63(4); 437–442.
12. Saranya R, Thirumalai T, Hemalatha M, Balaji R, David E. Pharmacognosy of Enicostemma littorale: a review. Asian Pac J Trop Biomed. 2013 Jan; 3(1); 79-84. https://doi.org/10.1016/S2221-1691(13)60028-3
13. Maroo J, Vasu VT, Aalinkeel R, Gupta S. Glucose lowering effect of aqueous extract of Enicostemma littorale Blume in diabetes: a possible mechanism of action. J Ethnopharmacol. 2002; 81(3); 317-20. https://doi.org/10.1016/S0378-8741(02)00095-8
14. Dadheech N, Soni S, Srivastava A, Dadheech S, Gupta S, Gopurappilly R, Bhonde RR, Gupta S. A Small Molecule Swertisin from Enicostemma littorale Differentiates NIH3T3 Cells into Islet-Like Clusters and Restores Normoglycemia upon Transplantation in Diabetic Balb/c Mice. Evid Based Complement Alternat Med. 2013; 280392. https://doi.org/10.1155/2013/280392
15. Tanna S, Shukla VJ, Prajapati PK. Physico-phytochemical evaluation of aqueous extract of Mamajjaka Enicostemma littorale. Int J Pharm Bio Arch. 2010; 1(3); 309–312.
16. Ghosal SS, Sharma AK, Chaudhuri PV. Chemical constituents of Gentianaceae IX: natural occurrence of Erythrocentaurin in Enicostemma hissopifolium and Swertia lawii. J Pharm Sci. 1974; 63; 944–945.
17. Jahan E, Perveen S, Malik A. Verticilliside, a new flavone C-glucoside from Enicostemma verticillatum. J Asian Nat Prod Res. 2009; 11(3); 257-60. https://doi.org/10.1080/10286020802675019
18. Desai PD, Ganguly AK, Govindachari TR, Joshi BS, Kamat VN, Manmade AH, et al. et al. Chemical investigation of some Indian medicinal plants: Part II. Ind J Chem. 1966; 4; 457–459.
19. Leelaprakash G, Mohan Dass S. Antimicrobial activity and phytochemical screening of methanol extract of Enicostemma axillare. Int J Pharm Pharm Sci. 2012; 4(1); 342–348.
20. Sathiskumar R, Lakshmi PTV, Annamalai A. Comparative analyses of non-enzymatic and enzymatic antioxidants of Enicostemma littorale Blume. Int J Pharma Bio Sci. 2010; 1(2); 1–16.
21. Jaishree V, Badami S, Krishnamurthy PT. Antioxidant and hepatoprotective effect of the ethyl acetate extract of Enicostemma axillare (Lam). Raynal against CCL4-induced liver injury in rats. Indian J Exp Biol. 2010; 48(9); 896-904.
22. Varghese A, Saboo P, Wairkar S. Bioactivity guided fractionation of methanolic extract of Terminalia arjuna for its CYP3A and CYP2D inhibition in rat liver microsomes. Biopharmaceutics & Drug Disposition. 2018 Mar; 39(3): 143-51. https://doi.org/10.1002/bdd.2121
23. Rolta R, Kumar V, Sourirajan A, Upadhyay NK, Dev K. Bioassay guided fractionation of rhizome extract of Rheum emodi wall as bio-availability enhancer of antibiotics against bacterial and fungal pathogens. Journal of ethnopharmacology. 2020 Jul 15; 257:112867. https://doi.org/10.1016/j.jep.2020.112867
24. Elsayed EW, El-Ashmawy AA, Mursi NM, Emara LH. Optimization of gliclazide loaded alginate-gelatin beads employing central composite design. Drug Dev Ind Pharm. 2019; 45(12); 1959-1972. https://doi.org/10.1080/03639045.2019.1689992
25. Toyota H, Asai T, Oku N. Process optimization by use of design of experiments: Application for liposomalization of FK506. Eur J Pharm Sci. 2017; 102; 196-202. https://doi.org/10.1016/j.ejps.2017.03.007
26. Abu-Dahab, R. & Afifi, F. Antiproliferative activity of selected medicinal plants of Jordan against a breast adenocarcinoma cell line (MCF7). Sci. Pharm. 2007; 75(3); 121–146.
27. Perez RM. Anti-inflammatory activity of compounds isolated from plants. Scientific World Journal. 2001; 1; 713-84. https://doi.org/10.1100/tsw.2001.77
28. Hwang ES, Thi ND. Effects of Extraction and Processing Methods on Antioxidant Compound Contents and Radical Scavenging Activities of Laver (Porphyra tenera). Prev Nutr Food Sci. 2014; 19(1); 40-8. https://doi.org/10.3746/pnf.2014.19.1.040
29. Ferreira SL, Bruns RE, Ferreira HS, Matos GD, David JM, Brandão GC, da Silva EG, Portugal LA, dos Reis PS, Souza AS, dos Santos WN. Box-Behnken design: an alternative for the optimization of analytical methods. Anal Chim Acta. 2007; 597(2); 179-86. https://doi.org/10.1016/j.aca.2007.07.011
30. Ahmad A, Alkharfy KM, Wani TA, Raish M. Application of Box-Behnken design for ultrasonic-assisted extraction of polysaccharides from Paeonia emodi. Int J Biol Macromol. 2015; 72; 990-7. https://doi.org/10.1016/j.ijbiomac.2014.10.011
31. Nalawade V, Vora A. Box–Behnken design directed optimization for sensitivity assessment of anti-platelet drugs. Drug Development and Industrial Pharmacy. 2019 Sep 2; 45(9): 1515-22. https://doi.org/10.1080/03639045.2019.1634092
32. R.H. A.C. Myers, Response Surface Methodology: Process and Product Optimization Using Designed Experiments. 4th+Edition, ISBN: 978-1-118-91601-8, Wiley, 2016; 865 pages.
33. Gullberg, P. Jonsson, A. Nordström, M. Sjöström, T. Moritz, Design of experiments: An efficient strategy to identify factors influencing extraction and derivatization of Arabidopsis thaliana samples in metabolomic studies with gas chromatography/mass spectrometry, Analytical Biochemistry. 2004; 283–295. https://doi.org/10.1016/j.ab.2004.04.037
34. Barmpalexis P, Grypioti A, Eleftheriadis GK, Fatouros DG. Development of a new aprepitant liquisolid formulation with the aid of artificial neural networks and genetic programming. Aaps Pharmscitech. 2018 Feb; 19; 741-52. https://doi.org/10.1208/s12249-017-0893-z
35. Yadav KS, Sawant KK. Formulation optimization of etoposide loaded PLGA nanoparticles by double factorial design and their evaluation. Current Drug Delivery. 2010 Jan 1; 7(1): 51-64. http://dx.doi.org/10.2174/156720110790396517
36. Nalawade VV, Peepliwal AL, Shidhaye SS, Pandey S. Formulation and evaluation of oral sustained release dry suspension of metformin hydrochloride. Eur J Pharm Med Res. 2016; 3; 447-57.
37. Ogbonna JD, Attama AA, Ofokansi KC, Patil SB, Basarkar GD. Optimization of formulation processes using Design Expert® Software for preparation of polymeric blends-artesunate-amodiaquine HCl microparticles. Journal of Drug Delivery Science and Technology. 2017 Jun 1; 39: 36-49. https://doi.org/10.1016/j.jddst.2017.02.011
38. Ganorkar SB, Dhumal DM, Shirkhedkar AA. Development and validation of simple RP-HPLC-PDA analytical protocol for zileuton assisted with design of experiments for robustness determination. Arabian Journal of Chemistry. 2017 Feb 1; 10(2): 273-82. https://doi.org/10.1016/j.arabjc.2014.03.009.
39. Moreiras G, Leão JM, Gago‐Martínez A. Design of experiments for the optimization of electrospray ionization in the LC‐MS/MS analysis of ciguatoxins. Journal of Mass Spectrometry. 2018 Nov; 53(11): 1059-69. https://doi.org/10.1002/jms.4281
40. Patel TP, Soni S, Parikh P, Gosai J, Chruvattil R, Gupta S. Swertiamarin: An Active Lead from Enicostemma littorale Regulates Hepatic and Adipose Tissue Gene Expression by Targeting PPAR- γ and Improves Insulin Sensitivity in Experimental NIDDM Rat Model. Evid Based Complement Alternat Med. 2013; 2013: 358673. https://doi.org/10.1155/2013/358673
41. Vishwakarma S, Bagul M, Rajani M, Goyal R. A sensitive HPTLC method for estimation of Swertiamarin in Enicostemma littorale Blume, Swertia chirata (Wall) Clarke, and in formulations containing E. littorale. JPC-Journal of Planar Chromatography-Modern TLC. 2004; 17(2); 128-31. https://doi.org/10.1556/jpc.17.2004.2.8
42. Vishwakarma S, Rajani M, Bagul M, Goyal R. A rapid method for the isolation of swertiamarin from Enicostemma littorale. Pharmaceutical Biology. 2004 Jan 1; 42(6); 400-3. https://doi.org/10.1080/13880200490885095
43. Bhandari P, Gupta AP, Singh B, Kaul VK. HPTLC determination of swertiamarin and amarogentin in Swertia species from the Western Himalayas. JPC–Journal of Planar Chromatography–Modern TLC. 2006 Jun; 19: 212-5. https://doi.org/10.1556/JPC.19.2006.3.8
44. Alam P, Ali M, Singh R, Shakeel F. A new HPTLC densitometric method for analysis of swertiamarin in Enicostemma littorale and commercial formulations. Natural Product Research. 2011; 25(1); 17-25. https://doi.org/10.1080/14786411003754348
45. Sawant L, Prabhakar B, Pandita N. A validated quantitative HPTLC method for analysis of biomarkers in Enicostemma littorale Blume. JPC-Journal of Planar Chromatography-Modern TLC. 2011; 24(6); 497-502. https://doi.org/10.1556/jpc.24.2011.6.8
46. Ahamad J, Hassan N, Amin S, Mir SR. Development and Validation of a High-Performance Thin-Layer Chromatographic—Densitometric Method for the Quantification of Swertiamarin in Traditional Bitters and Formulations. JPC–Journal of Planar Chromatography–Modern TLC. 2015; 28; 61-6. https://doi.org/10.1556/JPC.28.2015.1.10
47. Rana VS, Dhanani TU, Kumar SA. Improved and rapid HPLC-PDA method for identification and Quantification of swertiamarin in the aerial parts of Enicostemma axillare. Malaysian J. Pharma Sci. 2012; 10; 1-10.
48. Li HL, Peng XJ, He CJ, Feng EF, Xu GL, Rao GX. Development and validation of an LC-ESI-MS/MS method for the determination of swertiamarin in rat plasma and its application in pharmacokinetics. Journal of Chromatography B. 2011; 879; 1653–1658. https://doi.org/10.1016/j.jchromb.2011.04.003
49. Feng B, Zhu H, Guan J, Zhao L, Gu J, Yin L, Fawcett JP, Liu W. A rapid and sensitive UFLC-MS/MS method for the simultaneous determination of gentiopicroside and swertiamarin in rat plasma and its application in pharmacokinetics. J Pharm Pharmacol. 2014; 66(10); 1369-76. https://doi.org/10.1111/jphp.12266
50. Akei H, Nakauchi K, Yoshizaki F. Analysis of swertiamarin in swertia herb and preparations containing this crude drug by capillary electrophoresis. Anal Sci. 2001; 17(7); 885-8. https://doi.org/10.2116/analsci.17.885
51. Patel, M. B. and Mishra, S. H. Quantitative analysis of marker constituent swertisin in Enicostemma hyssopifolium Verdoon by RP-HPLC and HPTLC. Acta Chromatographica. 2012; 24(1); 2083-5736. https://doi.org/10.1556/achrom.24.2012.1.8