Enhanced Bioactive Compound Recovery, Heavy Metal Reduction, and Antifungal Activity of Acorus calamus Extracts Obtained by Optimized Supercritical CO2 Extraction

Yada  Nantiyagul; Thaval Rerksngarm; Thanvisith Charoenying

Authors

Yada Nantiyagul Faculty of Allied Health Sciences, Pathumthani University, Pathum Thani, Thailand
Thaval Rerksngarm Faculty of Allied Health Sciences, Pathumthani University, Pathum Thani, Thailand
Thanvisith Charoenying Faculty of Allied Health Sciences, Pathumthani University, Pathum Thani, Thailand https://orcid.org/0009-0007-6874-7388

Keywords:

Acorus calamus, Antifungal activity, Supercritical CO2 extraction, Heavy metal removal, Method validation, HPLC

Abstract

The rise of antifungal resistance has intensified the search for plant-derived alternatives with potent bioactivity. Acorus calamus, traditionally used for medicinal purposes, contains β-asarone, a compound with potential antifungal effects. Given that the rhizomes of A. calamus are prone to heavy metal accumulation, this study optimized supercritical CO₂ extraction (SCE) to enhance bioactive compound recovery, while reducing heavy metal contamination through its selective extraction capability. The HPLC method for β-asarone quantification was validated, exhibiting high accuracy, precision, and robustness. The detection and quantification limits (LOD and LOQ) were determined to be 0.378 mg/L and 1.149 mg/L, respectively. Under fixed extraction conditions (15 MPa, 45°C, 20 kg/h CO₂ flow rate, 120 min), SCE yielded significantly higher extraction efficiency (10.34% w/w) and β-asarone contents (193.64 g/L), compared to the 33 MPa condition (4.26% w/w and 10.52 g/L, respectively). Additionally, it effectively removed 75-97% of heavy metals, depending on the metal type. Antifungal activity testing against Candida albicans, Trichophyton rubrum, and Nannizzia gypsea using disk diffusion and broth macrodilution assays revealed that the extracts exhibited strong inhibitory and fungicidal activity, particularly against T. rubrum and N. gypsea, with inhibition zones exceeding those of Amphotericin B. Interestingly, despite the lower β-asarone concentration, the extracts obtained at 33 MPa exhibited superior antifungal activity in several aspects, suggesting the presence of additional bioactive compounds that enhance antifungal potency. These findings suggest that A. calamus extracts obtained via optimized SCE hold promise as natural antifungal agents, particularly for topical dermatophytosis treatments. However, further studies on detailed chemical profiling under different SCE parameters, mechanistic pathways, formulation stability, and in vivo safety are required to validate their therapeutic potential.

References

Rajput SB, Tonge MB, Karuppayil SM. An overview on traditional uses and pharmacological profile of Acorus calamus Linn. (Sweet flag) and other Acorus species. Phytomedicine 2014; 21: 268-76.

Zhao Y, Li J, Cao G, et al. Ethnic, Botanic, Phytochemistry and Pharmacology of the Acorus L. Genus: A Review. Molecules 2023; 28: 7117.

Sharma V, Singh I, Chaudhary P. Acorus calamus (The Healing Plant): a review on its medicinal potential, micropropagation and conservation. Nat Prod Res 2014; 28: 1454-66.

Herbal Product Division, Food and Drug Administration. National Herbal Medicine List of Thailand B.E. 2566 (2023). 1st ed. Pathum Thani, Thailand: Mini Group Co., Ltd.; 2023. (in Thai)

Ravindran PN, Pillai GS, Babu KN. Under-utilized herbs and spices. In: Peter KV, editor. Handbook of Herbs and Spices. Woodhead Publishing; 2004: p.53-103.

Miao JK, Shi RH, Li C, Li XW, Chen QX. Sweet Flag (Acorus calamus) Oils. In: Preedy VR, editor. Essential oils in food preservation, flavor and safety. Academic Press; 2016: p.775-82.

Kumar R, Prakash O, Pan AK, Hore SK, Chanotiya CS, Mathela CS. Compositional variations and antihelmentic activity of essential oils from rhizomes of different wild populations of Acorus calamus L. and its major component, beta-Asarone. Nat Prod Commun 2009; 4: 275-8.

Joshi RK. Acorus calamus Linn.: phytoconstituents and bactericidal property. World J Microbiol Biotechnol 2016; 32: 164.

Raina VK, Srivastava SK, Syamasunder K V. Essential oil composition of Acorus calamus L. from the lower region of the Himalayas. Flavour Fragr J 2003; 18: 18-20.

Liu XC, Zhou LG, Liu ZL, Du SS. Identification of Insecticidal constituents of the essential Oil of Acorus calamus rhizomes against Liposcelis bostrychophila Badonnel. Molecules 2013; 18: 5684.

Aryal S, Poudel A, Kafle K, Aryal LN. Insecticidal toxicity of essential oil of Nepalese Acorus calamus (Acorales: Acoraceae) against Sitophilus zeamais (Coleoptera: Curculionidae). Heliyon 2023; 9: e22130.

Yingjuan Y, Wanlun C, Changju Y, Hongyu Z, Hongxia H. Fumigant toxicity of β-asarone extracted from Acorus calamus against the adults and immature stages of Sitophilus zeamais. Chinese Bull Entomol 2009; 46: 453-60.

Yao Y, Cai W, Yang C, Hua H. Supercritical fluid CO2 extraction of Acorus calamus L. (Arales: Araceae) and its contact toxicity to Sitophilus zeamais Motschusky (Coleoptera: Curculionidae). Nat Prod Res 2012; 26: 1498-503.

Hannok P, Singhabumreung P, Thonnarak T, Kaewjai W, Agri Prod J. Preliminary effects of calamus powder (Acorus calamus L.) on storage pests infestation in corn seeds [in Thai]. Maejo J Agric Prod 2019; 1: 85-95.

Phongpaichit S, Pujenjob N, Rukachaisirikul V, Ongsakul M. Antimicrobial activities of the crude methanol extract of Acorus calamus Linn. Songklanakarin J Sci Technol 2004; 27: 517-23.

Begum J, Sohrab H, Yusuf M, et al. In vitro antifungal activity of Azaron isolated from the rhizome extract of Acorus calamus L. Pakistan J Biol Sci 2004; 7: 1376-9.

Rajput SB, Karuppayil SM. β-Asarone, an active principle of Acorus calamus rhizome, inhibits morphogenesis, biofilm formation and ergosterol biosynthesis in Candida albicans. Phytomedicine 2013; 20: 139-42.

Dhivya V, Nelson SJ, Nelson J, Paramasivam M. Comparing extraction efficacy of different solvents to extract Acorus calamus by using HPLC. J Pharmacogn Phytochem 2019; 8: 3074-8.

Vijayakumar KB, Bannimath G, Koganti VS, Iyer VB. Gas chromatographic method for analysis

β-Asarone in rhizome extracts of Acorus calamus and Their microbiological evaluation. Pharm Methods 2016; 7: 121-6.

Mejri J, Aydi A, Abderrabba M, Mejri M. Emerging extraction processes of essential oils: A review. Asian J Green Chem 2018; 2: 246-67.

Sánchez-Camargo AP, Mendiola JA, Ibáñez E, Herrero M. Supercritical fluid extraction. In: Reference module in chemistry, molecular sciences and chemical engineering. Elsevier; 2014.

Herrero M, Mendiola JA, Cifuentes A, Ibáñez E. Supercritical fluid extraction: recent advances and applications. J Chromatogr A 2010; 1217: 2495-511.

Mendiola JA, Herrero M, Cifuentes A, Ibañez E. Use of compressed fluids for sample preparation: food applications. J Chromatogr A 2007; 1152: 234-46.

Lin F, Liu D, Das SM, Prempeh N, Hua Y, Lu J. Recent progress in heavy metal extraction by supercritical CO₂ fluids. Ind Eng Chem Res 2014; 53: 1866-77.

Halili J, Mele A, Arbneshi T, Mazreku I. Supercritical CO₂ extraction of heavy metals Cu, Zn and Cd from aqueous solution using D ithizone as chelating agent. Am J Appl Sci 2015; 12: 284-9.

laxmi S, Sharanagouda H, Ramachandra CT, Roopa RS, Hanchinal SG. Supercritical fluid extraction of oil from sweet flag rhizome (Acorus calamus L.) and its insecticidal activity on pulse beetles (Callosobruchus maculatus). Int J Curr Microbiol Appl Sci 2017; 6: 3608-15.

Moskaluk AE, VandeWoude S. Current topics in dermatophyte classification and clinical diagnosis. Pathogens 2022; 11: 957.

Dias MFRG, Quaresma-Santos MVP, Bernardes-Filho F, Amorim AG da F, Schechtman RC, Azulay DR. Update on therapy for superficial mycoses: review article part I. An Bras Dermatol 2013; 88: 764-74.

Fisher MC, Alastruey-Izquierdo A, Berman J, et al. Tackling the emerging threat of antifungal resistance to human health. Nat Rev Microbiol 2022; 20: 557-71.

Rabaan AA, Sulaiman T, Al-Ahmed SH, et al. Potential Strategies to control the risk of antifungal resistance in humans: A comprehensive review. Antibiotics 2023; 12: 608.

United States Environmental Protection Agency (USEPA). Method 3051A: Microwave assisted acid digestion of sediments, sludges, and oils. Washington, DC; 2007.

AOAC INTERNATIONAL. Appendix K: Guidelines for Dietary Supplements and Botanicals. 20th ed.; 2013.

International Conference on Harmonization (ICH). Q2 (R1): Validation of analytical procedures-test and methodology. Geneva, Switzerland; 2005.

Boukhatem MN, Ferhat MA, Kameli A, Saidi F, Kebir HT. Lemon grass (Cymbopogon citratus) essential oil as a potent anti-inflammatory and antifungal drugs. Libyan J Med 2014; 9: 25431.

Espinel-Ingroff A, Chaturvedi V, Fothergill A, Rinaldi MG. Optimal testing conditions for determining MICs and Minimum Fungicidal Concentrations of new and established antifungal agents for uncommon molds: NCCLS Collaborative Study. J Clin Microbiol 2002; 40: 3776.

Ministry of Public Health [Thailand]. Standards for Purity and Other Quality Attributes for Registered Herbal Products 2021; 138: 6-7. (in Thai)

Brunner G. Supercritical fluids: technology and application to food processing. J Food Eng 2005; 67: 21-33.

Sugimoto N, Mikage M, Ohtsubo H, Kiuchi F, Tsuda Y. Pharmacognostical investigations of Acori Rhizomes (1) histological and chemical studies of Rhizomes of A. calamus and A. gramineus distributed in Japan. Nat Med 1997; 51: 259-64.

Rita WS, Kawuri R, Swantara IMD. Essential oil composition of Jeringau (Acorus calamus L.) rhizomes and their antifungal activity against Candida albicans. J Health Sci Med 2017; 1: 33-8.

Kim WJ, Hwang KH, Park DG, et al. Major constituents and antimicrobial activity of Korean herb Acorus calamus. Nat Prod Res 2011; 25: 1278-81.

Zhang AY, Camp WL, Elewski BE. Advances in tropical and systemic antifungals. Dermatol Clin 2007; 25: 165-83.

Dixon DM, Walsh TJ. Antifungal agents. In: Baron S, editor. Medical Microbiology. 4th ed. University of Texas Medical Branch at Galveston, 1996, Chapter 76.

Berg K, Bischoff R, Stegmüller S, Cartus A, Schrenk D. Comparative investigation of the mutagenicity of propenylic and allylic asarone isomers in the Ames fluctuation assay. Mutagenesis 2016; 31:

-51.

Uebel T, Wilken M, Vu Chi H, Esselen M. In vitro combinatory cytotoxicity of hepatocarcinogenic asarone isomers and flavonoids. Toxicol Vitr 2019; 60: 19-26.

Uebel T, Hermes L, Haupenthal S, Müller L, Esselen M. α-Asarone, β-asarone, and γ-asarone: Current status of toxicological evaluation. J Appl Toxicol 2021; 41: 1166-79.