Innovative strategy for extraction of green cardamom via super critical fluid extractor at different levels of pressure with its application against microorganisms in vitro and in silico

Abstract

The extraction of bioactive compounds from plants has emerged as a promising strategy for developing resource-efficient solutions that are both economically viable and value-driven. Supercritical fluid extraction (SFE), has become a popular technique for extraction significant plant-based compounds. Our investigation contrasted the yield, biological functions and phytochemical compositions of green cardamom extracts generated with SFE at 100, 200, and 300 bar of pressures. The maximal obtained weight was 0.279 gm upon applying 300 bar. There is a proportional elevation in the levels of most of phenolic compounds which detected using HPLC upon raising the pressure levels for extraction. Gallic acid had a significant increase (P ≤ 0.05) upon applying ascending pressure levels The extract obtained at 300 bar demonstrated the most potent antimicrobial activity against Bacillus subtilis and Candida albicans, with inhibition zones of 23.33 ± 0.58 mm and 22.17 ± 1.04 mm, respectively. Furthermore, antibiofilm and anti-hemolytic assays confirmed that higher extraction pressure enhanced the bioactivity of the extracts, with 300 bar showing the maximum effect. Time-kill kinetics demonstrated a progressive increase in microbial inhibition over time, with the 300-bar extract again displaying the most effective results. Transmission electron microscopy (TEM) revealed significant ultrastructural damage in B. subtilis and C. albicans treated with the 300-bar extract, indicating strong antimicrobial action at the cellular level. The molecular docking performance of the main constituents in green cardamom extracts gallic acid and syringic acid against B. subtilis (PDB ID: 5VX6) and S. aureus (PDB ID: 3V8J) using the molecular operating environment (MOE) software was evaluated. The docking scores (S), root mean square deviation (RMSD)_refine values, and energy terms (E_conf, E_place, E_score1, E_refine, E_score2) were analyzed to assess binding affinities. Key interactions, including hydrogen bonds, were identified, with distances and energies quantified. Syringic acid exhibited better binding (S = − 4.27 to − 5.04 kcal/mol) compared to gallic acid (S = − 4.11 to − 4.68 kcal/mol) across both targets. Interactions with residues like GLU 187 (Glutamic acid residue at position 187 in the protein sequence) and ARG 172 (Arginine residue at position 172) in 5VX6, and ASP 239 (ASP 239: Aspartic acid residue at position 239) in 3V8J, highlighted critical binding motifs. The findings concluded that green cardamom extracts, particularly those obtained at 300 bar, possess enhanced antimicrobial and antibiofilm properties, supported by both experimental and computational evidence. These results highlight the therapeutic potential of pressure-optimized SFE in maximizing the bioactivity of plant extracts.

Graphical abstract

Keywords

Super critical fluid extractor / Cardamom / Antimicrobial / Antihemolytic / Ultrastructure / Docking

References

[1] Abdel Ghany T, Hakamy OM. Juniperus procera as food safe additive, their antioxidant, anticancer and antimicrobial activity against some food-borne bacteria. J Biol Chem Res, 2014, 31: 668-677

[2] Abdelghany TMA, Ganash M, Alawlaqi MM, Al-Rajhi AMH. Antioxidant, antitumor, antimicrobial activities evaluation of Musa paradisiaca L. pseudostem exudate cultivated in Saudi Arabia. BioNanoScience, 2019, 9(1): 172-178.

[3] Abdelghany TM, Hassan MM, El-Naggar MA, Abd El-Mongy M. GC/MS analysis of Juniperus procera extract and its activity with silver nanoparticles against Aspergillus flavus growth and aflatoxins production. Biotechnol Rep, 2020, 27e00496

[4] Abdullah AA, Butt MS, Shahid M, Huang Q. Evaluating the antimicrobial potential of green cardamom essential oil focusing on quorum sensing inhibition of Chromobacterium violaceum. J Food Sci Technol, 2017, 54(8): 2306-2315.

[5] Abdullah AA, Asghar A, Huang Q, Mustfa W, Javed HU, Zehm S, Chikindas ML. Black cardamom essential oil prevents O157:H7 and Salmonella Typhimurium JSG 1748 biofilm formation through inhibition of quorum sensing. J Food Sci Technol, 2021, 58(8): 3183-3191.

[6] Abdullah N, Tian W, Zengliu S, Zou Y, Farooq S, Huang Q, Xiao J. Recent advances in the extraction, chemical composition, therapeutic potential, and delivery of cardamom phytochemicals. Front Nutr, 2022, 9: 1024820

[7] Ahmed HY, Kareem SM, Atef A, Safwat NA, Shehata RM, Yosri M, Youssef M, Baakdah MM, Sami R, Baty RS, Alsubhi NH, Alrefaei GI, Shati AA, Elsaid FG. Optimization of supercritical carbon dioxide extraction of Saussurea costus oil and its antimicrobial, antioxidant, and anticancer activities. Antioxidants, 2022, 11(101960

[8] Alawlaqi MM, Al-Rajhi AMH, Abdelghany TM, Ganash M, Moawad H. Evaluation of biomedical applications for linseed extract: antimicrobial, antioxidant, anti-diabetic, and anti-inflammatory activities in vitro. J Funct Biomater, 2023, 14(6300

[9] Alejo-Armijo A, Cobo A, Alejo-Armijo A, Altarejos J, Salido S, Ortega-Morente E. Evaluation of antibacterial and antibiofilm properties of phenolics with coumarin, naphthoquinone and pyranone moieties against foodborne microorganisms. Molecules, 2025, 304): 944

[10] Almehayawi MS, Almuhayawi MS, El-Fadl SRA, Nagshabandi MK, Tarabulsi MK, Selim S, Alruwaili YS, Mostafa EM, Al Jaouni SK, Abdelghany TM. Evaluating the anti-yeast, anti-diabetic, wound healing activities of Moringa oleifera extracted at different conditions of pressure via supercritical fluid extraction. Bioresour, 2024, 193): 5961-5977.

[11] Alqahtani TM, Ebrahim EG, Aloufi AS, El-Saadany SS, Almanzalawi EA, Awad A, El-Maghraby LM. Antibacterial and anticancer activities of cardamom volatile oil and its application in food covering. Italian J Food Sci, 2024

[12] Al-Rajhi AMH, Abdelghany TMA. Nanoemulsions of some edible oils and their antimicrobial, antioxidant, and anti-hemolytic activities. BioResources, 2023, 18: 1465-1481.

[13] Al-Rajhi AMH, Abdelghany TM. In vitro repress of breast cancer by bio-product of edible Pleurotus ostreatus loaded with Chitosan nanoparticles. Appl Biol Chem, 2023, 66: 33

[14] Al-Rajhi AMH, Yahya R, Abdelghany TM, Fareid MA, Mohamed AM, Amin BH, Masrahi AS. Anticancer, Anticoagulant, antioxidant and antimicrobial activities of Thevetia peruviana latex with molecular docking of antimicrobial and anticancer activities. Molecules, 2022, 27(10): 3165

[15] Al-Rajhi AMH, Bakri MM, Qanash H, Alzahrani HY, Halawani H, Algaydi MA, Abdelghany TM. Antimicrobial, antidiabetic, antioxidant, and anticoagulant activities of Cupressus sempervirens in vitro and in silico. Molecules, 2023, 28(21): 7402

[16] Al-Rajhi AMH, Qanash H, Almashjary MN, Hazzazi MS, Felemban HR, Abdelghany TM. Anti-Helicobacter pylori, antioxidant, antidiabetic, and anti-Alzheimer's activities of laurel leaf extract treated by moist heat and molecular docking of its flavonoid constituent, Naringenin, against Acetylcholinesterase and Butyrylcholinesterase. Life, 2023, 13(71512

[17] Al-Rajhi AMH, Qanash H, Bazaid AS, Binsaleh NK, Abdelghany TM. Pharmacological evaluation of Acacia nilotica flower extract against Helicobacter pylori and human hepatocellular carcinoma in vitro and in silico. J Funct Biomater, 2023, 144): 237

[18] Al-Rajhi AMH, Abdelghany TM, Almuhayawi MS, Alruhaili MH, Saddiq AA, Baghdadi AM, Al Jaouni SK, Albasri HM, Waznah MS, Alraddadi FA, Selim S. Effect of ozonation on the phytochemicals of black seed oil and its anti-microbial, anti-oxidant, anti-inflammatory, and anti-neoplastic activities in vitro. Sci Rep, 2025, 15(1): 3840

[19] Al-Rajhi AMH, Abdelghany TM, Selim S, et al. . Phytochemical characterization of peanut oil and its ozonized form to explore biological activities in vitro. AMB Expr, 2025, 15: 76

[20] Alsalamah SA, Alghonaim MI, Jusstaniah M, Abdelghany TM, Anti-Yeasts, . Antioxidant and healing properties of henna pre-treated by moist heat and molecular docking of its major constituents, chlorogenic and ellagic acids, with Candida albicans and Geotrichum candidum proteins. Life, 2023, 13(9): 1839

[21] Alsalamah SA, Alghonaim MI, Bakri MM, et al. . Zygnema sp. as creator of copper oxide nanoparticles and their application in controlling of microbial growth and photo-catalytic degradation of dyes. Appl Biol Chem, 2024, 6747

[22] Alsalamah SA, Alghonaim MI, Abdelghany TM, et al. . Effect of UV-C radiation on chemical profile and pharmaceutical application in vitro of Aloe vera oil. AMB Expr, 2025, 15: 83

[23] Appiah T, Boakye YD, Agyare C. Antimicrobial activities and time-kill kinetics of extracts of selected Ghanaian mushrooms. Evid Based Complement Alternat Med, 2017

[24] Ashokkumar K, Murugan M, Dhanya MK, Warkentin TD. Botany, traditional uses, phytochemistry and biological activities of cardamom [Elettaria cardamomum (L.) Maton]—a critical review. J Ethnopharmacol, 2020, 246112244

[25] Bazaid AS, Binsaleh NK, Barnawi H, et al. . Unveiling the in vitro activity of extracted Euphorbia trigona via supercritical fluid extraction against pathogenic yeasts, obesity, cancer, and its wound healing properties. Bioresour Bioprocess, 2025, 12: 28.

[26] Binsaleh NK, Bazaid AS, Barnawi H, Alharbi B, Alsolami A, Babsail AY, Qanash H. Chemical characterization, anticancer, antioxidant and anti-obesity activities with molecular docking studies of Pleurotus ostreatus biomass exposed to moist heat. J Food Meas Charact, 2025, 19: 2476-2495.

[27] Bouchelaghem S, Das S, Naorem RS, Czuni L, Papp G, Kocsis M. Evaluation of total phenolic and flavonoid contents, antibacterial and antibiofilm activities of Hungarian propolis ethanolic extract against Staphylococcus aureus. Molecules, 2022, 27(2): 574

[28] Cabañas CM, Hernández A, Martínez A, Tejero P, Vázquez-Hernández M, Martín A, Ruiz-Moyano S. Control of Penicillium glabrum by indigenous antagonistic yeast from vineyards. Foods, 2020, 9(12): 1864

[29] Castillo NET, Teresa-Martínez GD, Alonzo-Macías M, Téllez-Pérez C, Rodríguez-Rodríguez J, Sosa-Hernández JE, Parra-Saldívar R, Melchor-Martínez EM, Cardador-Martínez A. Antioxidant activity and GC-MS profile of cardamom (Elettaria cardamomum) essential oil obtained by a combined extraction method—instant controlled pressure drop technology coupled with sonication. Molecules, 2023, 28(3): 1093

[30] Chakraborty S, Paul K, Mallick P, Pradhan S, Das K, Chakrabarti S, et al. . Consortia of bioactives in supercritical carbon dioxide extracts of mustard and small cardamom seeds lower serum cholesterol levels in rats: new leads for hypocholesterolaemic supplements from spices. J Nutr Sci, 2019, 8: 1-15.

[31] Cho WI, Chung MS. Antimicrobial effect of a combination of herb extract and organic acid against Bacillus subtilis spores. Food Sci Biotechnol, 2017, 26(5): 1423-1428.

[32] Cucu A-A, Urcan AC, Bobiș O, Bonta V, Cornea-Cipcigan M, Moise AR, Dezsi Ș, Pașca C, Baci G-M, Dezmirean DS. Preliminary identification and quantification of individual polyphenols in Fallopia japonica plants and honey and their influence on antimicrobial and antibiofilm activities. Plants, 2024, 13(13): 1883

[33] Dzobo K. The role of natural products as sources of therapeutic agents for innovative drug discovery. Comp Pharmacol, 2022

[34] El-Baz AM, Mosbah RA, Goda RM, Mansour B, Sultana T, Dahms TES, El-Ganiny AM. Back to nature: combating Candida albicans biofilm, phospholipase and hemolysin using plant essential oils. Antibiotics, 2021, 10(1): 81

[35] Famuyide IM, Fasina FO, Eloff JN, McGaw LJ. The ultrastructural damage caused by Eugenia zeyheri and Syzygium legatii acetone leaf extracts on pathogenic Escherichia coli. BMC Vet Res, 2020, 16(1): 326

[36] Gopalakrishnan N, Narayanan SC. Supercritical carbon dioxide extraction of cardamom. J Agric Food Chem, 1991, 39(11): 1976-1978.

[37] Gumisiriza H, Olet EA, Mwikali L, Akatuhebwa R, Omara T, Lejju JB, Sesaazi DC. Antibacterial and antioxidant activities of flavonoids, phenolic and flavonoid glycosides from Gouania longispicata leaves. Microbiol Res, 2024, 15(4): 2085-2101.

[38] Gupta N, Bhattacharya S, Urbanová K, Dutta A, Hazra AK, Fernández-Cusimamani E, Leuner O. Systematic analysis of antimicrobial activity, phytochemistry, and in silico molecular interaction of selected essential oils and their formulations from different Indian spices against foodborne bacteria. Heliyon, 2023, 9(12e22480

[39] Guzmán E, Lucia A. Essential oils and their individual components in cosmetic products. Cosmet, 2021, 8: 114.

[40] Haido MH, Matti AH, Taher SM. Optimization of extraction conditions of bioactive compounds from Kurdistan species Urtica dioica. Cureus, 2024, 16(5e61146

[41] Hamdan S, Daood GH, Toth-Markus M, Illés V. Extraction of cardamom oil by supercritical carbon dioxide and sub-critical propane. J Supercrit Fluids, 2008, 441): 25-30.

[42] Hleba L, Hlebová M, Charousová I. In vitro evaluation of synergistic essential oils combination for enhanced antifungal activity against Candida spp. Life, 2024, 14(6): 693

[43] Hmidani A, Bouhlali EdT, Ajebli M, et al. . In vitro investigation of antioxidant and antihemolytic activities of three Lamiaceae species from Morocco. Beni-Suef Univ J Basic Appl Sci, 2021, 1027

[44] Huy TXN. Overcoming Klebsiella pneumoniae antibiotic resistance: new insights into mechanisms and drug discovery. Beni-Suef Univ J Basic Appl Sci, 2024, 13: 13.

[45] Ispiryan A, Atkociuniene V, Makstutiene N, Sarkinas A, Salaseviciene A, Urbonaviciene D, Viskelis J, Pakeltiene R, Raudone L. Correlation between antimicrobial activity values and total phenolic content/antioxidant activity in Rubus idaeus L. Plants, 2024, 134): 504

[46] Kaderides K, Papaoikonomou L, Serafim M, Goula AM. Microwave-assisted extraction of phenolics from pomegranate peels: optimization, kinetics, and comparison with ultrasounds extraction. Chem Eng Process Process Intensif, 2019, 137: 1-11.

[47] Kakouri E, Daferera D, Trigas P, Charalambous D, Pantelidou M, Tarantilis PA, Kanakis CD. Comparative study of the antibacterial activity, total phenolic and total flavonoid content of nine Hypericum species grown in Greece. Appl Sci, 2023, 13(53305

[48] Kashi M, Noei M, Chegini Z, Shariati A. Natural compounds in the fight against Staphylococcus aureus biofilms: a review of antibiofilm strategies. Front Pharmacol, 2024, 15: 1491363

[49] Khelfaoui H, Harkati D, Saleh BA. Molecular docking, molecular dynamics simulations and reactivity, studies on approved drugs library targeting ACE2 and SARS-CoV-2 binding with ACE2. J Biomol Struct Dyn, 2021, 39(18): 7246-7262.

[50] Makade CS, Shenoi PR, Bhongade BA, Shingane SA, Ambulkar PC, Shewale AM. Estimation of MBC: MIC ratio of herbal extracts against common endodontic pathogens. J Pharm Bioallied Sci, 2024, 16(Suppl 2): S1414-S1416.

[51] Mizzi L, Chatzitzika C, Gatt R, Valdramidis V. HPLC analysis of phenolic compounds and flavonoids with overlapping peaks. Food Technol Biotechnol, 2020, 58(1): 12-19.

[52] Nagashree S, Archana KK, Srinivas P, Srinivasan K, Sowbhagya HB. Antihypercholesterolemic influence of the spice cardamom (Elettaria cardamomum) in experimental rats. J Sci Food Agric, 2017, 97: 3204-3210.

[53] Nassar R, Hachim M, Nassar M, Kaklamanos EG, Jamal M, Williams D, et al. . Microbial metabolic genes crucial for S. aureus biofilms: an insight from re-analysis of publicly available microarray datasets. Front Microbiol, 2021, 11607002

[54] Neha K, Haider MR, Pathak A, Yar MS. Medicinal prospects of antioxidants: a review. Eur J Med Chem, 2019, 178: 687-704.

[55] Nguyen HT, O’Donovan LA, Venter H, Russell CC, McCluskey A, Page SW, Trott DJ, Ogunniyi AD. Comparison of two transmission electron microscopy methods to visualize drug-induced alterations of Gram-negative bacterial morphology. Antibiotics, 2021, 10(3): 307

[56] Noumi E, Snoussi M, Alreshidi MM, Rekha PD, Saptami K, Caputo L, De Martino L, Souza LF, Msaada K, Mancini E, et al. . Chemical and biological evaluation of essential oils from Cardamom species. Molecules, 2018, 2311): 2818

[57] Noumi E, Snoussi M, Alreshidi MM, Rekha P-D, Saptami K, Caputo L, De Martino L, Souza LF, Msaada K, Mancini E, et al. . Chemical and biological evaluation of essential oils from cardamom species. Molecules, 2018

[58] Nurcholis W, Alfadzrin R, Izzati N, Arianti R, Vinnai BÁ, Sabri F, Kristóf E, Artika IM. Effects of methods and durations of extraction on total flavonoid and phenolic contents and antioxidant activity of Java cardamom (Amomum compactum Soland Ex Maton) fruit. Plants, 2022, 11: 2221

[59] Pourkhosravani E, Dehghan Nayeri F, Mohammadi Bazargani M. Decoding antibacterial and antibiofilm properties of cinnamon and cardamom essential oils: a combined molecular docking and experimental study. AMB Express, 2021, 11(1): 143

[60] Qanash H, Al-Rajhi AMH, Almashjary MN, et al. . Inhibitory potential of Rutin and Rutin nano-crystals against Helicobacter pylori, colon cancer, hemolysis and butyrylcholinesterase in vitro and in Silico. Appl Biol Chem, 2023, 66: 79

[61] Qanash H, Alotaibi K, Aldarhami A, Bazaid AS, Ganash M, Saeedi NH, Abdelghany TA. Effectiveness of oil-based nanoemulsions with molecular docking of its antimicrobial potential. BioResources, 2023, 18: 1554-1576.

[62] Qanash H, Bazaid AS, Aldarhami A, Alharbi B, Almashjary MN, Hazzazi MS, Felemban HR, Abdelghany TM. Phytochemical characterization and efficacy of Artemisia judaica extract loaded chitosan nanoparticles as inhibitors of cancer proliferation and microbial growth. Polymers, 2023, 15: 391

[63] Qureshi KA, Imtiaz M, Parvez A, Rai PK, Jaremko M, Emwas AH, Bholay AD, Fatmi MQ. In vitro and in silico approaches for the evaluation of antimicrobial activity, time-kill kinetics, and anti-biofilm potential of Thymoquinone (2-Methyl-5-propan-2-ylcyclohexa-2,5-diene-1,4-dione) against selected human pathogens. Antibiotics (Basel), 2022, 11(1): 79

[64] Rossignol G, Merieau A, Guerillon J, Veron W, Lesouhaitier O, Feuilloley MGJ, Orange N. Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens. BMC Microbiol, 2008, 8: 189

[65] Selim S, Alruwaili Y, Ejaz H, Abdalla AE, Almuhayawi MS, Nagshabandi MK, Abdelghany TM. Estimation and action mechanisms of cinnamon bark via oxidative enzymes and ultrastructures as antimicrobial, anti-biofilm, antioxidant, anti-diabetic, and anticancer agents. BioResources, 2024, 19(4): 7019-7041.

[66] Selim S, Almuhayawi MS, Alruhaili MH, et al. . Extraction of mango seed kernels via super fluid extraction and their anti-H, pylori, anti-ovarian and anti-prostate cancer properties. Sci Rep, 2025, 15: 6359

[67] Silva LNA, Da Hora GCA, Soares TA, Bojer MS, Ingmer H, Macedo AJ, Trentin DS. Myricetin protects Galleria mellonella against Staphylococcus aureus infection and inhibits multiple virulence factors. Sci Rep, 2017, 7: 2823

[68] Slobodníková L, Fialová S, Rendeková K, Kováč J, Mučaji P. Antibiofilm activity of plant polyphenols. Molecules, 2016, 21(12): 1717

[69] Sobhy M, Elsamahy T, Abdelkarim EA, Khojah E, Cui H, Lin L. Cardamom essential oil-loaded zinc oxide nanoparticles: a sustainable antimicrobial strategy against multidrug-resistant foodborne pathogens. Microb Pathog, 2025, 205107661

[70] Stebuliauskaitė R, Liaudanskas M, Žvikas V, Čeksterytė V, Sutkevičienė N, Sorkytė Š, Bračiulienė A, Trumbeckaitė S. Changes in ascorbic acid, phenolic compound content, and antioxidant activity in vitro in bee pollen depending on storage conditions: impact of drying and freezing. Antioxidants, 2025, 14(4): 462.

[71] Sulieman AME, Abdallah EM, Alanazi NA, Ed-Dra A, Jamal A, Idriss H, Alshammari AS, Shommo SAM. Spices as sustainable food preservatives: a comprehensive review of their antimicrobial potential. Pharmaceuticals, 2023, 16(101451

[72] Techaoei S. Time-kill kinetics and antimicrobial activities of Thai medical plant extracts against fish pathogenic bacteria. J Adv Pharm Technol Res, 2022, 131): 25-29.

[73] Teresa-Martínez GD, Cardador-Martínez A, Téllez-Pérez C, Allaf K, Jiménez-Martínez C, Alonzo-Macías M. Effect of the instant controlled pressure drop technology in cardamom (Elettaria cardamomum) essential oil extraction and antioxidant activity. Molecules, 2022, 27: 3433.

[74] Upadhya V, Pai SR, Hegde HV. Effect of method and time of extraction on total phenolic content in comparison with antioxidant activities in different parts of Achyranthes aspera. J King Saud Univ Sci, 2015, 27: 204-208.

[75] Veiko AG, Olchowik-Grabarek E, Sekowski S, Roszkowska A, Lapshina EA, Dobrzynska I, Zamaraeva M, Zavodnik IB. Antimicrobial activity of quercetin, naringenin and catechin: flavonoids inhibit Staphylococcus aureus-induced hemolysis and modify membranes of bacteria and erythrocytes. Molecules, 2023, 28: 1252

[76] Yahya R, Al-Rajhi AMH, Alzaid SZ, Al Abboud MA, Almuhayawi MS, Al Jaouni SK, Selim S, Ismail KS, Abdelghany TM. Molecular docking and efficacy of aloe vera gel based on chitosan nanoparticles against helicobacter pylori and its antioxidant and anti-inflammatory activities. Polymers, 2022, 142994

[77] Yousefi M, Rahimi-Nasrabadi M, Pourmortazavi SM, Wysokowski M, Jesionowski T, Ehrlich H, et al. . Supercritical fluid extraction of essential oils. Trends Anal Chem, 2019, 118: 182-193.

[78] Zhao X, Zeng J, Gao H, Wang Y. Optimization and composition of volatile oil from Polygonatum odoratum (Mill Druce) using supercritical fluid extraction. Trop J Pharm Res, 2014, 13: 779-786.

Funding

Al-Imam Muhammad Ibn Saud Islamic University(IMSIU-DDRSP2501)

RIGHTS & PERMISSIONS

The Author(s)