Cryoethanolic Extraction, Purification of Allicin from Allium sativum and HPTLC analysis

Hemanth Kumar Manikyam *

Department of Chemistry, Faculty of Science, North East Frontier Technical University- Aalo Post Office, West Siang Distt, National Highway 229, Aalo, Arunachal Pradesh, 791001, India.

Dhanaseelan J

Department of Biotechnology, Adhiyamaan College of Engineering, Hosur, Tamil Nadu, 635109, India.

Jayaraj Mani

Department of Biotechnology, Plant Genetic Engineering and Molecular Biology Lab, Periyar University, Salem, Tamil Nadu-636011, India.

*Author to whom correspondence should be addressed.


The significance of this research extends beyond the mere extraction and purification of allicin, encompassing its potential applications across diverse industries. Allicin exhibits a plethora of biological activities, including antimicrobial, antifungal, antiviral, and antiprotozoal properties. Its mechanism of action involves the inhibition of thiol-containing enzymes in microorganisms, rendering it effective against a wide array of pathogens. Moreover, allicin has demonstrated promising anticancer properties, eliciting apoptosis and inhibiting cell proliferation in various cancer cell lines. Additionally, its anti-inflammatory and cardioprotective effects underscore its potential therapeutic utility in mitigating cardiovascular diseases and other inflammatory conditions. This study presents a novel method for extracting and purifying allicin from raw garlic cloves. Dry ice ensures the ethanol to reach its cryo temperature (hilled to sub-zero temperatures ranging from -40°C to -80°C). The process involves the use of ethanol, dry ice and ascorbic acid as solvents, vacuum stirring, and subsequent crystallization. The final product, a hygroscopic powder, was analyzed using High-Performance Thin-Layer Chromatography (HPTLC) to determine allicin content. The extraction yielded 5 grams of whitish hygroscopic powder from 21 grams of crude extract, with an allicin content of 5%. Chromatographic conditions included a mobile phase of acetonitrile: water: Formic acid (30:8:2) and derivatization with ninhydrin, with detection under UV light at 366 nm. This method provides an efficient way to isolate and purify allicin for various applications.

Keywords: Allicin, allium sativum, HPTLC, extraction, purification, crystallization, cryoethanolic extraction

How to Cite

Manikyam , Hemanth Kumar, Dhanaseelan J, and Jayaraj Mani. 2024. “Cryoethanolic Extraction, Purification of Allicin from Allium Sativum and HPTLC Analysis”. International Journal of Biochemistry Research & Review 33 (6):43-51.


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Borlinghaus J, Albrecht F, Gruhlke MC, Nwachukwu ID, Slusarenko AJ. Allicin: Chemistry and biological properties. Molecules. 2014;19(8):12591-618. DOI: 10.3390/molecules190812591

PMID: 25153873; PMCID: PMC6271412.

Borlinghaus J, Foerster Née Reiter J, Kappler U, Antelmann H, Noll U, Gruhlke MCH, Slusarenko AJ. Allicin, the Odor of Freshly Crushed Garlic: A Review of Recent Progress in Understanding Allicin's Effects on Cells. Molecules. 2021;26 (6):1505. DOI: 10.3390/molecules26061505

PMID: 33801955; PMCID: PMC8001868.

Sasi M, Kumar S, Kumar M, Thapa S, Prajapati U, Tak Y, Changan S, Saurabh V, Kumari S, Kumar A, Hasan M, Chandran D, Radha, Bangar SP, Dhumal S, Senapathy M, Thiyagarajan A, Alhariri A, Dey A, Singh S, Prakash S, Pandiselvam R, Mekhemar M. Garlic (Allium sativum L.) Bioactives and Its Role in Alleviating Oral Pathologies. Antioxidants(Basel). 2021;10(11):1847. DOI: 10.3390/antiox10111847

PMID: 34829718; PMCID: PMC8614839.

El-Saber Batiha G, Magdy Beshbishy A, G Wasef L, Elewa YHA, A Al-Sagan A, Abd El-Hack ME, Taha AE, M Abd-Elhakim Y, Prasad Devkota H. Chemical Constituents and Pharmacological Activities of Garlic (Allium sativum L.): A Review. Nutrients. 2020;12(3):872. DOI: 10.3390/nu12030872. PMID: 32213941; PMCID: PMC7146530.

Muller A, Eller J, Albrecht F, Prochnow P, Kuhlmann K, Bandow JE, Slusarenko AJ, Leichert LI. Allicin Induces Thiol Stress in Bacteria through S-Allylmercapto Modification of Protein Cysteines. J Biol Chem. 2016;291(22):11477-90. DOI: 10.1074/jbc.M115.702308

Epub 2016 Mar 23. PMID: 27008862; PMCID: PMC4882420.

Stepien AE, Trojniak J, Tabarkiewicz J. Anti-Cancer and Anti-Inflammatory Properties of Black Garlic. Int J Mol Sci. 2024;25(3):1801. DOI: 10.3390/ijms25031801

PMID: 38339077; PMCID: PMC10855366.

Shang A, Cao SY, Xu XY, Gan RY, Tang GY, Corke H, Mavumengwana V, Li HB. Bioactive Compounds and Biological Functions of Garlic (Allium sativum L.). Foods. 2019;8(7):246. DOI: 10.3390/foods8070246

PMID: 31284512; PMCID: PMC6678835.

Narayan UL, Garnaik B, Patro SK, Sahu S. HPTLC Methods for Determination of Gemifloxacin Mesylate in Rabbit Plasma. J. Pharm. Res. Int. 2014;4(14):1707-14. Available:

[Accessed on: 2024 Jun. 8]

Zakirova UM. Mamatkulova NU. Khodjaniyazov KK. Khidyrova NM. Shakhidoyatov K. Polyprenols of Grape Vitis vinifera L. Leaves. Int. J. Biochem. Res. Rev. 2013 [cited 2024 Jun. 8];3(2):97-106. Available:

Shewiyo DH, Kaale EA, Risha PG, Dejaegher B, Smeyers-Verbeke J, Vander Heyden Y. HPTLC methods to assay active ingredients in pharmaceutical formulations: A review of the method development and validation steps. Journal of pharmaceutical and biomedical analysis. 2012;66:11-23.

Variyar PS, Chatterjee S, Sharma A. Fundamentals and theory of HPTLC- based separation. High-performance thin-layer chromatography (HPTLC). 2011:27-39.

Catanzaro E, Canistro D, Pellicioni V, Vivarelli F, Fimognari C. Anticancer potential of allicin: A review. Pharmacological research. 2022; 177:106118.

Puri V, Nagpal M, Singh I, Singh M, Dhingra GA, Huanbutta K, Dheer D, Sharma A, Sangnim T. A Comprehensive Review on Nutraceuticals: Therapy Support and Formulation Challenges. Nutrients. 2022;14(21):4637. DOI: 10.3390/nu14214637

PMID: 36364899;

PMCID: PMC9654660.

Sunanta P, Kontogiorgos V, Pankasemsuk T, Jantanasakulwong K, Rachtanapun P, Seesuriyachan P, Sommano SR. The nutritional value, bioactive availability and functional properties of garlic and its related products during processing. Front Nutr. 2023;10:1142784. DOI: 10.3389/fnut.2023.1142784. PMID: 37560057; PMCID: PMC10409574.

Reiter J, Hubbers AM, Albrecht F, Leichert LIO, Slusarenko AJ. Allicin, a natural antimicrobial defence substance from garlic, inhibits DNA gyrase activity in bacteria. Int J Med Microbiol. 2020; 310(1):151359. DOI: 10.1016/j.ijmm.2019.151359

Epub 2019 Sep 25. PMID: 31585716.

Bar M, Binduga UE, Szychowski KA. Methods of Isolation of Active Substances from Garlic (Allium sativum L.) and Its Impact on the Composition and Biological Properties of Garlic Extracts. Antioxidants (Basel). 2022;11(7):1345. DOI: 10.3390/antiox11071345

PMID: 35883836; PMCID: PMC9312217

Zaini AS, Putra NR, Idham Z, Mohd Faizal AN, Che Yunus MA, Mamat H, Abdul Aziz AH. Comparison of Alliin Recovery from Allium sativum L. Using Soxhlet Extraction and Subcritical Water Extraction. ChemEngineering. 2022;6:73. Avaialble:

Jose M. del Valle, Veronica Glatzel, Jose L. Martínez, Supercritical CO2 extraction of allicin from garlic flakes: Screening and kinetic studies, Food Research International. 2012;45(1):216-224 Available:

Zhou C, Hu XY, Chao C, Li H, Zhang SY, Yan XM, Yang FM, Li QH. Quantitation of Allicin in Garlic-based Products: Comparisons among Spectrophotometry, GC and HPLC. Advance Journal of Food Science and Technology. 2015;9(4):269-277.Available:

Blania G, Spangenberg B. Allicin-Freisetzung aus getrocknetem Knoblauch (Allium sativum): Eine einfach durchzuführende HPTLC-Simultanbestimmung von Allicin und Ajoen in Knoblauchpulver und daraus hergestellten Fertigarzneimitteln [Formation of allicin from dried garlic (Allium sativum): A simple HPTLC method for simultaneous determination of allicin and ajoene in dried garlic and garlic preparations]. Planta Med. 1991;57(4):371-5. DOI: 10.1055/s-2006-960120

PMID: 1775580.

Itakura YM. Ichikaw, Y, Mori R. Okino M. Udayama T. Morita, how to Distinguish Garlic from the Other Allium Vegetables, The Journal of Nutrition. 2001; 131(3).

Pages 963S-967S, ISSN 0022-3166, Available:

Bosca B, Mot AC. Novel simultaneous determination of alliin and allicin in Allium sp. using digital subtraction HPTLC. J Chromatogr B Analyt Technol Biomed Life Sci. 2023;1222:123700. DOI: 10.1016/j.jchromb.2023.123700.

Epub 2023 Apr 5. PMID: 37031569

Niranjan S Kanaki, M Rajani, Development and Validation of a Thin-Layer Chromatography-Densitometric Method for the Quantitation of Alliin from Garlic (Allium sativum) and Its Formulations, Journal of AOAC International. 2005;88(5):1568–1570, Available:

Dhwani S, Pushparaj Poojitha, Pa, Parameswaran. A review on different extraction and quantification methods of allicin from garlic; 2021.DOI: 10.37896/jxu15.6/020.

Xu DP, Li Y, Meng X, Zhou T, Zhou Y, Zheng J, Zhang JJ, Li HB. Natural Antioxidants in Foods and Medicinal Plants: Extraction, Assessment and Resources. Int J Mol Sci. 2017;18(1):96. DOI: 10.3390/ijms18010096

PMID: 28067795; PMCID: PMC5297730

Manikyam, Hemanth Kumar. Medicinal plants and alternative therapies for reproductive system health. Nutraceuticals: A Holistic Approach to Disease Prevention. 2024;237.

Nadeem MS, Kazmi I, Ullah I, Muhammad K, Anwar F. Allicin, an Antioxidant and Neuroprotective Agent, Ameliorates Cognitive Impairment. Antioxidants. 2022; 11:87. Available:

Manikyam, Hemanth Kumar, et al. Microwave assisted Subcritical water extraction of Berberine hydrochloride from the roots of Berberis Aristata using Harmony search algorithm. Journal of Herbal Medicine Research. 2017;2: 1-9.

Kumar, Mr Hemanth, and M. Spandana. Method of Extraction and Determination of Forskolin from Coleus forskolii of Nepal. Journal of Pharmacognosy and Phytochemistry 2013;2(4):37-39.

Manikyam HK, Tripathi P, Patil SB, Lamichhane J, Chaitanya M, et al. Extraction, purification, and quantification of hesperidin from the immature Citrus grandis/maxima fruitNepal cultivar. Asian Journal of Natural Product Biochemistry. 2020;20(1).

Manikyam Hemanth Kumar, J. Dhanaseelan, Jayaraj Mani. Extraction and Purification of Squalene from Virgin Olive Oil via Catalytic Transesterification and Molecular Distillation. European Journal of Medicinal Plants. 2024;35(3):50-61.

Manikyam HKP, Tripathi J, Joshi J, Balasubramanian S, Patil B, Lamichhane T, Lamichhane J. Microwave Assisted Extraction of Berberine and Preparation of Berberine Hydrochloride from Berberis Aristata Variety of Nepal, and Quantification Using RP-HPLC and HPTLC Methods. European Journal of Medicinal Plants. 2021;32(12):46-53. DOI: 10.9734/ejmp/2021/v32i1230434

Manikyam Hemanth Kumar, Joshi Sunil, Tripathi, Prathibha, Joshi Jyoti, Balasubramanian, Jayaram Udhyog, Jadibuti Assistant. Arabian Journal of Medicinal & Aromatic Plants Extraction, Purification and quantification of Ursolic acid Ursolic extraction process from Rosemary officinalis leaves of Nepal Cultivar. 2023;9:54-64. Available:

Mr. Hemanth Kumar, M. Spandana. Simultaneous Extraction, determination and analysis of Adenosine, Cordycepin and other derivatives of Cordyceps sinensis of Nepal by new validated HPLC method. J Pharmacogn Phytochem. 2013;2(4):43-45.29.

Balkrishna, Acharya, M. Hemanth Kumar, Ashish Kumar Gupta. Comparative analysis of HPTLC, secondary metabolites and antioxidant activities of Tinospora cordifolia stem powders. International Journal of Pharmaceutical Sciences and Research. 2016;7(10):4263-4271.