Antioxidant and Anticancer Activities of Lactobacillus Hilgardii Strain AG12a

Zahra Pourramezan, Mana Oloomi, Rouha KasraKermanshahi


Background: It has been proven that probiotic Lactobacillus bacteria have inhibitory effects on human cancer cell lines. The aim of this study is to isolate and characterize the antioxidant probiotic Lactobacillus and determine the possible anticancer activities of the selected strain.

Methods: One of the Lactobacillus strain isolated from camel doogh sample showed the high antioxidant activity by using of different methods such as resistance to hydrogen peroxide, hydroxyl radical and superoxide anions. The antioxidant strain was characterized by sequencing of 16S rRNA V2-V3 regions and the 16S-23S intergenic spacer (ITS). The methanol extract of this strain supernatant was fractionated using thin layer chromatography (TLC) and antioxidant activity of fractions was detected by 0.1% of DPPH through TLC-DPPH bioautography. In vitro anticancer activity of each fraction was investigated by using MTT and flow cytometry methods.

Results: According to the phylogenetic results, the antioxidant Lactobacillus strain was closely related to Lactobacillus hilgardii strain E91 (Accession No. EF536365). After fractionation and anti-proliferation assessments of Lactobacillus hilgardii strain AG12a extracellular materials, one of the antioxidant fraction (F4) showed maximum DPPH radical scavenging activity (IC50 of 535.27 µg/mL). MTT assay of the F4 fraction demonstrated cytotoxic activity against Caco-2 with the IC50 value of 299.05 µg/mL. The cell death activity of the fraction was confirmed by flow cytometry with 30.925.

Conclusions: In this study, the anticancer and apoptotic properties of Lactobacillus hilgardii against Caco-2 cell line was reported for the first time. The isolated bioactive fraction from the extracellular methanol extract needs to be further investigated in human studies of cancer therapy.


Apoptosis; cancer; Lactobacillus; thin layer chromatograghy

Full Text:



Kim HS, Chae HS, Jeong SG, Ham JS, Im SK, Ahn CN, Lee JM.

In vitro antioxidative properties of Lactobacilli. Asian Australas J

Anim Sci 2006;19:262-5.

Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants

and functional foods: impact on human health. Pharmacogn Rev


Reid G, Jass J, Sebulsky MT, McCormick JK. Potential

uses of probiotics in clinical practice. Clin Microbiol Rev


Woo JY, Gu W, Kim KA, Jang SE, Han MJ, Kim DH.

Lactobacillus pentosus var. plantarum C29 ameliorates memory

impairment and inflammaging in a D-galactose-induced

accelerated aging mouse model. Anaerobe 2014;27:22-6.

Patel A, Prajapati JB. Food and health applications of

exopolysaccharides produced by lactic acid bacteria. Adv Dairy

Res 2013;1:1-7.

Abedin-Do A, Taherian-Esfahani Z, Ghafouri-Fard S,

Ghafouri-Fard S, Motevaseli E. Immunomodulatory effects of

Lactobacillus strains: Emphasis on their effects on cancer cells.

Immunotherapy 2015;7:1307-29.

Amer MN, Mansour NM, El-Diwany AI, Dawoud IE,

Rashad FM. Isolation of probiotic lactobacilli strains harboring

l-asparaginase and arginine deiminase genes from human infant

feces for their potential application in cancer prevention. Ann.

Microbiol 2013;63:1121-9.

Wang Y, Waters J, Leung ML, Unruh A, Roh W, Shi X, et al.

Clonal evolution in breast cancer revealed by single nucleus

genome sequencing. Nature 2014;512:155-60.

Rawla P, Sunkara T, Barsouk A. Epidemiology of colorectal

cancer: Incidence, mortality, survival, and risk factors. Prz

Gastroenterol 2019;14:89-103.

Uccello M, Malaguarnera G, Basile F, D’agata V,

Malaguarnera M, Bertino G, et al. Potential role of probiotics on

colorectal cancer prevention. BMC Surgery 2012;12:S35.

Altonsy MO, Andrews SC, Tuohy KM. Differential induction

of apoptosis in human colonic carcinoma cells (Caco-2) by

Atopobium, and commensal, probiotic and enteropathogenic

bacteria: Mediation by the mitochondrial pathway. Int J Food

Microbiol 2010;137:190-203.

Fallah Z, Feizi A, Hashemipour M, Kelishadi R. Effect of

fermented camel milk on glucose metabolism, insulin resistance,

and inflammatory biomarkers of adolescents with metabolic

syndrome: A double-blind, randomized, crossover trial. J Res

Med Sci 2018;23:32.

Pourramezan Z, Kasra Kermanshahi R, Oloomi M,

Aliahmadi A, Rezadoost H. In vitro study of antioxidant and antibacterial activities of Lactobacillus probiotic spp. Folia

Microbiol 2017;6331-42.

Wegkamp A, Teusink B, De Vos WM, Smid EJ. Development

of a minimal growth medium for Lactobacillus Plantarum. Lett

Appl Microbiol 2010;50:57-64.

Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA,

et al. Bergey’s Manual of Systematic Bacteriology. New York:

Springer; 2009.

Ryan KA, Jayaraman T, Daly P, Canchaya C, Curran S, Fang F,

et al. Cytotoxic effects of various lactic acid bacteria on Caco2 cells. Turk J Biol 2015;39:23-30.

Jose NM, Bunt CR, Hussain MA. Comparison of microbiological

and probiotic characteristics of lactobacilli isolates from dairy

food products and animal rumen contents. Microorganisms


Tamura K, Peterson D, Peterson N, Stecher G, Nei M,

Kumar S. MEGA5: Molecular evolutionary genetics analysis

using maximum likelihood, evolutionary distance, and maximum

parsimony methods. Mol Biol Evol 2011;28:2731-9.

Saadatzadeh A, Fazeli MR, Jamalifar H, Dinarvand R. Probiotic

properties of lyophilized cell free extract of Lactobacillus casei.

Jundishapur J Nat Pharm Prod 2013;8:131-7.

Kedare SB, Singh RP. Genesis and development of DPPH

method of antioxidant assay. J Food Sci Technol 2011;48:412-22.

Li WJ, Cheng XL, Liu J, Lin RC, Wang GL, Du SS, Liu ZL.

Phenolic compounds and antioxidant activities of Liriope

muscari. Molecules 2012;17:1797-808.

Randerath K, Libman D. Thin-Layer Chromatography. Jstor;

Arullappan S, Rajamanickam P, Thevar N, Narayanasamy D,

Yee HY, Kaur P. Cytotoxic effect and antioxidant activity of

bioassay- Guided fractions from solanum nigrum extracts. Trop

J harm. Res. 2015;14:1199-205.

Longo-Sorbello Giuseppe SA, Saydam G, Banerjee D, Bertino JR.

Cytotoxicity and cell growth assays. Elsevier Sci 2006;315-24.

Chaudhary S, Chandrashekar KS, Sreedhara K, Pai R, Setty MM,

Devkar RA, et al. Evaluation of antioxidant and anticancer

activity of extract and fractions of Nardostachys jatamansi DC in

breast carcinoma. BMC Complement Altern Med 2015;10:15-50.

Jayashree D. Phytochemical analysis and TLC fingerprinting of

methanolic extracts of three medicinal plants. Int Res J Pharm


Srisawat T, Chumkaew P, Heed-Chim W, Sukpondma Y,

Kanokwiroon K. Phytochemical screening and cytotoxicity of

crude extracts of vatica diospyroides symington type LS. Trop J

Pharm Res 2013;12:71-6.

Miraghajani M, Rafie N, Hajianfar H, Larijani B, Azadbakht L.

Aged garlic and cancer: A systematic review. Int J Prev Med


Jamilian M, Khademi L, Vahedpoor Z, Bahmani F,

Mahmoodi S, Taghizadeh M, et al. Effects of flaxseed oil

omega-3 fatty acids supplementation on regression and metabolic

status in endometrial hyperplasia: A randomized, double-blind,

placebo-controlled trial. Int J Prev Med 2019;10:61.

Górska A, Przystupski D, Niemczura MJ, Kulbacka J. Probiotic

bacteria: A promising tool in cancer prevention and therapy. Curr

Microbiol 2019;76:939-49.

Ewaschuk JB, Walker JW, Diaz H, Madsen KL. Bioproduction

of conjugated linoleic acid by probiotic bacteria occurs in vitro

and in vivo in mice. J Nutr 2006;136:1483-7.

Sevda ER, Koparal AT, Kivanḉ M. Cytotoxic effects of various

lactic acid bacteria on Caco-2 cells. Turk J Biol 2015;39:23-30.

Awaisheh SS, Obeidat MM, Al-Tamimi HJ, Assaf AM,

EL-Qudah JM, Al-khaza’leh JM, et al. In vitro cytotoxic

activity of probiotic bacterial cell extracts against Caco-2 and

HRT-18 colorectal cancer cells. Milk Dairy Prod Hum Nutr


Kim Y, Lee D, Kim D, Cho J, Yang J, Chung M, et al. Inhibition

of proliferation in colon cancer cell lines and harmful enzyme

activity of colon bacteria by Bifidobacterium adolescentis

SPM0212. Arch Pharm Res 2008;31:468-73.

Vamanu A, Vamanu E, Drugulescu M, Popa O, Campeanu G.

Identification of a lactic bacterium strain used for obtaining a

pollen-based probiotic product. Turk J Biol 2006;30:75-80.

Lee NK, Park JS, Park E, Paik HD. Adherence and

anticarcinogenic effects of Bacillus polyfermenticus SCD in the

large intestine. Lett Appl Microbiol 2007;44:274-8.

Kumar M, Kumar A, Nagpal R, Mohania D, Behare P, Verma V,

et al. Cancer-preventing attributes of probiotics: An update. Int J

Food Sci Nutr 2010;61:473-96.