IHJPAS. 36(1)2023 52 Creative Commons Attribution 4.0 International LicenseThis work is licensed under a Abstract The study is designed to evaluate the effect of the aqueous extract of the P. lanceolata plant, as well as to know the effect of the drug CCl4 on the formation of micronucleus in vivo 48 female albino mice. In the study mice were separated into eight groups treated intraperitoneally for seven day first group Negative control, second positive control( CCl4 0.02%), third group aqueous extract (250 mg/kg), fourth group aqueous extract (500 mg/kg), fifth group (CCl4 0.02%) plus aqueous extract (250 mg/kg), sixth group (CCl4 0.02%) plus aqueous extract (500 mg/kg), seventh group aqueous extract (250 mg/kg) plus (CCl4 0.02%), and eighth group aqueous extract (500 mg/kg) plus (CCl4 0.02%). The genetic-cellular aspect involved measuring the coefficient of micronucleus formation in bone marrow cells in mice treated with CCl4 and plant aqueous extract. The results showed that the treatment of mice with the drug led to a rise in the coefficient of micronucleus formation compared to the negative control group. In addition, it showed the plant's ability to reduce the drug CCl4 effect in the totals of overlaps between the plant extract and the drug at the concentrations used for the plant 250 and 500 μg/ml and reduce the formation of micronucleus. The cellular toxicity of the plant’s aqueous extract on the liver cancer cell line was assessed in HepG2 (liver cancer cell line) and the WRL68 (hepatic human cell line) using concentrations (25, 50, 100, 200, and 400 μg /ml) from the plant’s aqueous extract on the HepG2 liver cancer cell line. The results showed a decrease in cell viability depending on aqueous extract concentration. The vitality of cancer cells decreased with the increase in concentration; the viability of the aqueous extract of the plant on cancer cells reached the minimum at concentration 400 μg/ml 45.34±4.44, while it reached the maximum when concentration 25 μg/ml 84.53±2.41. Keywords: Plantago lanceolata, CCL4,Micronucleous , HepG2 cell line. Doi.org/10.30526/36.1.2953 Article history: Received 25 July 2022, Accepted 31 August 2022, Published in January 2023. Ibn Al-Haitham Journal for Pure and Applied Sciences Journal homepage: jih.uobaghdad.edu.iq Anti-tumor Activity of Plantago lanceolata Aqueous Extract In Vitro and Genotoxicity by Micronucleus Assay In Vivo Eman Ali Hussein Al-Mosawie Department of Biology, College of Education for Pure Sciences (Ibn- Al- Haitham) University of Baghdad, Baghdad,Iraq. eman.ali1202a@ihcoedu.uobaghdad.edu.iq Worood Kamil Shalash Al-Maliky Department of Biology, College of Education for Pure Sciences (Ibn- Al- Haitham) University of Baghdad, Baghdad,Iraq. worood_almaliky@yahoo.com mailto:eman.ali1202a@ihcoedu.uobaghdad.edu.iq mailto:worood_almaliky@yahoo.com IHJPAS. 36(1)2023 53 1.Introduction Plantago lanceolata is a perennial plant that is extensively distributed. Plantago lanceolata is used to treat several malignant conditions and to enhance the immune, respiratory, digestive, urinary, and reproductive systems. [1,2]. Numerous active chemicals in the plant, such as flavonoids, which stop the oxidation of low-density lipoprotein and eliminate free radicals, give the plant its medicinal potential [3]. Flavonoids that are anti-oxidant and anti-mutagenic are present in plants [4]. Use test for micronucleus: It is one procedure used to investigate mammals' in vivo genetic toxicity as one of the most effective tests to analyze the induction of chromosomal abnormalities, one of the most significant causes of mutations, and assess the hazards of substances [5]. Numerous chemical, physical, and biological agents can kill cells, but a class of agents known as genetic toxins cans also damage genetic material without killing cells. These agents are not necessarily toxic to cells. Environmental pollutants expose modern humans to a variety of genotoxic substances. To determine this exposure, genotoxicity tests are used, the most significant and well- known of which are micronuclei tests performed on organisms both inside and outside their bodies [6]. 2.Materials and Methods Plantago lanceolata gathering and Identification Plant sample collection The dried leaves of Plantago lanceolata were obtained from the local market in Baghdad / Herb Al-Razi in August of 2021 and the plant classifies by the College of Pharmacy, Al-Mustansiriya University, and Baghdad. Preparation of plant aqueous extract After drying, the plant leaves were chopped, and 150 grams of them were extracted for four hours in 250 ml of distilled water using the Soxhlet equipment. A water bath provided heating at 45°C. The leaf extract solution was put in plates and incubated in the oven at 37°C to prepare plant extract powder. The powder was added to create the necessary concentration for the laboratory mice [7]. Dose of plant aqueous extract Two doses of plant extract were given based on LC50 (half lethal concentration) to the plant, to albino female mice, the first dose being 250 mg/kg and the second increased to 500 mg\kg.LC50 of Plantago lanceolata 2,940 mg/kg[8]. Dose of drug carbon tetrachloride (CCL4) The drug tetrachloride carbon CCL4 was used by Thomas Baker / India at 0.02 ml and olive oil was added at 99.98 [9]. Laboratory animals This study utilized female white mice (Mus musuls) from the Biotechnology Research Center Al- Nahrain University. They ranged from 8-12 weeks and weighed 23-27 grams. They were placed in dedicated cages, each set in a cage. Each cage contained six female white rats while providing similar conditions for all groups of water, food (standard pellets ), temperature, and lighting. Scheme of Experiment IHJPAS. 36(1)2023 54 Forty eight mice were divided into 8 groups. The first group was a negative that received treatment with distal water, and the second group was received one dosage of the medication CCl4 intraperitoneally (i. p.) daily for seven days. For a week, Third and fourth groups received 250 mg/kg and 500 mg/kg of plant extract, respectively. On the first day, CCl4 was administered to groups fifth and sixth, and over the next six days, post treatment consisted of 250 mg/kg and 500 mg/kg of plant extract. Groups seven and eight received CCl4 (i. p.) for the last day after receiving plant extract treatments of 250 mg/kg and 500 mg/kg i. p. for six days. 0.1 ml of the dosage was administered i. p. to the groups. Micronucleus Formation Assay in vivo To valuation of micronucleus formation, the method of [10] depended on the following steps: The mice were sacrificed and then dissected to obtain the femur. Then cutting, both ends of the bone were gripped from the middle with forceps in a vertical position over the edge of a test tube. Then the cellular content was collected with heat-inactivated human AB plasma 2 ml using a disposable insulin syringe. 1. After gentle mixing, the test tube was centrifuged (1000 rpm) for 10 minutes, and the supernatant was discarded. The cellular deposit was gently mixed; a thin smear was made on a clean slide, and air-dried at room temperature. 2. The smear was fixed with absolute methanol for 5 minutes, air-dried at room temperature, stained with Giemsa stain for 15 minutes, and rinsed with distilled water. The slides were examined under an oil immersion lens (100X), and at least 1000 polychromatic erythrocytes (PCE) were examined for the presence of micronucleus formation. The micronucleus index was obtained using the subsequent equation: 100 x PCE ofCount Total iMicronucle ofNumber eus/cell)(micronuclIndex usMicronucle        Cytotoxicity MTT assay in vitro This study used the Liver cancer cell line HepG2 obtained from the University of Malaya / College of Medicine / Department of Pharmacology / Center for Investigation of New Therapies in Malaysia. A-The Kit Contents • The MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) Solution: Vials 10 x 1ml. • The Solubilization Solution: 50 ml × 2 bottle. B. Assay procedure The procedure consists of the following steps [11]. • Cells were planted at a density (1×104 to 1 x 106 cell/ml) 100 microliters of HepG2 cells were added to 96 pits in the microliter plate with repetition of each concentration as well as control. • Covered the plate and incubated at 37°C for 4-2 hours with 5% C02 after which the culture medium (RPMI-1640) Roswell Park Memorial Institute was discarded. • One hundred μL of Plantago lanceolata aqueous extract (25, 50,100,200, and 400 μg/ml) were added to each treated pit well in the plate for 48 hours at 37°C. Three duplicates for all concentrations were prepared. After the treatment with compounds, ten microliters of MTT solution and incubate for 4 hours at 37 °C. one hundred microliters of Solubilization solution per hole for 5 minutes. The results were read using the ELIZA reader (ASYS/Austria) to check the absorption at a wavelength of 575 nanometers. Percentage Growth inhibition (GI) for each concentration of the extract was calculated by the subsequent equation method: Viability (%)=(Optical density of sample / Optical density of control) x100 Statistical analysis IHJPAS. 36(1)2023 55 One mode examination of variance ANOVA (Duncan) was made to test statistical significance was defined as (p≤ 0.05). Data were expressed as mean± standard error, and statistical significances were carried out using Graph Pad Prism version 6 (Graph Pad Software Inc., La Jolla). 3.Results and Discussion 1-Micronucleus assay in vivo The results showed that the rate of micronucleus in the negative control group was 0.011±0.001 MN/Cell, while the positive control group treated with CCl4 increased significantly (P ≤ 0.05) 0.062±0.008 MN/cell. While in groups treated with P. lanceolata aqueous extract 250 and 500 mg/kg the rate of micronuclei was 0.016±0.001 and 0.015±0.005, respectively where the difference was non-significant compared to a negative control. The two groups treated on the first day with CCl4 and the remaining six days with plant aqueous extract 250 and 500 mg/kg were 0.025±0.004 and 0.020±0.001 respectively. The two groups were treated in the first six days with 250 and 500 mg/kg plant extract, and the seventh day with CCl4 was 0.030±0.001 and 0.027±0.004, respectively. This indicated that the plant has a protective effect as the proportion of micronuclei formation was significantly reduced (P ≤ 0.05) when compared to the positive control group, as shown in Table( 1-1) and Figure(1-1). Table (1-1): The effect of P. lanceolata aqueous extract the formation of micronucleus in albino mice treatment with CCl4 Group Concentration Micronucleus Count (Mean ±SD) 0.011± 0.015D Negative control 0.062±0.008A 0.02 % Positive control 0.016±0.001 CD 250 mg/kg P. lanceolata extract 0.015±0.005CD 500 mg/kg P. lanceolata extract 0.025±0.004 BC 250mg/kg+0.02 P. lanceolata +CCl4 0.020±0.001 C 500mg/kg+0.02 P. lanceolata + CCl4 0.027±0.004BC 250mg/kg+0.02 CCl4+ P. lanceolata 0.030±0.001B 500mg/kg+0.02 CCl4+ P. lanceolata Different letters: marked variation (P ≤ 0.05) among means Figure (1-1): Two bone marrow cells of mice treated with CCl4 show the formation of micronucleus using Giemsa stain magnification force (X100). Micronucleus testing is a reliable application to many cells, such as red and white blood cells and epithelial cells. It is essential for assessing DNA damage and defects during cell division [12]. A study proved that the Sagittarius plant is rich in flavonoids, with an estimated ratio of IHJPAS. 36(1)2023 56 66.9 μg/ml[13]. The plant has an apparent effect on the decrease in the rate of micronucleus formation in the fifth, sixth, seventh, and eighth mice groups. Interactions between aqueous extract and ccl4 drug compared with the positive control group as shown in Table 1-1. Significant differences and different letters indicate significant differences between groups of mice. 2-Cytotoxicity MTT assay Cytotoxicity screening was used to assess the toxic effects of the aqueous extract P. lanceolata in the cell line of liver cancer HepG2. Cell viability and cancer cell inhibition ratio were estimated by the MTT assay using different concentrations of the plant’s aqueous extract ranging from 25-400 μg/ml. As for the effect of the plant's aqueous extract on the HepG2 liver cancer cell line, the results showed a decrease in cell viability depending on concentration. The vitality of cancer cells has declined as concentration has increased. The viability of the aqueous extract on cancer cells reached a minimum concentration of 400 μg/ml 45.34±4.44, while it reached the maximum concentration of 25 μg/ml 84.53±2.41 as shown in Table (2-1). The inhibition efficacy of the plant's aqueous extract on the natural human cell line WRL68 withlC50 was 129.7 μg/ml. The inhibition efficacy of the liver cancer cell line HepG2 was compared with lC50 of 105.0 μg/ml (Figure 2-1). Table (2-1) :The cytotoxicity effect of P. lanceolata aqueous extract in the HepG2 and WRL68 cell lines Viable cell count WRL68 Mean ±SD Viable cell count of HepG2 Mean ±SD Concentration mg/ml 64.30±1.79 D 45.34±4.44 E 400 72.49±1.54 C 53.08±1.91 D 200 82.48±0.55 B 65.98±6.04 C 100 93.52±0.53 A 75.54±3.13 B 50 95.02±0.72 A 84.53±2.41 A 25 Different letters: marked variation (P ≤ 0.05) among means Figure 2-1: The effect P .lanceolata aqueous extract in the HepG2 and WRL68 cell lines HepG2 is a common liver cancer cell line used in many studies to examine the cytotoxicity of substances in the liver [14]. P. lanceolata has a toxic effect on the HepG2 cell line in higher doses than in low doses. Through the results, the high dose was more toxic to cancer cells. This is IHJPAS. 36(1)2023 57 consistent with a study [15] that showed that the inhibition of cancer cells in plant extracts depends on the dose. A study has proven that the plant P. lanceolata is anticancer by possessing antioxidant properties and has a toxic effect on genes in all concentrations in HepG2 cells[16]. Plant toxicity to cancer cells is attributed to its high content of many anticancer compounds, such as glycosides and flavonoid compounds, the most important of which are rutin, myrictin, quarcetin, and kaempferol [17]. 4.Conclusion Plantago lanceolata modulates the mutagenic effect of the CCl4 drug through the reduced formation of a micronucleus. It has toxic activity on the cell line HepG2(liver cancer cell line). References 1.Tutel, B.; Kandemir ,I.; Kufi ,S.; Kence, A. Classification of Turkish Plantago L. species using numerical taxonomy, Turk J Bot. 2005, 29, 51-61. 2.Kurt, B .;Bilge, N.; Sozmen, M.,; Adyin, U.; Ö nyay, T.; Ozaydin ,I. Effects of Plantago lanceolata L. extract on full-thickness excisional wound healing in a mouse model, Biotech Histochem. 2018,93(4),249-257. 3.Ji, X.; Hou ,C.and Guo, X. 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