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ORIGINAL ARTICLE
Year : 2021  |  Volume : 12  |  Issue : 3  |  Page : 232-235  

Antimutagenic activity of nanoparticles of Rhaphidophora pinnata leaves in mice using micronucleus assay


1 Department of Pharmaceutical Chemistry, Universitas Sumatera Utara; Nanomedicine Centre, Universitas Sumatera Utara, Medan, 20155, Indonesia
2 Nanomedicine Centre; Department of Pharmacology Pharmacy Faculty of Pharmacy, Universitas Sumatera Utara, Medan, 20155, Indonesia
3 Department of Pharmaceutical Chemistry, Universitas Sumatera Utara, Medan, 20155, Indonesia

Date of Submission31-Dec-2020
Date of Decision17-Feb-2021
Date of Acceptance28-Apr-2021
Date of Web Publication16-Jul-2021

Correspondence Address:
Prof. Masfria Masfria
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Sumatera Utara, Medan 20155
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/japtr.JAPTR_380_20

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  Abstract 


Cancer is one of the deadliest diseases in the world. Cancer may occur due to gene mutation. Rhaphidophora pinnata is a plant that has many benefits, especially in the leaves which have been used traditionally to treat cancer. The aim of this research is to test the antimutagenic activity of nanoparticles R. pinnata using the micronucleus method. The mice were induced with cyclophosphamide and then followed with the administration of nanoparticles of R. pinnata at the doses of 50, 100, 200 mg/kg for 7 days. The antimutagenic activity was evaluated at the decrease in the number of micronucleus in 200 polychromatic erythrocytes (PCE) cells of mice bone marrow. The result showed that the reduction of amount of micronucleus in PCE of a negative control group, treatment groups, and normal group is 22.65%, 60.3%, 79.6%, 93.8%, and 100%. These results indicate that the antimutagenic activity of nanoparticle of R. pinnata increases proportionally as the doses were increased. It can be concluded that nanoparticles R. pinnata at the doses of 50, 100, and 200 mg/kg have antimutagenic activity.

Keywords: Antimutagenic, micronucleus, Rhaphidophora pinnata


How to cite this article:
Masfria M, Marianne M, Permata YM, Octavio S, Mulyani S. Antimutagenic activity of nanoparticles of Rhaphidophora pinnata leaves in mice using micronucleus assay. J Adv Pharm Technol Res 2021;12:232-5

How to cite this URL:
Masfria M, Marianne M, Permata YM, Octavio S, Mulyani S. Antimutagenic activity of nanoparticles of Rhaphidophora pinnata leaves in mice using micronucleus assay. J Adv Pharm Technol Res [serial online] 2021 [cited 2021 Sep 21];12:232-5. Available from: https://www.japtr.org/text.asp?2021/12/3/232/321518




  Introduction Top


Mutation is a change that occurs in a gene so that it can cause changes to the products encoded by the gene. Mutations can be associated with the emergence of various disorders, including obesity, diabetes, cardiovascular disease, cancer, and also neurodegenerative diseases mainly Alzheimer's disease and Parkinson's disease.[1],[2],[3] Micronucleus is one indicator of mutation and it has become the most prevalent method to assess genotoxicity of different chemical and physical factors.[1],[4]

The micronucleus is a small nucleus outside the main nucleus in the cytoplasm. This is an abnormal nucleus due to the cleavage of chromosomes caused by mutagenic compounds. The micronucleus is easily observed in polychromatic erythrocytes (PCE). The number of micronucleus in PCE indicates the degree of genetic damage to the erythropoietic system of living things.[1]

The antimutagenic test was conducted to determine the possibility of compounds having antimutagenic properties. One of the test methods is cytogenetic antimutagenic tested by in vivo is the micronucleus test method.[5] One of the plants that is promising as antimutagenic is Rhaphidophora pinnata (L.f.) Schott. This plant is included in the Araceae family and its leaves have been used traditionally both in Indonesia and Singapore to treat cancer. The anticancer activity of this plant has also been tested against MCF-7 cells and T47D cells.[6],[7],[8] However, research on its antimutagenic activity has never been carried out. Therefore, this study is to determine the antimutagenic activity of nanoparticle of R. pinnata leaves in mice (Mus musculus L.) using the micronucleus test method.


  Materials and Methods Top


Materials

The leaves of R. pinnata were collected from Medan, North Sumatera, Indonesia, in July 2008. The plant authentication was carried out by Dr. Eko Baroto Walujo, APU from Research Center for Biology, Indonesian Institute of Sciences, Indonesia with the result is R. pinnata (L.f.) Schott, family Araceae (the reference number is 1052/IPH.1.02/If. 8/2008).

The leaves were dried and mashed in PT. Nanotech Herbal Indonesia, Bogor, Indonesia. The leaves were mashed by top-down method using high-speed milling until gained the particle with size 616.0 ± 128.0 nm. The determination of particle size using Beckman Coulter Delsa nanoparticle analyzer which measured in temperature of 25°C with water as the solvent of the particle.

The chemical used were methanol (Merck, Germany), Giemsa solution (Sigma-Aldrich, United States), immersion oil, 0.9% NaCl (PT. Widatra Bhakti, Indonesia), bovine blood serum, and cyclophosphamide (Cyclovid®, PT Novell Pharmaceutical Laboratories).

The apparatus used in this study were surgical apparatus (Wells Spencer), microscope (Boeco, BM-180, halogen lamp), centrifuge (Dynamica, velocity 18R), microtube, and digital camera.

Animals

The animals used in this research are mice (Mus musculus of Swiss Webster strain) obtained from the Animal House of Faculty of Pharmacy, Universitas Sumatera Utara, Medan, Indonesia. The animal is 3-months-old and has never given birth with bodyweight about 20–30 g. The protocol of this research has been approved by the Ethic Committee of Animal Research of Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara. The approval number is 0606/KEPH-FMIPA/2019.

Testing of antimutagenic effects in mice

The mice were divided into five groups and each group consists of 5 mice. The experimental design is shown in [Table 1].
Table 1: Experimental design

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On the 8th day, the animal was sacrificed then the femur was taken and cleaned. The end of the proximal bone was cut and then the femoral bone marrow was taken using a syringe containing 0.1 ml of bovine serum and phosphate buffer (1:1 v/v). The syringe was inserted into the open bone marrow channel to aspirate the marrow fluid so that it mixes with serum bovine and phosphate buffer. After that, the mixture was put in a microtube.[9],[10],[11]

Preparation of femoral bone marrow smears

The mixture of bone marrow and serum bovine-buffer phosphate (1:1 v/v) in a microtube was centrifuged at 2000 rpm for 5 min, then the supernatant was discarded. The precipitate was resuspended with two drops of serum bovine-buffer phosphate (1:1 v/v). Then one drop of cell suspension was taken and placed on the slide until formed a thin layer. Then, the slide was dried and fixed with methanol for 3 min subsequently stained with May-Gruenwald dye. The staining was carried out for 3 min. After that, the sample was soaked again in May-Gruenwald dye which had been diluted with aqua dest in a ratio of 1:1 for 2 min, then washed with aqua dest as much as two times. The next step, the samples were immersed in a glass filled with Giemsa dye solution mixed with phosphate buffer in a ratio of 1:23 for 10 min. The samples were washed with aqua dest and then dried. Furthermore, the samples were dipped in a glass containing a xylol solution for 10 min then washed with aqua dest. After drying, it was covered with a deck glass and observed under a microscope with a magnification of 10 × 100 with the addition of oil immersion. The number of micronucleus cells in 200 PCE was counted.[12],[13]

Data analysis

Data were analyzed using the Statistical Product and Service Solutions (SPSS) 18 software. To determine the difference in antimutagenic activity in each treatment was carried out using one-way analysis of variance test.


  Results Top


The result of amount of micronucleus existed in the erythrocyte femur is shown in [Figure 1] and [Table 2].
Figure 1: The cells observed in the smear of femoral bone marrow of mice. (a) Normal control; (b) Negative control; (c) 50 mg/kg; (d) 100 mg/kg; (e) 200 mg/kg

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Table 2: The number of micronucleus per 200 peritoneal exudate cells in the bone marrow of mice for 7 days

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Based on the result in [Figure 1] and [Table 2], the percentage reduction in the number of micronucleus per 200 PCE cells in the negative control, doses of 50, 100, and 200 mg/kg, as well as normal control respectively, is 22.65%, 60.3%, 79.6%, 93.8%, and 100%. These results indicate that the increase of the dose of nanoparticle of R. pinnata is directly followed by the decrease in the number of micronucleus.


  Discussion Top


Cyclophosphamide is mostly used to treat various malignancy and nonmalignancy disorder, however, it has a wide spectrum of toxicity.[14] Cyclophosphamide can cause toxicity in the bone marrow and produce micronucleus.[15]

Micronucleus is one indicator of mutation. Micronucleus is the result of mutations from intact chromosomes that are broken and then appear as a small nucleus in a cell. Micronucleus is easily observed in erythrocyte polychromatic cells in the bone marrow. The number of micronucleus PCE indicates the level of genetic damage in the erythropoietic system of a living creature.[1],[16]

Theoretically, the prevention of carcinogenesis/mutagenesis can occur through inhibition of promotion until the progression phase. The initiation process can be inhibited by compounds that decrease the metabolic activation of carcinogens, increase the detoxification of carcinogens, or prevent the bonding between carcinogens and cellular targets.[17]

The antimutagenic mechanism of R. pinnata needs further research, but so far it is suspected related to its antioxidant activity.[18] Previous research showed that the antioxidant activity of R. pinnata had a value of 74.2413 μg/ml that is classified as a strong antioxidant. The antimutagenic activity shown by R. pinnata nanoparticles was stronger than the ethanol extract of R. pinnata leaves.[19] This result showed that by reducing the particle size, it is thought that absorption in the intestine will be better because there is an increase in solubility, an increase in the permeability of the enterocyte membrane, and the opening of the paracellular tight junctions between enterocytes.[20]


  Conclusions Top


Nanoparticles of R. pinnata at the doses of 50, 100, and 200 mg/kg have antimutagenic activity by reducing the amount of micronucleus in PCE. Increasing the dose of nanoparticles of R. pinnata increases antimutagenic activity.

Financial support and sponsorship

This research was funded by Directorate Research and Community Service, Ministry of Research, Technology and Higher Education of the Republic of Indonesia with contract number: 201/UN5.2.3.1/PPM/KP-DRPM/2019.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Fenech M. The in vitro micronucleus technique. Mutat Res Fund Mol M 2000;20:455:81-95.  Back to cited text no. 1
    
2.
Andreassi MG, Barale R, Iozzo P, Picano E. The association of micronucleus frequency with obesity, diabetes and cardiovascular disease. Mutagenesis 2011;26:77-83.  Back to cited text no. 2
    
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Migliore L, Coppedè F, Fenech M, Thomas P. Association of micronucleus frequency with neurodegenerative diseases. Mutagenesis 2011;26:85-92.  Back to cited text no. 3
    
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Sommer S, Buraczewska I, Kruszewski M. Micronucleus assay: The state of art, and future directions. Int J Mol Sci 2020;21:1534.  Back to cited text no. 4
    
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Vilar JB, Ferreira FL, Ferri PH, Guillo LA, Chen Chen L. Assessment of the mutagenic, antimutagenic and cytotoxic activities of ethanolic extract of araticum (Annona crassiflora Mart. 1841) by micronucleus test in mice. Braz J Biol 2008;68:141-7.  Back to cited text no. 5
    
6.
Masfria, Harahap U, Nasution MP, Ilyas S. The activity of Rhaphidophora pinnta L.f. Schott leaf on MCF-7 cell line. Adv Biol Chem 2013;3:397-402.  Back to cited text no. 6
    
7.
Masfria, Harahap U, Nasution MP, Ilyas S. Cytotoxic activity, proliferation inhibition and apoptosis induction of Rhaphidophora pinnata ( L.f.) Schott chloroform fraction to MCF-7 cell line. Int J Pharmtech Res 2014;6:1327-33.  Back to cited text no. 7
    
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Masfria, Hafni A. Cytotoxicity of “Ekor naga” leaf (Rhaphidophora pinnata (L.f.) Schott) chloroform extract against T47D cancer cells. Int J Pharmtech Res 2015;7:238-42.  Back to cited text no. 8
    
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Kalil IC, Gibson BA, Ribeiro CA, Benincá LS, Brasil GA, Andrade TU, et al. Antimutagenic activity of Carica papaya L. assayed in vivo by micronucleus test. Rev Ciênc Farm Básica Apl 2011;32:419-23.  Back to cited text no. 9
    
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Devi KR, Vani S, Minny JP. Protective effects of Solanum lycopersicum fruit extract on cyclophosphamide induced micronuclei in bone marrow cells of mice. Innov J Med Health Sci 2014;4:67-70.  Back to cited text no. 10
    
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Jain RA, Agarwall RC, Jain AP. Evaluation of Argemone mexicana fruits extract using micronucleus assay in mouse bone marrow cells. Bull Pharm Res 2011;1:22-4.  Back to cited text no. 11
    
12.
Busk L, Sjöström B, Ahlborg UG. Effects of vitamin A on cyclophosphamide mutagenicity in vitro (Ames test) and in vivo (mouse micronucleus test). Food Chem Toxicol 1984;22:725-30.  Back to cited text no. 12
    
13.
Hayashi M, Tice RR, MacGregor JT, Anderson D, Blakey DH, Kirsh-Volders M, et al. In vivo rodent erythrocyte micronucleus assay. Mutat Res Envir Muta 1994;312:293-304.  Back to cited text no. 13
    
14.
Fraiser LH, Kanekal S, Kehrer JP. Cyclophosphamide toxicity. Characterising and avoiding the problem. Drugs 1991;42:781-95.  Back to cited text no. 14
    
15.
Goldberg MT, Blakey DH, Bruce WR. Comparison of the effects of 1,2-dimethylhydrazine and cyclophosphamide on micronucleus incidence in bone marrow and colon. Mutat Res 1983;109:91-8.  Back to cited text no. 15
    
16.
Suzuki Y, Nagae Y, Li J, Sakaba H, Mozawa K, Takahashi A, et al. The micronucleus test and erythropoiesis. Effects of erythropoietin and a mutagen on the ratio of polychromatic to normochromatic erythrocytes (P/N ratio). Mutagenesis 1989;4:420-4.  Back to cited text no. 16
    
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Ruddon RW. Cancer Biology. England: Oxford University Press; 2007.  Back to cited text no. 17
    
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Yen GC, Chen HY. Antioxidant activity of various tea extracts in relation to their antimutagenicity. J Agric Food Chem 1995;43:27-32.  Back to cited text no. 18
    
19.
Masfria, Sumaiyah, Dalimunthe A. Antimutagenic activity of ethanol extract of Rhaphidophora pinnata (L.f.) Schott leaves on mice. Sci Pharm 2017;85:7.  Back to cited text no. 19
    
20.
Zhao Q, Luan X, Zheng M, Tian XH, Zhao J, Zhang WD, et al. Synergistic mechanisms of constituents in herbal extracts during intestinal absorption: Focus on natural occurring nanoparticles. Pharmaceutics 2020;12:128.  Back to cited text no. 20
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2]



 

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