Darsanaki R. K, Rokhi M. L, Mohammadi M, Raeisi G, Nourbakhsh M, Aliabadi M. A. Antimutagenic Properties of Nettle Leaf Aqueous Extract (Urtica dioica L.). Biomed Pharmacol J 2012;5(2)

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Reza Kazemi Darsanaki¹, Mahdiyeh Laleh Rokhi², Mahya Mohammadi³, Golnaz Raeisi³, Maryam Nourbakhsh³ and Morteza Azizollahi Aliabadi ³*

¹Young Researchers Club, Lahijan Branch, Islamic Azad University, Lahijan, Iran.

²Department of Microbiology, Falavarjan Branch, Islamic Azad University, Falavarjan, Iran.

³Department of Microbiology, Faculty of Basic Science, Lahijan Branch, Islamic Azad University, Lahijan, Iran.

Abstract

Natural Products, especially plants, have been used for the treatment of various diseases for thousands of years. Cancer is a major public health burden in both developed and developing countries. Plant derived agents are being used for the treatment of cancer. Nettle (Urtica dioica L.) is widely grown in different parts of the world and has been used to promote health. In this study, aqueous extract of nettle leaf (Urtica dioica L.) was studied for antimutagenic properties against sodium azide and acrylic amide by Ames test in presence and absence of rat microsomal liver enzyme (S9). The results showed that nettle leaf aqueous extracts can inhibit mutagenic agents of sodium azide and acrylic amide. Nettle leaf aqueous extracts with the inhibition of 40.39% sodium azide and 43.34%, acrylic amide showed high potential in decreasing mutagenic agents. Antimutagenic activity was increased significantly when there were liver microsome extract (S9).

Keywords

Nettle leaf; Ames test; Antimutagenic activity

Introduction

Cancer is one of the main causes of death in the world and mutagens cause death in millions of patients. Noticing the side effects of the drugs used to treat cancer, scientists are looking for drugs with fewer side effects and more therapeutic effects. Accordingly, the number of studies in this field is rapidly increasing. An abundance of data indicates that diets high in fruits and vegetables are effective in protecting humans from cancer. The search for anticancer agents from plant sources started in earnest in the 1950s with the discovery and development of the vinca alkaloids, vinblastine and vincristine, and the isolation of the cytotoxic podophyllotoxins^1-2. Urtica dioica, nettle or stinging nettle belongs to family Urticaceae. It occurs as a perennial plant in temperate zones of Asia, America and Europe. It is of great medicinal value but the plant is undervalued by almost all of us ³. Urtica dioica herbs are used to treat stomachache in Turkish folk medicine. In addition, this herb is used to treat rheumatic pain and for colds and cough and is used against liver insufficiency ⁴. Each factor that causes removal, inhibition and inactivation of mutagen substances is rewarding. Today, bacteria are being used for the assessment of antimutagenic activities of different compounds in a short-time with excellent results⁵. One of the methods used for assessing the mutation prevention properties of a compound in bacteria is the Ames test. Ames test is a worldwide short-term bacterial reverse mutation test specifically designed for screening a variety of new chemical substances and drugs that can produce genetic damage that leads to gene mutations ^6-7. The Salmonella strains used in the test have different mutations in various genes in the histidine operon, each of these mutations is designed to be responsive to mutagens that act via different mechanisms ^7-8. The aim of the present study was to investigate antimutagenic activity of nettle leaf aqueous extracts against sodium azide and acrylic amide by Ames test in presence and absence of S₉.

Material and Methods

Salmonella typhimurium TA100

Salmonella typhimurium TA100 was from Dr. Ames of the University of California, Berkeley, USA. Salmonella typhimurium TA100 was activated at 37ºC for 48h on a plate containing minimal glucose agar supplemented with biotin, histidine and ampicillin. Tests of histidine requirement, rfa mutation, uvrB mutation and R- factor were carried out to confirm the genotypes of S. typhimurium TA100. Prior to each mutagenicity test, S. typhimurium TA100 was grown in Nutrient broth (Merck; Germany) at 37ºC overnight⁹.

Preparation Aqueous Extract of nettle leaf

Nettle leaves used in this study were collected in winter 2011 from Gilan province (Northern Iran). Then, nettle was left on a bench to dry. The dried sample was chopped into small parts with a blender. For water extraction, 20 g dried aerial parts of nettle ground into a fine powder in a mill and was mixed with 400 ml boiling water by magnetic stirrer during fifteen minutes. Finally, obtained solution was passed through filter⁴.

Preparation of the rat microsomal liver enzyme (S₉) and mutagen substances

A broad range of carcinogenic agents require metabolic activation for recognition. In this investigation, 5 male rats (body weight~200g), were used. Rats were starved for 24 hours in order to get the titer of the liver enzymes to their highest levels. Animals were sacrificed by cervical dislocation and the livers were collected, homogenized in 0.15 M KCl. Livers were cut into pieces using sterile scissors and smashed prior to a 10 min centrifugation at 9000g. The supernatant (S₉) was stored at -80°C. The antimutagenic assay was performed in the presence and absence of S₉. Two chemical mutagen, sodium azide and acrylic amide were purchased from Merck company and dissolved in dimethyl sulfoxide (DMSO), a final concentration of 5 μg/ml ^{7, 8-10}.

Ames test

Antimutagenic properties of of nettle leaf aqueous extracts against sodium azide and acrylic amide, were evaluated by a pre-incubation method of Maron and Ames (1983) in presence and absence of liver microsome extract (S₉) [8]. Each assay was performed in triplicates simultaneously and the percentage of inhibition was determined using the formula [1-T/M] ×100 ¹¹.

Procedure in presence of liver microsome (S₉)

In this assay 0.5 ml of nettle leaf aqueous extract is mixed 0.1 ml of the overnight culture S. Typhimurium TA100 and 0.1 ml of our mutagenic substances including sodium azide and acrylic amide in test-tube containing 3ml top agar. Then, 0.1ml of histidine and biotin 0.5 mM solution and 0.5ml of liver microsome extract (S₉) were added. After were poured on glucose minimal medium and incubated for 24h at 37ºC.

Positive control: The mixture of 0.1 ml of overnight cultured S. typhimurium TA100, 0.1 ml of mutagenic substances including sodium azide and acrylic amide were prepared and were poured in test-tube containing 3ml top agar. Then, 0.1ml of histidine and biotine 0.5 mM solution and 0.5ml of liver microsome extract (S₉) were added, after shaking for 3 minutes, the test-tube contents was poured on glucose minimal medium and incubated for 24h at 37ºC.

Negative control: The mixture of 0.1ml of overnight cultured S. typhimurium TA100, 0.1ml of DMSO, 0.1 ml of histidine and biotine 0.5 mM solution and 0.5 ml of liver microsome extract (S₉) were added to 3ml of top agar. After shaking for 3 minutes, it was poured on glucose minimal medium and incubated for 24h at 37ºC.

Procedure in absence of liver extract (S₉)

All the steps in this stage are the same as previous part. But, here, it was not used from liver microsome extract (S₉).

Results and Discussion

Strain TA100 identity

In accordance with the Salmonella typhimurium TA100 strain genotype, the presence of colony in biotin-histidine medium and absence one in control biotin medium show that these strains are dependent to histidine. The existence of inhibitory zone around the disk indicates that the bacteria do not grow and the Rfa mutation was occurred. This mutation can causes relative decreasing of lipopolysaccharide barriers and then, increase cell wall permeability for bigger molecules. If the inhibitory zone is not presence around the disk, the bacterium has R-factor plasmid and also, lack of growth in radiated culture region indicates that uvr B mutation was ocurred.

Determination antimutagenic activity

The results showed that nettle leaf aqueous extracts can inhibit mutagenic agents of sodium azide and acrylic amide (Table 1, 2). Antimutagenic activity was increased significantly when there were S₉. Olive leaf extract with the inhibition of 40.39% sodium azide and 43.34% acrylic amide showed high potential in decreasing mutagenic agents.

Table 1: Antimutagenic effect of nettle leaf aqueous extracts against sodium azide.

Table 2: Antimutagenic effect of nettle leaf aqueous extracts against acrylic amide.

Cancer is the second leading cause of death in the United States, where one in four deaths is due to cancer. Plants have long been used in the treatment of cancer ¹². The use of antimutagens and anticarcinogens in everyday life is the most effective procedure for preventing human cancer and genetic disease ¹³. Ruan et al, reported that antimutagenic substances may prevent cancer because they can destroy mutagens both inside and outside body cells, and block mutagens that damage DNA and cause mutations in cells ¹⁴. Natural products discovered from medicinal plants have played an important role in the treatment of cancer ¹². Nettle has great medicinal potential. Nettle is nutritionally high in vitamins A, C and D, also minerals iron, manganese, potassium and calcium. It is also beneficial during pregnancy ³. Clinical evidence shows that freeze-dried extracts of nettle reduce allergy symptoms ¹⁵. The study undertaken by Gulcin et al, showed that nettle water extract had an appropriate antioxidant power so that the its inhibitor power of free radicals in DPPH test ¹⁶. Also, the investigations on other plants antimutagenic properties ^17,18-19. Ames test was used to determine antimutagenic of olive leaf. This method is very fast and economical and used to identify antimutagenic and mutagenicity of agents. In this study, consideration of antimutagenic and anti-carcinogenic of nettle leaf aqueous extracts compared with positive controls (sodium azide and acrylic amide) indicates good antimutagenic properties nettle leaf extracts. It can be concluded that nettle leaf should have more effective place in treatment because of antimutagenic and anti-carcinogenic. Of course, more comprehensive researches are needed for indicating scope and exact mechanism of this function. Extract of this plant can be purified and used for food and pharmaceutical industries.

References

1. Dixon. R. A., Canovas, P., Guo, Z., He, X., Lamb, C., McAlister, F. Molecular controls for isoflavanoid biosynthesis in relation to plant and human health. Recent Adv Phytochem., 1999; 33:133–159.
2. Cragg, G.M., Newman, D.J. Plants as a source of anti-cancer agents. Journal of Ethnopharmacology., 2005; 100: 72–79.
3. Bisht, S., Bhandari, S., Bisht, N.S. Urtica dioica (L): an undervalued, economically important plant. Agricultural Science Research Journals.,2012; 2: 250-252.
4. Gülçin, I., Küfrevioˇglu, Ö. I., Oktay, M., Büyükokuroˇglu, M.E. Antioxidant, antimicrobial, antiulcer and analgesic activities of nettle (Urtica dioica L.). Journal of Ethnopharmacology., 2004; 90: 205–215.
5. Issazadeh, K., Azizollahi Aliabadi, M., Kazemi Darsanaki, R., Khoshkholgh Pahlaviani, M.R.M. Antimutagenic Activity of Olive Leaf Aqueous Extract by Ames Test. Advanced Studies in Biology., 2012; 4:397 – 405.
6. Hakura, A., Suzuki, S., Satoh, T. Advantage of the use of human liver S₉ in the Ames test. Mutation Research., 1999;438: 29-36.
7. Mortelmans, K., Zeiger, E. The Ames Salmonella/microsome mutagenicity assay. Mutation Research., 2000;455: 29- 60.
8. Maron, D. R., Ames, B.N. Revised methods for the Salmonella mutagenicity test. Mutation Research., 1983;113: 173-215.
9. Pei-Ren, L., Roch-Chui, Y., Cheng-Chun, C. Ya-Hui, T. Determinations of the antimutagenic activities of several probiotic bifidobacteria under acidic and bile conditions against benzo[a]pyrene by a modified Ames test. International Journal of Food Microbiology., 2004;93: 249-257.
10. Garner, R.C., Miller, E. C., Miller, J.A. Liver microsomal metabolism of aflatoxin B1 to a reactive derivative toxic to S. typhimurium TA1530. Cancer Res., 1972; 32: 2058- 2066.
11. Ong, T., Wong, W., Stwart, J.D. Chlorophyllin a potent antimutagen against environmental and dietary complex mixture. Nutr Res.,1986; 173: 111-115.
12. Shoeb, M. Anticancer agents from medicinal plants. Bangladesh J Pharmaco.,2006; 1: 35-41.
13. Kim, S.Y., Shon, Y.H., Lee, J. S., Kim, C.H., & Nam, K.S. Antimutagenic activity of soybeans fermented with basidiomycetes in Ames/Salmonella test. Biotechnology Letters., 2000;22: 1197-1202.
14. Ruan, C., Liang, Y., & Liu, Z. (1989). Inhibition of twelve Chinese traditional medicinal herbs on mutagenic effect induced by aflatoxin B1. Chin. J. Cancer.,1989; 1: 29–31.
15. Mittman, P. Randomized, Double-Blind Study of Freeze- Dried Urtica dioica in the Treatment of Allergic Rhinitis. Planta Medica., 1990;56: 44–47.

16. Cuendet, M., Ltostettmann, K., Potterat, O. Iridoid glucosides with free radical scavenging properties from Fagraea blumei. Helvetica Chimica Acta., 1997;80: 1144-1152.
17. Emad, S. Antioxidant effect of extracts from red grape seed and peel on lipid oxidation in oils of sunflower. LWT., 2006;39: 883-892.
18. Loots, D.T., Van der Westhuizen, F.H., Botes, L. Aloe ferox leaf gel phytochemical content, antioxidant capacity, and possible health benefits, J Agric Food Chem., 2007;55: 6891-6896.
19. Adhami, V., Ahmad, N., Mukhtar, H. Molecular targets for green tea in prostate cancer prevention. J. Nutr., 2003;133: 2417- 2424.