Mutagenicity of Makahiya (Mimosa Pudica) Decoction

Mutagenicity of Makahiya (Mimosa Pudica) Decoction Chapter I BACKGROUND OF THE STUDY Plants have been a valuable source of natural products for maintaining human health, and the use of herbal plants as pharmacological treatments of diseases began long ago. A lot of people who are below the poverty line cannot afford the high cost of commercial synthetic medicines from drugstores and thus opt to use local plants within their reach since these can be obtained readily, cheaply and entail easy preparation and application (Racadio, 2008). Mimosa pudica Linn, which is known as â€Å"Makahiya† in Filipino, is a popular ornamental plant, as its leaves fold up when stimulated by touch, heat or wind (Balag-ey, 2009). It is considered as a common weed that abundantly grows here in the Philippines. Aside from ornamental purposes, local folks use the decoction of the plant for relief from common illnesses, such as asthma, dysentery, dysmenorrhea, rheumatoid arthritis, cough, fever, sore throat and a lot more (Castillo, et. al. 2005). One of the major concerns these days on the safety of plant extracts, is their mutagenicity. Mutagenicity refers to a chemical or physical agent’s capacity to cause mutations or genetic alterations. The identification of plant extracts with chemicals or compounds capable of inducing mutations is crucial since mutagenic compounds can potentially induce cancer (Ming, 2014). Makahiya has been discovered to contain mimosine. Mimosine is a toxic alkaloid that possesses an antimitotic activity that blocks the cell cycle and inhibits DNA synthesis (Xuan, et. al., 2013) thus might cause genomic instability. This may be a factor that leads to the onset of genetic mutations. So far, a study done by Jadhav et.al in 2013 shows that Mimosa pudica seeds, after S9 metabolic activation were found to be mutagenic and significant. Based on this premise, the researchers conducted a similar study, but instead of focusing on just the seeds of the plant, the researchers used the whole plant. Most importantly, this study utilized the decoction extract of the plant since this is the one being used by the local folks. Furthermore, the Muta-chromoplate kit was used instead of the standard Ames test used by Jadhav et.al. Objectives of the Study This study aimed to identify the mutagenicity of Makahiya (Mimosa pudica) decoction and fresh ethanolic extract using Muta-ChromoplateTM kit. Specific Objectives 1. To determine the number of wells with color change per plate of Salmonella typhimurium TA100 strain using: a. decoction with S-9 activation b. decoction without S-9 activation c. fresh ethanolic extract with S-9 activation d. fresh ethanolic extract without S-9 activation 2. To interpret the number of wells with color change per plate of Salmonella typhimurium TA100 strain utilizing 0.05 level of significance in the following preparations: a. decoction with S-9 activation b. decoction without S-9 activation c. fresh ethanolic extract with S-9 activation d. fresh ethanolic extract without S-9 activation Null Hypothesis No Hypothesis No Hypothesis Significance of the Study This study is essential and beneficial to the following: Society. The people will be equipped with more information and be aware of the mutagenicity of Makahiya as herbal medicine. Most specifically, they will be aware of the safety of the decoction preparation of the said herb as it is commonly used in a decocted form. Department of Health. With the understanding of the study, the Department of Health will be able to utilize the findings and disseminate information to the people in the City of Davao regarding the mutagenicity of the usage of Makahiya. Researchers. The study may serve as an avenue in enhancing the skills of the researcher, specifically on scientific inquiry. Moreover, this study may guide the researchers to explore further studies on the mutagenicity of Makahiya. Future Researchers. This study may provide invaluable knowledge, values, and expertise in performing research studies among future researchers if and when they plan to conduct a similar study. This study will be helpful when conducting a scientific inquiry on the problems or issues related to the mutagenicity of Makahiya. Scope and Limitations The scope of this experimental study was the determination of the mutagenicity of the decoction and fresh ethanolic extract of Makahiya. The herb was gathered in Davao City and the extracts were prepared in Davao Medical School Foundation, Inc. laboratory, Dr. A. Gahol Street, Bajada, Davao City. There was no utilization of animals as testing subjects. Rather, Muta-Chromo Plate Kit with Salmonella tyhimirium TA100 strain, which was based on the reverse-mutation test, known as â€Å"Ames Test† was used and provided by the EBPI Company. Also, the S9 enzyme activation was through the use of a crude liver extract provided in the kit. Theoretical Framework The theoretical framework of this study was based on a test developed by Bruce Ames, commonly known as the Ames test. In the study conducted by Ames et al. in 1975 (as cited in Razak et al, 2007) they used a test that utilizes a bacterial strain of the Salmonella typhimurium as tool to detect mutations. These S. typhimurium strains also have other characteristics that enhance their ability to detect mutations. Mutation in the histidine biosynthesis gene makes the cell wall of the bacteria more permeable to large molecules. Mutation in a gene responsible for proper excision and repair of DNA damage increases their sensitivity to mutagens. The strains of S. typhimurium are known as auxotrophs which mean that they are unable to produce a required nutrient. The test organism cannot synthesize the amino acid histidine, thus, it will not grow unless the nutrient is supplied in the growth media.Auxotrophs are usually produced as a result of a mutation that occurs in a prototroph, a bacterium that is able to synthesize the particular nutrient. The Ames’ test determines the ability of a tested substance to cause a reversal, also called a back-mutation of these auxotrophs to the original prototrophic state. During the test, auxotrophs are grown in glucose-minimal salts agar plates that contained all required nutrients but only trace amounts of histidine and biotin. The auxotrophs are able to grow for several generations until the histidine in the media was exhausted. This time they will stop growing unless they have sustained a back-mutation that has restored their ability to synthesize histidine. Suspected mutagenic substances were tested for their ability to stimulate back- mutations by placement set on the surface of the minimal agar plates previously inoculated with the auxotroph. The test substance diffused into the surrounding media and, if mutagenic, would induce back-mutations which would then allow growth into visible colonies. The more revertants observed near the test substance, relative to experimental controls, the more mutagenic the substance was likely to be. Conceptual Framework The study aimed to determine the mutagenic activity of decoction and fresh ethanolic extract of Makahiya on Salmonella typhimurium TA100 strain which involved substitution, addition, or deletion of one or a few DNA base pairs. The independent variables were the decoction and fresh ethanolic extract of Makahiya. The dependent variable was the number of wells with color change per plate of Salmonella typhimurium TA100 strain, with and without the addition of the S9 activation enzyme. Independent Variables Dependent Variables Makahiya (M. pudica) Decoction Fresh Ethanolic Extract Number of wells with color change per plate of Salmonella typhimurium TA100 strain: With S-9 enzyme activation Without S-9 enzyme activation Figure 1.1 Conceptual Framework of the study Definition of Terms Ames test.  A test used to determine the mutagenic potential of a substance based on the mutation rate of bacteria that were exposed to the substance. Decoction.  A method that involves boiling in order to extract a plant substance. Fresh ethanolic extract. Extract collected from M. pudica through the use of dilution with 95% ethyl alcohol followed by rotary evaporation process. Makahiya.  Scientific name,Mimosa pudica. The plant sample used in this study. Muta-ChromePlateTM. A 96-well micro-plate version of the Salmonella typhimurium â€Å"Ames Test Mutagenicity.  The capacity to induce mutation. Salmonella typhimurium.  The strain used for the Ames test. It carries mutant gene that prevents them from synthesizing the essential amino acid histidine from the ingredients in standard bacterial culture medium S-9 enzyme. S-9 is a crude liver enzyme extract utilized to mimic mammalian metabolism. This enzyme is purchased together with the kit.