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Medicinal plant can be defined as a plant that has therapeutic properties which can be used to cure sickness or improve wellness. As mentioned by Farnsworth and Soejarto (1991), medicinal plant is a higher plant that suggested to have medicinal properties which can give effect to health or being use as useful drugs and even contain properties that same as the used drugs. Material inside of a plants has being used for many proposes. Some being used directly as a medicine, in which the substances from the plant will extracted and use as material in producing purified drugs. For example, the usage of extracted Papaver somniferum in morphine and extracted Catharanthus roseus in vincristine (Lewwington, 1993). Medicinal plants also act as a blue-prints in the manufacturing of synthetic drugs that have a similar structure. Alkid cocaine that being extracted from Erythroxyham coca has provided a chemical structure in the production of procaine and other related anesthetics. Furthermore, by using medicinal plants as guide, it given us the understanding in physiological and pharmalogical mechanisms, drug development and testing
In Malaysia, there are about 15,000 species of flowering plants and about 10% has been said to have therapeutic properties (Hanum et, al, 2001). Moreover, Malaysia has been identified as one of the 12 mega diversity countries of the world (Eswani et.,al, 2010). For many years, plants had been used as traditional medicine and had become part of Malaysian culture. Some of medicinal plant that can be found in Malaysia is Labisia pumila (Fig. 2.1) or known as Kacip Fatimah. L.pumila is a popular herb among traditional medicinal practitioner and from Myrsinaceae family. L.pumila has been scientifically proven can be used as facilitate in childbirth likewise as a post mortem medication that will help in the contraction of birth channel as well as to tone abdominal muscles and can be consume for the purpose of regaining body strength (Wan Ezumi et al., 2007).
Fig. 2.1: Labisia pumila (Kacip Fatimah)
2.2 Genomic DNA isolation
DNA is a building blocks consist of four different nucleotides subunits, designated T, C, A, and G. The DNA molecules consist of two strands that wrap around each other to form a double helix, the order of its building blocks provide for the storage of genetic information. Every plant, animals, mammalian, and any organism, there is a copy of the organismâ€™s genetic material at the center of the organism. This DNA carries a complete blueprint of the organism itself. That genetic material is what transfers characteristics from one generation to the next. In 1920s, evidence began to accumulate that DNA is hereditary material (Solomon et al, 2008). Therefore, DNA can be used as a tool to identify organisms between their species.
The basic purpose of DNA extraction is to obtain relatively purified DNA in which can be used later for further investigation such as PCR. Therefore, it is important to use DNA extraction method that can produce a good quality DNA. There are varieties of DNA extraction method that can be used. But different method may yield different result. Jasbeer et al, (2009) research on different DNA extraction method on Zea mays has given different DNA purity and also quantity for each method. Moreover, DNA extraction of animal and plant is quite different whereby in plant tissue there is cell wall making the DNA extraction of a plant requiring the usage of traditional method. The nucleus in plant is guarded within nucleus membrane which surrounded by cell membrane and cell wall. In order to recover the DNA inside the nucleus, there are four basic steps to remove and purify the DNA. They are cell lysis, precipitation, washing, and resuspension. Step 1 and 2 may use different reagents and chemicals for different method whereby step 3 and 4 practically using the same chemical, ethanol for washing and TE buffer for resuspension of the DNA.
2.2.1 Cetyltrimethyl Ammonium Bromide (CTAB)
A good DNA isolation method is the one that are simple, rapid and efficient thus yielding appreciable level of high quality DNA which can be used for further analysis. Based on Moyo et al. (2008) research, the CTAB method was being optimize for DNA extraction of medicinal plant species namely Sclerocarya birrea (tree), Barleria greenii (shrub), Aloe polyphylla and Huernia hystrix (both succulent plants). There are several reagents and chemicals that being used in this method. They are CTAB extraction buffer, CTAB/NaCl solution, CTAB precipitation solution, high salt TE buffer, TE buffer, chloroform: isoamyl alcohol, iso-propanol, absolute ethanol, 80% ethanol; and polyvinylpolypyrrolidone (PVPP).
CTAB extraction buffer will act as detergent and dissolve membrane bound organelles including nucleus, mitochondria and chloroplast. It also will protect the DNA from the endogenous nucleases along with EDTA that exist inside the buffer. EDTA is a chelating agent that will binds with magnesium2+ and a cofactor for most nucleases. As for the PVP, it will to inhibit the polyphenol oxidase activity responsible for the coloration. Later the protein will be separated from the DNA using chloroform: isoamyl alcohol and the supernatant will be collected and high salt TE buffer is added to discard the CTAB since the CTAB has becomes complexes with DNA. As for absolute ethanol, it is needed to precipitate the DNA and washing of the DNA is done by using 80% ethanol. During washing step, CTAB residue, salt and other contaminants will be removes. In Moyo et al. (2008) research has produce a result that the quantity of DNA yield using modified CTAB method is higher if the amount of the sample per 500 Î¼l of extraction buffer is lower. For DNA quantification using UV-Vis spectrophotometer has given the purity ranged between 1.76 to 2.14.
2.3 DNA barcoding
Even though an efficient and fast method can be use to utilize the DNA, obtaining the genetic information within the plant is quite challenging because it highly complex and abundant. One of the methods for plant identification is DNA barcoding which used a certain region in DNA of the plant as a specific identifier. The purpose of DNA barcoding is to be applied as identified individuals as belonging to a species. DNA barcoding also used to form a universal barcode it demands a standardized region in the plants genome; and this region should make it possible to identify even a small piece of tissue from an unidentified organism (Kress et al, 2007). DNA barcoding can be of great use to taxonomists, for instance acting as a sieve to filter large collections into groups to expedite inventories and analyses and particularly to identify species when morphological features are no longer present (Schindel and Miller, 2005).
DNA barcoding has many usages especially as a research tool for taxonomists which will aid the identification of the plant species. It also can expand the species diagnosis to all life history stages, including fruits, seeds, dimorphic sexes, and even damaged specimens. DNA barcoding can also help to test the consistent of species definitions with a DNA measure of variability. It also can used to identify potential new species, especially undecided and cryptic species. In order to obtain the specific region as identifier in DNA barcoding, there are several steps that need to be done first. The DNA need to be extract first from the samples and must undergo PCR process to obtain the specific region and also to replicate the region.