The use of plant extracts for health purposes has a traditional history. In China and other Far East countries like Korea, plant like pine tree is well known since ancient times and it was used to prepare drinks, which have been taken as anti-hypertension medicine (Kim and Chung, 2000). Ancient Chinese believed that pine tree as emblem of longevity and immortality. It is also believed that the air in a pine forest is clear, non-allergic and filled with pine smell and hence, breathing this air is helpful for people suffered from lung diseases like tuberculosis. However, the isolation of their bioactive components involved did not gain respected speed till 19th century. Epidemiological reports showed that about 64% of the world's population use drug extracted from plants to fight against health problems.
Pine tree which is an evergreen needle-leaf tree belong to the family pinaceae has global distribution and has been considered as an important natural resource contributing to natural medicines and human health. One among the reasons of selecting this plant for this study is that every part of plant is useful. It is possible to get extracts from its cones, barks, needles and even from its dust and all extracts can have beneficial to living organisms (M. Pinelo, M. Rubilar, J. Sineiro, M. J. Nunez, 2004).
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There are more than 115 different species of pine tree known occupying different geographical position depending on the weather and other favourable conditions. Among them are Pinus morrisonicola Hay (Taiwanese white), Pinus sylvestris L (Scotch pine), Pinus strobus L, Pinus densiflora, Pinus nigra (black pine, Austrian pine), pinus aristata,(bristle pine), Pinus banksiana (Jack pine), Pinus bungeana (Lace bark pine common in China) ( Michael Frankis, 2002).
Recently, because of high costs in drug production and hazardous side effects, the exploration of pine tree in the scientific community for diseases treatment has gained momentum. A number of pine trees extracts from needles, barks and cones have been tested its efficacy on the body of model organism and it has indicated that numerous bioactive compounds present in it can be potentially useful for disease treatments and for nutritional value (Kada and Namid, 1981). On the other hands, excessive drinking or eating pine needle (Ponderosa pine) or needle extracts can bring side effects to some organisms like cattle (Panter K.E et al; 1992, Kim I.H, Choi K.C; .2003).
The extraction procedure together with solvent of extraction chosen has greater influences in the yield of expected phenolic components. This choice should base on the chemical characteristics and polarities of the extracts as well as the safety of the people engaged in the experiments. Formerly, dichloromethane and dichlorobenzene were used in the extraction. Studies have been shown that the above mentioned solvents can harm peripheral nervous system, damage liver (hepatocarcinogens) as well as has a cardiovascular effect (Romuldo Benigni; 1992, Jean Q et al; 2000).
Currently, acetone, water, ethanol and hexane are commonly used. In other cases two solvents (ethanol: water, acetone: water) can be combined together in appropriate ratios for extraction of required extracts. Other studies showed the use of different solvents in the same species of pine can yield different amount of pine needle extracts. For example, in Pinus densiflora, extraction by the use of hot water-hexane( HWH) showed high yield of proanthocyanidins (93.8%), while the ethanol extract showed only 44.4% of proanthocyanidins (Nam-Young Kim et al; 2010). This indicated that proanthocyanidins is favored by hexane extraction while other components rather than this condensed tannins are much favored by the ethanol extraction.
One among the most challenging problem in extraction of pine needle extracts is low yield of extracts. A number of research showed that a huge amount of needle is required to obtain measurable amount of extracts. In one extraction, 10kg was homogenized with methanol and extracted for 24 hours, only 721.78gms was obtained (Kim, Keun-Young et al; 2000). Hot solvent procedure under reflux state is efficient for recovery of phenolic components, thus offering higher extract yield (Shon et al. 2004). Other study showed that high yield of phenolic components is favored by shaker extraction (Bushra et al; 2009). There fore it is important to optimize an extraction method to obtain maximum yields of needle extracts especially phenolic contents.
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Stability of pine needle extracts under thermal condition was studied. It has observed that pine needle extracts are stable under thermal conditions. This was proved by subjecting the extracts at 90C and 100C for 30 minutes (Yong-Mu Kim et al; 2005).
Because natural products provide a diversity of structure compared to the synthetic one, it is important to search even new compounds from pine needle extracts. These new compounds possibly would possess remarkable functions compared to known substances.
Currently, unicellular organisms emerged to be useful models for studying different aspects of biological changes that takes place in higher organisms. The use of yeast Saccharomyces cerevisiae as a model is much attractive to scientific community due to ease of genetic manipulation, availability of genome sequence information, being more homology to human systems and other required features for particular interest (Rodriquez, C.E. et al; 2004).
The hypothesis of this study is that pine needle extracts can be used to inhibit growth of yeast Saccharomyces cerevisiae.
The overall goal of this study was to determine bioactivity of pine needle extracts on The specific objectives are to:
Determine the simplest technique and harmless solvent used to extract pine needle analytes.
Identify chemical composition of pine needle extracts by the use of GC-MS.
Investigate the inhibitory property of pine needle extracts on yeast proteins.
1.2. REVIEW OF THE LITERATURE
1.2.1 Pine Needles.
Pine needles are the leaves of coniferous trees that occupied in different parts of the world. These needles possess a valuable extracts including phenols and other components. Phenolic components nowadays became the subject of attention due to present data indicating that consumption in the diet may prevent some diseases like cancer and other degenerative diseases (Cozzi et al; 1997). Plant synthesizes phenolic compounds in response to defensive mechanisms against infection and injury (S. Karakaya, S.N. EL, A.A. Ta; 2001). So it is believed that substances in the plants are able to protect other organisms against invaders.
Classification of pine needle components is great subject to concern. But in many studies flavonoids have been pointed as a largest class of pine needle extract containing polyphenols. Phenolic compounds which are present in pine needle extracts are +(-) catechin, -(-) epicatechin, taxifolin, dihydroquercetin, flavonoid like proanthocyanidins, numerous phenolic acids like gallic acid, caffeic acid, and hydroxycinnamic acid ( J.E. Wood et al; 2002, Porter, L.J; 1986). Apart from phenolic constituents, pine needles have other components like vitamin C, fatty acids, aliphatic acids and resin.
Structurally, polyphenols from plant extracts consist of two benzene rings that are connected to a third heterocyclic ring. There fore, it is possible to distinguish one from another by looking substitution group attached to heterocyclic ring (B.A. Bohm, 1998).
Chemical structures of some common phenolic compounds found in plants.
1.2.2 Bioactivity and health benefits of pine needle extracts.
Although pine needles are non-nutritive compounds, their extracts having phenolic components have many health benefits (HERTOG et al; 1992a). Many research has been pointed their function as antioxidants, anti-obesity, antimicrobial, antimutagenic as well as anti-carcinogenic and anti-inflammatory compounds. However, other report showed that excessive and uncontrolled intake of polyphenols may resulted into mutagenic and also inhibition of important enzymes (SKIBOLA and SMITH; 2000).
1.2.3. Antioxidant activity of pine needle extracts.
Our cells live in the environment which always subjected to free radicals. A free radical is any atom or molecule having at least one unpaired electron in the outermost shell, capable to exist independently. Free radicals are highly reactive due to the presence of unpaired electron. Among the common free radicals are superoxide radical anions (O2), hydroxyl radical (HO), reactive nitrogen species like nitric oxide radical (NO), peroxynitrite (ONOO) and lipid peroxyl radical. Superoxide radicals are produced in the body through biological processes like electron transport chain in mitochondrion in which oxygen act as terminal electron acceptor (Sjodin, T. et al; 1990, B. Frei, 1994). Hydroxyl radicals which are the most damaging radicals within the body can be form from O2 and H2O2 via the Haber-Weiss reaction or Fenton reaction (Koppenol, W.H.2001). When a hydroxyl radical formed in the mitochondrion during the electron transport chain removes hydrogen atom from carbon atoms in the unsaturated fatty acids, a molecule of water is formed leaving behind fatty acid carbon atom with unpaired electron. This carbon centered fatty acid radical reacts with oxygen molecule to form peroxyl radical.
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Under normal circumstances, the body antioxidant defense system can play its role to handle free radicals produced. Under pathological effects, for example, excessive increased in NO radical in the body can bring problems in the cells and tissues (Kim et al; 1999). There fore much attention focused on how to reduce or balance these radicals without cause other effects to the cells. The search for use of antioxidants from out side of the body is an important.
By definition from many scholars, the term antioxidant can be defined as any compound which is capable of quenching reactive oxygen species without itself being converted to destructive radicals (Nashikimi and Yagi; 1996). In short, these are reducing agents which can terminate chain reactions caused by free radicals by being oxidized themselves. There are two categories of antioxidants namely, synthetic and natural ones. Some scholars also put forward the characteristics of effective antioxidants. These including, the ability of competition with substrate for reactive intermediate, able to be repaired by the body system, be inert to the substrate and act as catalyst in quenching mechanism (Boskou and Elmadfa, 1999).
Phenolic components from pine needle extracts nowadays considered as natural antioxidant compounds. This is possible due to the structures they possess. Most of phenolic compounds in these needles contained one or more aromatic rings having one or more hydroxyl groups. By having these structures it's possible to quench free radicals by forming stable phenoxyl radicals (RICE-EVANS et al; 1995).
Variety of research reports confirms the antioxidant property of pine needle extracts. In one experiment, the extracts from water extracted pine needle (WEPN) was studied and revealed the ability of extracts to remove hydroxyl radicals depending on their concentrations, i.e. as concentration increases the percentage of removing hydroxyl radical increases(J.B. Jeong et al; 2009).
Iron is major component of red blood cells and has many significance roles in the body of human being and other organisms. But this metal can gain or lose electrons. Because of this ability of gain or loss of electron, there is great possibility of breaking down of RBCs and releasing free Iron during the stress and the body system to become overloaded with Iron. It is investigated that needle extracts in concentration dependent can act as metal chelators by chelating Fe+2 in similar manner as Deferoxamine, the only iron chelator which is used to remove over loaded iron in human body (J.B. Jeong et al; 2009, Nam-Young Kim et al; 2009). In another experiment which is called FIC assay, the chelating capacities of various extracts were investigated by quantifying in competitive manner with ferrozine for the Fe+2, hexane pine needle extract showed high chelating capacity (Nam-Young Kim et al; 2010). In other report demonstrated that the chelating capacity of needle extracts is due to quercetin (R. A. Walgren et al; 2000).
In other study which called cytochrome c test was conducted. Cytochrome c test is the system which always employed to test drugs which possess anti-oxidant property where xanthine/xanthine (X/XO) system is used as generator of superoxide anions. Pine needle extracts in lowest IC50 show highest scavenging ability compared to other parts of pine tree like cones and barks (T.-Y. Hsu et al; 2005).
Food products which containing lipids can be oxidized easily by oxygen attack and produce unpleasant smell. This is major concern because the products will not be accepted by consumer due to the fact that is no longer safe. (F, Shahid, 1997). Reports showed that pine needle extracts can inhibit Iron induced peroxidation in rat liver and hence can be used as suitable as antioxidant in food preservation to replace the synthetic ones which seemed to have side effects.
1.2.4. Anti-inflammatory property.
Inflammation is defined as a localized reaction of tissue to irritation, injury, or infection. Symptoms of inflammation include pain, swelling, red coloration to the area, and some times loss of movement or function. Inflammation is promoted by Prostaglandins which is produced by enzymes cyclooxygenase 1 and 2 (COX-1 and COX-2). Inflammation is also a component of chronic diseases, such as heart disease and strokes.
Foods rich in sugar have also been related with inflammation, obesity and chronic disease such as diabetes. So it is important to eliminate high-sugar foods such as sodas, soft drinks, pastries, pre-sweetened cereals and candy.
The use of synthetic anti-inflammatory drugs like Aspirin, diclofenac and others for relief has been reported to have side effects including kidney failure, liver failure, ulcers and heart failure. The search for plant constituents to replace these synthetic drugs is globally concerns. Pine needle extracts have shown good results for being used as anti-inflammatory compound.
Neutrophils are white blood cells from granulocyte when activated they can produce reactive oxygen species (ROS). Excessive of ROS lead to inflammation and damage the tissue it occupied. It has been shown that pine needle extracts are able to modulate cytotoxic effects produced by activated human neutrophils (W. K. KIM-PARK et al; 2003). In another study on ethyl acetate extraction pine needle, it was observed that epicatechin and p-coumaric acid as the constituents of needle extracts were able to inhibit inducible nitric oxide synthase and cyclooxygenase 2 (COX-2), protein expressions in Lipopolysaccharides-stimulated RAW 264.7 cells (Yen GC et al; 2007).
Anti-inflammatory property of needle extracts was also studied on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) subjected to the cytokines such as IFN-y and TNF-alpha, and to the lipopolysaccharide (LPS) stimulated RAW 264.7 cells. Needle extracts from Pinus densiflora inhibit expressions of inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) and no nitric oxide and prostaglandins produced (Joo Hee Kwon et al; 2010).
Pine needle extract is potent analgesic compound and act as anti-inflammatory agent for people with joint pain, arthritis and rheumatism. It works by cleaning toxins from the body.
1.2.5. Anti-obesity and anti-diabetic activity of pine needle
Obesity is the abnormal or accumulation of excessive fats to extend that lead to risk of an individuals health. One of its consequences is disease called diabetes. According to WHO, 17million people die due to diabetes. Synthetic anti-diabetic drugs like Insulin glargine, Pioglitazone and Glimepiride have been used to regulate amount of blood sugar in diabetic patients. The drugs have shown many side effects including headache, loss of appetite and diarrhea. The search for the safe drugs for treatment of diabetes is globally concerns. Bioactive components from pine needle extracts have shown positive results during clinical trials.
In one experimental report indicated that, pine needles and bark ethanol extracts have the ability to inhibit several carbohydrate-hydrolysing enzymes like alpha-amylase and alpha-glucosidase in Saccharomyces cerevisiae in combination of non-competitive and un-competitive mode of inhibition(Yong-Mu Kim et al; 2005).
In study conducted on rats as animal model, anti-obesity of pine needle extracts have been revealed by inhibition of glycerol-3-phosphate dehydrogenase (GPDH) activity, an enzyme which responsible for differentiation of 3T3-L1 preadipocytes into adipocytes. This means needle extracts have the ability to reduce body weight caused by food rich in fat (Jeong-Ryae Jeong et al; 2006).
Study of serum and liver lipid composition in rat fed with food which is highly oxidized was conducted. The rats were subjected to the food diet with normal, normal diet and oxidized food, normal diet plus oxidized food and powdered pine needle. The needle tends to decrease the level of plasma cholesterol in rats. This proves the anti-obesity activity of needle extracts (Lee E. 2003).
1.2.6. Antimutagenic and anticancer activity of pine needle extracts.
Cancer is the one among the global burden disease affecting a number of people. Statistically, about 7.6 million people suffering from cancer and cancer related diseases in the year 2007. In one way, cancer is formed when human body produces cells in which its growth can not controlled by normal body mechanisms and hence these cells can not die. These cells can manage to move through out of the body using blood or lymph as a transport agents, manage to divide and producing new blood vessels for its own food. On the other hand, these uncontrolled growth cells are caused by endogenous substances which damage genetic materials. In other word, cancer is formed as a result of alteration that prevent oncogene and tumor suppressor gene function.
Recently, much attention has been directed towards discovering chemotherapy from the plant extracts especially pine needle extracts which can prevent formation of cancer cells with few side effects. Polyphenols isolated from pine needle extracts showed promising results on preventing cancer cell formation (Yang and Wang, 1993). Anticancer activity of polyphenols is difficult to defend. Some reports related this property with one component present in polyphenol. For example, terpenes and their derivatives have been found to be useful in prevention of cancer (R. Paduch et al; 2007). In another study from red pine leaves, it is found that epigallocatechin gallate and catechin gallate inhibit invasion of SK-Hep-1 human hepatocellular carcinoma cells (SK-Hep-1 cells) (Sang, Jun Lee et al; 2007). Other report indicated that tannins and their derivatives like tannic acid has shown interesting as antimutagenic and antitumourigenic compounds (Mukhtar et al; 1988). Other study indicated that flavone can inhibit growth and initiate apoptosis reaction in human colon carcinoma cells (Wenzel et al; 2000 ).
Because pine needle extracts are galanicals, it is untrue to relate only one component of extracts with a certain function in a tested system. Anticancer property of polyphenol depends on the kind of cancer it concern and the concentration of needle extract applied to the cancer cells.
Leukemia is a cancer of blood or bone marrow in which bone marrow produces abnormal Leukocytes (leukemia cells). These abnormal cells are hard to die as the normal process of cells. Due to this situation it prevents normal blood cells to perform its work. Pine needle extract has cytotoxic effect on leukemia cells. Study from Pinus morrisonicola Hay revealed that water extract from this tree has highest inhibition capacity against leukemia cell line U937 (T.-Y. Hsu et al; 2005).
Aflatoxin B1 (AFB1) is a toxin chemical produced by fungus Aspergillus spp has considered as a causative agent of cancer. Mechanisms in which this chemical (AFB1) induces carcinogenicity is still a subject to learn, although some authors relate with production of ROS in intracellular components. Pine needle extracts from Pinus densiflora had shown it is ability by decreasing the level of intracellular ROS in human hepatoma cell line (HepG2) treated with Aflatoxin B1. This result confirms the anti carcinogenicity of needle extracts (Joon-Kyoung Lee et al; 2010).
In the case study which engage micronucleus and sister chromatin exchange indicated that pine needle extracts are ideal compounds for antimugenicity. Cyclophosphamide as a mutagent was inhibited by pine needle extracts (Kong Z, Liu Z, Ding B; 1995).
Anticancer activity of needle extract have been supported by results of Kwak, CS, Moon SC, Lee MS (2006), in which experimental rats was treated with mammary carcinogen, 7,12-dimethyl benz[a]anthracene. Formation of tumor cells was suppressed by pine needle diet given to this organism. This indicated that needle extract is anticarcinogenic compound.
N-nitroso compounds (NOC) are much more used as food additives. But these compounds are cancer causative agent in humans and animals. Report from experiments which involved mutagenicity compound like N-methyl- N-nitro - N-Nitroguanidine (MNNG) on microorganism Salmonella typhimurium TA98 and TA100 revealed that pinus densiflora ethanol extract is suitable for being antimutagenic compound. Thus, it inhibits mutagenicity induced by MNNG on these organisms (Kim, eun Jeong; 2007).
1.2.7. Antimicrobial activity of needle extracts.
Plants have been used as a good source for treatment of diseases since ancient time. Alkaloids and other related compounds even now are used as the source of drugs which have plant origin to treat diseases which are caused by variety of microorganisms. Quinine is the one of them which despite of being used to treat malaria, also can be used to relieve nocturnal leg cramps. One noticed fact on the need of plant extracts is that usually they have multiple effects on the body. For example, Hydrastis Canadensis is not only has antimicrobial property, but also has ability to increase blood supply to the spleen causing promotion of its activity to release mediating compounds (Murray, 1995). Although one half of all pharmaceuticals used in these current world originated from plants, a negligible amounts were intended for use as antimicrobials, because of most antimicrobial drugs extracted from fungi and bacteria sources.
Current emerging of anti resistance microbes worldwide is the subject of concerns. Many bacteria and fungi are no longer sensitive to most of synthetic antibiotic drugs. There fore, there is in need of finding new alternatives for combating these microbes.
Plant synthesizes secondary metabolites like polyphenols for different purposes including pigmentation, reproduction and for resistance to pathogens like fungal and bacteria. It is true to say plant synthesize greater secondary metabolites compared to animals because they cant move physically to escape the invaders, and these metabolites acts as defenders against herbivores, microbes and viruses (Swan, T. 1977, Kutchan, T.M; 2001). Because polyphenols have seen to protect plants from pathogens, it is believed that it can do so in other systems like human body and other animals.
Most of current studies support this fact that polyphenols from plant extracts have cytotoxic effects on microbes like bacteria and fungi. Few report talks about antiviral activity (Perez, R.M; 2003).
There are contradiction reports on the reactivity of polyphenols on relation to their chemical structures. Some reports showed that polyphenols which are less polar i. e polyphenols lacking hydroxyl groups on their B ring have high ability to engulf microbial compared to those having hydroxyl groups in B ring (CHABOT et al; 1992). Report from other study support this phenomenon by showing, substitution of hydroxyl groups by methyl group in the nucleus of polyphenols increases antimicrobial activity in some bacteria like staphylococcus aureus (IBEWUIKE et al; 1997).
In contrary, other authors relates the site and the number of hydroxyl groups present in polyphenols. As the number of hydroxyl group increases, the toxicity of polyphenols on microbial also increases (Ionela Daciana et al; 2007). This idea was supported by other author who clamed that polyhydroxyl groups on A and B rings together with substitution on the A ring are factors for determination of antimicrobial activity on polyphenol component like flavones on bacteria like actinomyces viscosus and streptococcus (Sato et al; 1996). Another study investigated that methylated phenols are not suitable enough for being protective compounds against microbial. This was proved by observing antimicrobial activity of polyphenol components having glycosides like quercetin which shows antimicrobial property against variety of microbes (Tereschuk et al; 1997)
Also it has been shown that oxidation of phenols have positive effects on inhibition of microbial activity. This oxidized phenolic has been related to the necrotic reaction which resulted to the formation of barrier which hinders pathogen to penetrate to the cell of tested organism. Also this reaction can decrease essential nutrients for fungal development.
Although some reports indicated that tannins is antimicrobial compounds, several species still have ability to grow in tannins materials (SCALBERT. 1991). This abnormality is due to the capability of these bacteria to secrete tannins binding polymers. Example of this fact is penicillin species. But all in all, many reports support the antimicrobial activity of tannins. Study from tannins extracted from pine needles using water and acetone as extractants have shown inhibitory activity in most of agricultural microbes. The mode of inhibition is due of delaying decomposition process of organic matter in the soil. The inhibitory activity depends on species of microbes and concentration of needle tannins. For examples, Azotobacter species (VL-A2) can tolerate up to 1000ppm tannins concentration without showing any growth inhibition. In contrary, only 50ppm concentration is enough to inhibit growth of Rhzobium species (VL-R1). Also 100ppm concentration is suitable for inhibition of Bacillus halodurans (MTCC-7181). Fungal shows more resistance behavior when subjected with tannins extract. Up to 1000ppm concentrations fungal like Pleurotus djmor can still grow. Other fungal like Trichoderma virescens (MTCC-6321) and Trichoderma reesii (VL-T21) can tolerates up to 3000ppm concentration in both water and acetone extracted tannins (G. Selvakumar et al; 2007).
In another study on antimicrobial activity of pine needle extracts, it is investigated that the antimicrobial activity depends on pH value. The aqueous ethanolic pine needle extracts under acidic condition showed better antimicrobial effect and can hinders growth of spoilage bacteria (Zeng Wei-Cai et al; 2009). To support the effect of pH on the antimicrobial activity, other study has been done under pH 3.6 on diethyl ether extracts tested in six bacteria species. The results showed that pine needle extracts have strong antimicrobial property against E. coli bacteria depending on time extension on the needle extraction (Yong- Suk Kim et al; 2004).
In separate study, antimicrobial property of needle extracts was related to fermentation process. A self fermented needle extracts from Pinus morrisonicola which kept for 7 years showed inhibitory activity by inhibiting growth of bacteria like Salmonella typhimurin, Staphylococcus aureus, E. coli and Bacillus subtilis (Gayoung Park et al; 2008). With almost the same results, Pinus massoniana was tested against antimicrobial activity by disc diffusion method. Aqueous pine needle extracts demonstrates its effects by inhibiting growth of bacteria like Bacillus subtilis, staphylococcus aureus, Bacillus cereus and E. coli (Su Feng et al; 2009).
Fungi are unicellular eukaryotic organisms which are worldwide known due to it is benefits to human and plants. There are about 100000 species of known fungi according to taxonomists (Kirk et al; 2001) which were divided into seven phylum. These are phylum Microspodia, Chytridiomycota, Neocalli mastigomycota, Glomeromycota, Ascomycota and Basidiomycota
Among the economic importance of fungi to human being is the use of these organisms in fermentation industries like brewing, in biomedical research like cancer research and drug metabolism, in biological research like molecular biology and biochemistry, in environmental technologies like bioremediation and waste utilization.
The most and well studied phyla in fungi is Ascomycota phylum which comprises of yeast like Saccharomyces cerevisiae. Currently, scientific world turns the eyes on research application of yeast rather than brewing and baking. The exceptional properties of the yeast, Saccharomyces cerevisiae, among several 700 yeast species and its huge hidden potential which has been exploited for several thousands of years, made it to be an ideal organism also for research.
Yeast has been introduced as a model organism in the 20th century (Roman, 1981). Currently, the use of this model organism received special attention due to being the first eukaryotic organism of which the whole genome sequence is available (Goffeau et al; 1996., Dujon, 1996). The richness of information obtained from DNA sequence of complete genome is much useful as a reference against which sequence of human is compared. Also knowing the number of genes or chromosomes, appropriate size and place, with selection of required conditions and methods; it is possible to isolate chromosome-specific DNA (Carle and Olson; 1985).
Yeast cells have capability of being transformed through recombination process either by deletion or addition of new genes. This characteristic made yeast accessible for gene cloning and genetic engineering methods. For example, the yeast genes which still have ability to be expressed are fused to green fluorescent (GFP) and allowed to place gene products in living cell by using fluorescent microscopy (Niedenthal et al; 1996).
Saccharomyces cerevisiae exists in both a stable haploid and diploid state. In this characteristic feature, recessive mutations which are usually masked in diploid state can be phenotypically analyzed in haploid state.
Short generation time, having a lot of mutants and accessibility to grow in defined media also made scientists to select this organism as a model. Yeast has a generation time of almost 90 minutes for completion of life cycle, easily cultured in the presence of fermented carbon source, so it is easily to manage generation time and culturing procedure in the laboratory. The mutant strains of yeast can be used as makers. For examples are auxotrphic makers, drug makers (Sherman, F., P. Waken., 1991; mapping yeast genes. Methods enzymol. 194: 38-57)
Several reports also demonstrating the antifungal activity of pine needle extract. Study of composition of ethanolic and hexane extracts of Pinus taiwanensis and Pinus morrisonicola needles indicated that its effectively antifungal compounds on decay fungi and plant pathogen fungi like Alternaria mali. The antifungal property of these two needles was caused by having essential oils and caryophyllene oxide (Tsao, Chun-Wei. 2005, Kyung-Hwan Jung et al; 2007). In another study from Pinus koraiensis water extracted by distillation method indicated that, the oil which composed of alpha pinene from its needles has antifungal activity by inhibition growth of fungus Candida albicans (Eui - Ju Hong et al; 2004). Fungicidal properties have been studied in air pollutant microorganisms. It is observed that some microorganisms like Aspergillus flavus, A. oryzea and their toxins are able to cause problem in breathing and headache. Pine needle extract from Pinus sylvestris in lowest minimum inhibition concentration showed to be active against yeast and other fungi (Ona Motie Junaite et al; 2004).