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CHAPTER 2REVIEW OF RELATED LITERATURECONCEPTUAL LITERATUREMus musculusAccording to the Foundation for Biomedical Research (FBR), 95 percent of all lab animals are mice and rats. Scientists and researchers rely on mice and rats for several reasons. One is convenience: rodents are small, easily housed and maintained, and adapt well to new surroundings. They also reproduce quickly and have a short lifespan of two to three years, so several generations of mice can be observed in a relatively short period of time. Mice and rats are also relatively inexpensive and can be bought in large quantities from commercial producers that breed rodents specifically for research. The rodents are also generally mild-tempered and docile, making them easy for researchers to handle, although some types of mice and rats can be more difficult to restrain than others.

Most of the mice and rats used in medical trials are inbred so that, other than sex differences, they are almost identical genetically. This helps make the results of medical trials more uniform, according to the National Human Genome Research Institute. As a minimum requirement, mice used in experiments must be of the same purebred species. Another reason rodents are used as models in medical testing is that their genetic, biological and behavioral characteristics closely resemble those of humans, and many symptoms of human conditions can be replicated in mice and rats. “Rats and mice are mammals that share many processes with humans and are appropriate for use to answer many research questions,” said Jenny Haliski, a representative for the National Institutes of Health (NIH) Office of Laboratory Animal Welfare. Over the last two decades, those similarities have become even stronger. Scientists can now breed genetically-altered mice called “transgenic mice” that carry genes that are similar to those that cause human diseases. Likewise, select genes can be turned off or made inactive, creating “knockout mice,” which can be used to evaluate the effects of cancer-causing chemicals (carcinogens) and assess drug safety, according to the FBR.

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Rodents also make efficient research animals because their anatomy, physiology, and genetics are well-understood by researchers, making it easier to tell what changes in the mice’s behaviors or characteristics are caused by (Melina,2012).Physical DescriptionMus musculus or also known as house mice are from 65 to 95 mm long from the tip of their nose to the end of their body; their tails are 60 to 105 mm long. Their fur ranges in color from light brown to black, and they generally have white or buffy bellies. They have long tails that have very little fur and have circular rows of scales (annulations). The house mouse is native to Eurasia, but it now has a worldwide distribution because of accidental introductions. This species was not known in the United States until about the time of the American Revolution when it is believed to have arrived as a stowaway aboard transatlantic ships. It is believed originally to have been transported to the southern United States along shipping lanes from the Iberian peninsula (Schwarz and Schwarz, 1943).History as a Biological ModelMice have been used in biomedical research since the 16th Century when William Harvey used them for his studies on reproduction and blood circulation and Robert Hooke used them to investigate the biological consequences of an increase in air pressure.

During the 18th Century, Joseph Priestley and Antoine Lavoisier both used mice to study respiration. In the 19th Century, Gregor Mendel carried out his early investigations of inheritance on mouse coat color but was asked by his superior to stop breeding in his cell “smelly creatures that, in addition, copulated and had sex”. He then switched his investigations to peas but, as his observations were published in a somewhat obscure botanical journal, they were virtually ignored for over 35 years until they were rediscovered in the early 20th Century. In 1902 Lucien Cuénot published the results of his experiments using mice which showed that Mendel’s laws of inheritance were also valid for animals — results that were soon confirmed and extended to other species.In the early part of the 20th century Clarence Cook Little, a Harvard undergraduate was conducting studies on mouse genetics in the laboratory of William Ernest Castle. Little and Castle collaborated closely with Abbie Lathrop who was a breeder of fancy mice and rats which she marketed to rodent hobbyists and keepers of exotic pets and later began selling in large numbers to scientific researchers.

Together they generated the DBA (Dilute, Brown, and non-Agouti) inbred mouse strain and initiated the systematic generation of inbred strains. The mouse has since been used extensively as a model organism and is associated with many important biological discoveries of the 20th and 21st Centuries.The Jackson Laboratory in Bar Harbor, Maine is currently one of the world’s largest suppliers of laboratory mice, with around 3 million mice a year. The laboratory is also the world’s source for more than 8,000 strains of genetically defined mice and is home to the Mouse Genome Informatics database.As stated by An Introduction to the Laboratory Mouse: Mus musculus. Mice (Mus musculus) are an important research tool for modeling human disease progression and development in the lab.

Despite differences in their size and appearance, mice share a distinct genetic similarity to humans, and their ability to reproduce and mature quickly make them efficient and economical candidate mammals for scientific study. Caulerpa lentilliferaCaulerpa lentillifera is a kind of edible seaweed, known as ‘sea grape’ or ‘green caviar’. (Kudaka, 2008).  It belongs to the genus of Caulerpa and the family of Caulerpaceae. The genus Caulerpa is common seaweed in tropical and subtropical water. Within this genus, Caulerpa lentillifera is one of the favored species due to its grass-green in color, soft, and succulent texture and usually consumed in the form of fresh vegetable or salad.

It can be cultivated in ponds and open lagoon in the Philippines. The seaweed resembles bunches of little grapes. This seaweed is usually tightly packed on a vertical ‘stem’, often forming a sausage-like shape with the length of 2-10cm long. This species is distinguished by the distinct constriction where the ‘grape’ attaches to the stalk.

The ‘stems’ emerge from a long horizontal ‘root’ that creeps over the surface. Its colors range from bright green to bluish and olive green. Round sea grapes are a popular edible species in some places. In the Philippines, the seaweed is eaten fresh as a salad or salted so it can be eaten later. Small quantities are also exported to Japan. It is also eaten in Malaysia and Indonesia. This seaweed is commercially farmed in Cebu, Philippines. Cuttings are planted by hand in muddy mangrove ponds and harvested about two months later.

The seaweed is also fed to livestock and fish. The seaweed is high in minerals and is said to taste refreshing. It is also reported to have antibacterial and antifungal properties and to be used to treat high blood pressure and rheumatism.Caulerpa lentillifera is highly nourishing as it contains vast proportions of minerals and vitamins. Asian people believe that if they eat this seaweed they can recover from serious illnesses as it contains high amounts of vitamin A, Vitamin C, and minerals.

  Caulerpa lentillifera is also a good source of magnesium that is essential in reducing high blood pressure and preventing the heart attack. This category of seaweed also helps in suppressing cancer cell effect. And contains a large amount of iodine which makes it useful for people suffering from thyroid problems. The chemical composition of seaweeds varies with the species, habitat, maturity and environmental conditions it acquires. In general, seaweeds are rich in non-starch polysaccharides, vitamins, and minerals. As seaweed polysaccharides cannot be easily digested by a human, they are considered as a new source of dietary fiber and food ingredient.

Since seaweed produces low lipid content, they also provide a very low amount of energy. Thus consumption of seaweeds can increase the intake of dietary fiber and lower the occurrence of chronic diseases such as cardiovascular diseases.Cardiovascular Disease and HypercholesterolemiaHypercholesterolemia is defined as excessively high plasma cholesterol levels and is a strong risk factor for many negative cardiovascular events. (Stapleton et. Al, 2010). People with hypercholesterolemia have a high risk of developing a form of heart disease called coronary artery disease. This condition occurs when excess cholesterol in the bloodstream is deposited in the walls of blood vessels, particularly in the arteries that supply blood to the heart (coronary arteries). The abnormal buildup of cholesterol forms clumps (plaque) that narrow and harden artery walls.

As the clumps get bigger, they can clog the arteries and restrict the flow of blood to the heart. The buildup of plaque in coronary arteries causes a form of chest pain called angina and greatly increases a person’s risk of having a heart attack. Modern primary care practitioners spend considerable time and effort on preventative medicine. Diagnosing and managing hypercholesterolemia as a way to prevent cardiovascular disease (CVD) is a common activity for primary care physicians.

According to Centers for Disease Control data from a survey of 1,492 physicians who provide ambulatory care in non-government settings, hypercholesterolemia is second only to hypertension in the list of the 10 most common chronic conditions that were seen. The fact that hypercholesterolemia is a strong risk factor for cardiovascular disease (CVD) is well established. The problem can be due solely to hereditary factors, but more commonly it is an acquired condition (R.H, Nelson (2013).Hypercholesterolemia is a dominant risk factor for the development and progression of atherosclerosis and cardiovascular diseases. Natural compounds have been proved to be useful in lowering serum cholesterol to slow down the progression of cardiovascular diseases (LP, Yan 2006) such as seaweeds.

Chemical Composition of Caulerpa lentillifera    The chemical composition of the Caulerpa lentillifera that helps in preventing hypercholesterolaemia and peroxidation in rats were studied via evaluating the plasma lipids and, plasma and organs malondialdehyde (MDA) concentrations. The rats were fed on high-cholesterol/high-fat (HCF) diets in order to increase their cholesterol levels. The seaweed has also antioxidant components but the main focus is on lowering the blood pressure. Chemical analysis of seaweeds comprised of a proximate composition, dietary fiber, vitamin C, vitamin E (?-tocopherol), minerals, carotenoids, chlorophylls, fatty acids and amino acids. Also, there are tests for liver, heart and kidney damage in order to check if there is necrosis with the said organs upon intake of the seaweed. Presence of biological markers such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), ?-glutamyltransferase (GGT), creatinine kinase (CK), CK-MB isoenzyme, urea, creatinine and uric acid indicates that the patient is positive for that certain disease.     According to a test done with the Malaysia Seaweed by Matanjun, Patricia (2008) her results showed that the administration of C.

lentillifera and another seaweed reduced (P<0.05) plasma low-density lipoprotein cholesterol and triglyceride, and increased (P<0.05) plasma high-density lipoprotein cholesterol thus improving the atherogenic index of rats fed an HCF diet. The seaweed reduces body weight gain in rats fed an HCF diet.

    Caulerpa lentillifera has hypolipidemic effects and can apply a protective effect in reducing the cardiac, hepatic, renal and brain abnormalities in rats fed HCF diet. The presence of high dietary fiber especially soluble fiber, omega-3 fatty acids such as eicosapentaenoic acid (C20:5?3) in C. lentillifera can possibly contribute to lowering the cholesterol level in rats.According to Wresdiyati et al. (2006a,b)  hypercholes-terolemic conditions altered the liver and kidney tissues of rats, and decreased the level of intracellular antioxidant copper, zinc-superoxide dismutase (Cu,Zn-SOD) in these organs. These alterations may account for the hypercholesterolemic condition, arousing the production of reactive oxygen species-free radical.

The increased levels of the reactive oxygen species (ROS), reactive nitrogen species (RNS), and other free radicals may create a situation known as oxidative stress. Human body cells can rust when breathing due to oxidative stress, a process caused by free radicals. The condition may lead to metabolic impairment and cell death.

Free radicals are the natural byproducts of chemical processes, such as metabolism. According to Dr. Lauri Wright, a registered dietitian and an assistant professor of nutrition at the University of South Florida, free radicals as waste products from various chemical reactions in the cell that when built up, harm the cells of the body. Yet, free radicals are essential to life (Wanjek 2006).Seaweed draws an extraordinary wealth of mineral elements from the sea that can account for up to 36% of its dry mass. The mineral macronutrients include sodium, calcium, magnesium, potassium, chlorine, sulfur and phosphorus; the micronutrients include iodine, iron, zinc, copper, selenium, molybdenum, fluoride, manganese, boron, nickel and cobalt. Seaweeds have a salty taste that is an indication that the material can disperse phlegm accumulation, particularly as it forms soft masses, include goiter, the thyroid swelling that indicates severe iodine deficiency.

Saccharina japonica, Sargassum, and Porphyra are medicinal seaweeds that are being used in China. Saccharina japonica and  Sargassum are salty and cold and enter the liver, lung, and kidney meridians. Both can clear heat, transform phlegm, soften hardness, and dissipate nodules. According to Yang Yifan who wrote about the differences between the two seaweeds, Sargassum is stronger in transforming phlegm and dissipating nodules, and it is more suitable for treating goiter and scrofula.

While Saccharina japonica is stronger in softening hardness and reducing congealed blood; it is more suitable for treating liver-spleen enlargement, liver cirrhosis, and tumors.Caulerpa lentillifera is widely distributed in tropical areas of the Indian and Pacific Oceans. In South-East Asia it has been recorded in Thailand, Vietnam, Peninsular Malaysia, Singapore, Indonesia, the Philippines and Papua New Guinea.

Caulerpa lentillifera is generally found growing on sandy to muddy substrates on reef flats. The seaweed is stenohaline and cannot thrive in areas where salinity is less than 25‰. Caulerpa lentillifera is mainly used in a raw vegetable salad and a potential source of protein and shows haemagglutinin activity. It usually contains small quantities of terpenoids. As a medicine, it has antibacterial and antifungal properties and is used to lower blood pressure and to treat rheumatism.

RESEARCH LITERATURE    In a study conducted by Mark Colocado of De La Salle University-Dasmariñas entitled “Effect Caulerpa lentillifera (Lato) Extract in Blood Cholesterol Level of  Mus musculus (Albino mice)”, this study was undertaken to determine the possible effects of Caulerpa lentillifera (lato) extracts on lowering the blood cholesterol level of albino mice. It showed that various concentrations of lato extracts were prepared and orally administered to the mice two times a day for four weeks. Eighteen (18) albino mice were divided into 3 treatment groups, T1 (50%) lato extract, T2 (75%) lato extract, and T3 (100%) lato extract. Each treatment was done in duplicate.

The mice were acclimatized for one whole week (seven days) before administration of high-fat diet. After acclimatization, two weeks was given for the induction of pellets with commercial margarine to obtain hypercholesterolemia on the mice. Thereafter, four weeks were allotted for the administration of treatments. Three sets of blood samples were collected: after acclimatization, after high-fat diet, and after treatment. Blood samples were analyzed using a Kernel multi check device. Results show that each concentration significantly decreased the blood cholesterol levels in albino mice (p= 0.000655).

Similarly, the efficiency of each treatment to reduce the blood cholesterol level manifested significant difference (p = 0.023). The results are indicative of efficiency range of T3>T2>T1. The ranking shows that the more the concentrations, the more it is effective in lowering the blood cholesterol level. The ratio 1:1 (part water, part lato extract) which was the 50% lato extract was the least effective, 3:1 ratio which was the 75% lato extract was the second most effective, and the pure 100% lato extract was the most effective of all the treatment. This may be attributed to its bioactive compounds such as dietary fibers, essential fatty acids, vitamins, and minerals present in different dosages. Thus, the higher the dosage of the seaweed extracts, the more it is effective.

Another study performed by Ma. Corazon S. Loquellano, et. al “Cholesterol Lowering Activity Of Formulated Green Caviar (Caulerpa Lentillifera J. Agardh.,Caulerpaceae) Seaweed Extract Tablet in Hypercholesterolemia-Induced Rabbits” a study  was designed to investigate the effects of the formulated Green Caviar (Caulerpa lentillifera J. Agardh) seaweed extract in lowering total cholesterol, triglyceride and low-density lipoprotein levels in 15 rabbits fed on high-cholesterol/high-fat diets. Ethanolic extract was determined on its Acute Oral Toxicity using female albino rabbits and Approximate Effective Dose (AED) using male rabbits.

Meanwhile, Effective Dose (ED90) was determined within AED range using Algebraic Probit analysis using hypercholesterolemia-induced rabbits. Bioassay for the formulated tablet was obtained by dividing 3 groups of rabbits. Group 1 was administered with the formulated Green caviar tablet, group 2 with placebo and 5 mg/kg dose of Simvastatin (positive control) for group 3. Rabbits on the high cholesterol fat diet had significantly increased plasma total cholesterol (TC), plasma low-density lipoprotein cholesterol (LDL-C), plasma triglycerides (TG) in 30 days. Animals treated with Simvastatin showed a decrease in total cholesterol, triglycerides and LDL level at 1.

1475± 0.7030, 1.0775 ± 01.2224 and 0.76 ± 0.7109 respectively.Those that received the formulated test drug have shown positive response towards the treatment as indicated in the decreased of values in these parameters. Total cholesterol, triglycerides, and LDL lowered by the experimental group was found to be at 1.

36 ± 0.1627, 1.1725 ± 0.7444 and 0.13 ± 0.4567.

Statistical analysis showed that there is no significant difference in the total cholesterol, triglyceride and low-density lipoprotein lowered by the formulated Green Caviar tablet and Simvastatin.It was revealed in the study of “The Potential Health Benefits of Seaweed and Seaweed Extracts” the in vitro antioxidant activity and total phenolic screenings of eight species of Malaysia seaweeds (Kappaphycus alvarezii, Eucheuma denticulatum, Halymenia durvillaei, Caulerpa lentillifera, Caulerpa racemosa, Dicyota dichotoma, Sargassum polycystum and Padina spp.), determine chemical composition of three selected edible seaweeds and investigate effects of these seaweeds on antioxidative, cholesterol-lowering, and their effects on biochemical, morphological and histological characteristics of selected tissues of rats fed on high-cholesterol/high-fat (HCF) diets.

In vitro, antioxidant activities of the eight species of seaweeds were evaluated using TEAC (Trolox equivalent antioxidant capacity) and FRAP (ferric reducing antioxidant power) assays. Total phenolic contents of these seaweeds were determined using Folin-Ciocalteu assay. Red seaweed K. alvarezii, green seaweed C. lentillifera and brown seaweed S. polycystum were selected based on their high in vitro antioxidant activity and further evaluated for their chemical composition, in vivo antioxidant activity and cholesterol-lowering effects in Sprague Dawley rats fed with HCF diet for 16 weeks. Chemical analysis of seaweeds comprised of the proximate composition, dietary fiber, vitamin C, vitamin E (?-tocopherol), minerals, carotenoids, chlorophylls, fatty acids and amino acids. Animal experimental diets comprised of eight groups: normal diet (N, control group), HCF diet (HCF group), a normal diet supplemented with 5% seaweeds (N+KA, N+CL and N+SP groups), and HCF diet supplemented with 5% seaweed (HCF+KA, HCF+CL and HCF+SP groups).

Effects of seaweeds in preventing hypercholesterolemia and peroxidation in rats were studied via assessing the plasma lipids and, plasma and organs malondialdehyde (MDA) concentrations. Likewise, activities of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were accessed as indices of oxidative stress. Biochemical markers for liver, heart, and kidney damage such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), ?-glutamyltransferase (GGT), creatinine kinase (CK), CK-MB isoenzyme, urea, creatinine and uric acid were measured. Somatic index and descriptive histological changes in the liver, heart, kidney, brain, spleen, and eye of the experimental rats were also performed, while quantitative histology was restricted only to necrosis in the liver, kidney and brain.

The results showed that administration of K. alvarezii and C. lentillifera reduced (P<0.05) plasma low-density lipoprotein cholesterol and triglyceride, and increased (P<0.05) plasma high-density lipoprotein cholesterol thus improving the atherogenic index of rats fed an HCF diet. These seaweeds were shown to reduce body weight gain in rats fed an HCF diet in the following order S. polycystum>C.

lentillifera>K. alvarezii. However, K. alvarezii and C. lentillifera were more effective than S.

polycystum in improving the antioxidant status by reducing (P<0.05) lipid peroxidation and increasing (P<0.05) antioxidant enzymes in liver, heart, and kidney of rats fed the HCF diet. Histological examinations demonstrated consumption of all three seaweeds did not exert any damage to the liver, heart, kidney, brain, spleen, and eyes in normal rats.

In conclusion, K. alvarezii and C. lentillifera showed hypolipidaemic effects improve the antioxidant status and exert a protective effect in mitigating the cardiac, hepatic, renal, and brain abnormalities in rats fed HCF diet. The presence of high dietary fiber especially soluble fiber, omega-3 fatty acids such as eicosapentaenoic acid (C20:5?3), and antioxidant compounds such as polyphenols, vitamin C, ?-tocopherol, carotenoids, and selenium may probably be contributed to the cholesterol-lowering and antioxidant efficacy of these seaweeds.In the study entitled The Analysis of  Fatty Acid Composition and Heavy Metals in Edible Seaweeds Kappaphycus alvarezii and Caulerpa lentillifera using Gas-Chromatography-Mass Spectrometry and Atomic Absorption Spectrometry, Sea weeds were collected from wet markets in metro manila during the month of August and September. Lipid extraction of the freeze-dried sample was done using a modified version of Bligh & Dyer. The fatty acid composition was analyzed by converting the lipid extract into fatty acid methyl, esters, and analyzed using gas chromatography mass spectrometry.

For analysis of the concentration of cadmium and lead, the freeze-dried sample was subjected to nitric acid and the acid digestion and analyze via atomic absorption spectroscopy together with the prepared standard solutions.It is revealed that the result of fatty acid composition analysis shows the presence of both saturated and unsaturated fatty acids ranging from C8-C24. Among the saturated fatty acid, myristic acid was found in all samples. Oleic acid, a mono-unsaturated fatty acid and arachidonic acid, a polyunsaturated fatty acid were also common among the samples.

However, omega-3 fatty acids were only detected on the fish oil sample. The result of the heavy metal analysis on the freeze-dried samples shows that the concentration of the cadmium and lead on the freeze-dried samples which represents proximately 10% of the actual mass of seaweed when consumed as the ingredient in salad and other delicacies are within the safe levels set by the World Health Organization. Further studies are currently undertaken to confirm these results. Results of the study can be used to evaluate the nutritional value and help hazard of seaweed consumption.


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