Effects Of Trogopogon Porrifolius Intake Biology Essay

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The sensation of feeling full under normal physiological conditions is a voluntary act induced by a conscious satisfaction known as satiety.We have been able to study the effect of a plant called Tragopogon Porrifolius on short and long term satiety,body weight and blood lipid value.Sixty-four adult male Sprague-Dawley were randomly allocated into 8 groups of 8 rats each,according to the type of diet and whether or not they are given the plant extract (in their water) for 4 weeks.Different methods and procedures were used including Human Cholecystokinin, Enzyme Immunoassay Kit, centrifugation and spectrophotometer for stools analysis, to test for CH, TG & CCK levels . Our results showed that tragopogon plant have a significant effect in controlling satiety by decreasing appetite, body weight as well as CH & TG levels.

Keyword: Tragopogon Porrifolius; satiety; cholecystokinin; Cholesterol; Triglycerides.

1.Introduction

Satiety essentially means "satisfied" especially when refering to eating and implies to the individual who will stay satisfied for awhile.(Dolson.2007) .It is a complex psycho-physiological mechanism that allows the adjustment of energy intake to expenditures. As such, it plays an important role among the numerous interacting mechanisms contributing to body weight control.( as cited in Appetite. 2010).Satiety is also defined as the internal state of body that lead voluntarily to the cessation of eating under normal physiological conditions. Due to some impairement of central mechanisms regulating food intake ,causing obesity also,appetite increase. in contrast,there is a loss of appetite or anorexia caused by pathological situations.(Kidwai and Upreti,1993)

What controls satiety in the body

Many theories of hunger are historically discussed from the biological component. Cannon and Washburn (as cited in Coon, 1995) came up with the stomach contraction theory which states that we know we are hungry when our stomach contracts. In the notorious balloon study, Washburn trained himself to swallow a balloon which was attached to a tube, then the balloon was inflated inside of his stomach. When the balloon was inflated, he did not feel hungry. Later this theory was opposed by the fact that people whose stomach was removed still felt hungry. Glucose theory states that we feel hungry when our blood glucose level is low. Bash (as cited in Franken, 1994) conducted an experiment transfusing blood from a satiated dog to a starved dog. The transfusion resulted in termination of stomach contraction in the starved dog, and supported the glucose theory. But as LeMagnen (as cited in Kalat, 1995) suggests that blood glucose level does not change much under normal conditions. Insulin theory states that we feel hungry when our insulin level increases suddenly in our bodies (Heller, & Heller, 1991). However, this theory seems to indicate that we have to eat to increase our insulin level in order to feel hungry. Fatty acid theory states that our bodies have receptors that detect an increase in the level of fatty acid. Activation of the receptor for fatty acid triggers hunger (Dole, 1956, Klein et al., 1960 cited in Franken, 1994). Heat-Production theory suggested by Brobeck (as cited in Franken, 1994) states that we feel hungry when our body temperature drops, and when it rises, the hunger decreases. This might explain that we tend to eat more during winter. The mechanism of hunger and satiety are not necessarily the same.What is known about control of food intake is often discussed in terms of short-term and long-term controls. This discussion will focus on the following areas:

Role of the central nervous system

At the brain level, the other is at the gastrointestinal tract level. There are two places in the hypothalamus, part of the brain, that controls hunger and eating. The Ventromedial Nuclei gives a signal when to stop eating, and the Lateral hypothalamus gives a signal to start eating (e.g.,Coon 1995). We feel satiety at the brain level because of the function of the Ventromedial Nuclei.

Pregastric factors

We all know of "environmental" conditions that can dramatically affect food intake. Appearance of food, Taste and/or odor of food, Learned preferences and aversions and psychologic factors affect food intake. (Bowen.1999)

Gastrointestinal and postabsorptive factors

The degree of gastrointestinal fill is the most important signal from the digestive tract per se - a full stomach and intestine induce satiety, probably via the vagus nerve relaying that fact back to the hypothalamus. Additionally, the enteric hormone cholecystokinin is well documented to induce satiety in experimental settings, while the hormone ghrelin seems to be a potent stimulator of appetite.

As nutrients such as glucose and amino acids are absorbed, their concentrations in blood rise, as do the concentration of several hormones (cholecystokinin as mentioned above, but also insulin and glucagon). These changes also have been linked to the sensation of hunger or satiety.( Bowen.1999)

Long-term controls

The satiety factor studied most extensively to date is the hormone leptin, which has the following basic characteristics:

Leptin is synthesized and secreted predominantly by fat cells (adipocytes).

A major site of leptin receptors is in the hypothalamus, which is known to play an important role in control of food intake and metabolic rate.

Plasma levels of leptin rise and fall in parallel with body fat content - as body fat mass increases, so does the concentration of leptin in blood.

Injection of leptin into leptin-deficient animals leads to reduction in body weight by suppressing food intake and increasing metabolic rate and energy expenditure.

Several other genes have been isolated that encode proteins that affect food intake, energy metabolism and body weight. Right now it is difficult to predict their future role in the pharmaceutical control of obesity, but needless to say, a number of companies are betting multimillions that one or more of these proteins will become the miracle drug for treatment of obesity.( Bowen.1999)

Therapeutic plants

The use of plants as medicines has played an important role in nearly every culture, including Asia, Africa, Europe and the Americas (Wargovich et al., 2001). Therapeutic plants contain natural substances that can promote health and alleviate illness. Several plants can help to reduce blood sugar, high blood cholesterol, provide some protection against cancer and stimulate the immune system. Furthermore, a diet in which culinary plants are used generously to flavor food provides a variety of active photochemical that promotes health and protective against chronic diseases. Additionally, several commonly used plants have been identified by the National Cancer Institute in USA as possessing cancer-preventive properties. Furthermore, several plants can help to reduce blood sugar, high blood cholesterol, provide some protection against cancer and stimulate the immune system. These plants include members of the Allium sp. (garlic, onions and chives), members of the Labiatae (mint) family (basil, mints, oregano, rosemary, sage and thyme), members of Zingiberacae family (turmeric and ginger). (Steinmetz and Potter, 1991).

Tragopogon Porrifolius is a Mediterranean biennial that belongs to the same family of plants as chicory and dandelion. It is annually grown with culture similar to that for parsnips or carrots. This plant also called 'salsify' has long, slender, pointed, smooth, flat leaves that are about 1 inch in diameter at the crown and are 10-12 inches long. Roots are 8-12 inches long, cylindrical, 1 inch or less in diameter, and salmon or brown in color. Older roots possess a white, milky white sap. Salsify requires a long growing season of 120 to 150 days from seeding to harvest. Salsify will withstand frost.The seedlings should be thinned to 3-4 inches apart in rows 18-24 inches apart. Cover the seed ½ inches deep. Salsify is used in soups and stews; it can be boiled, cut into small pieces, and creamed like asparagus. It can be cut into long strips, boiled, and then fried in butter or mashed.(Stephens.2009)

Importance of blood lipid values

Table1: Level of total-cholesterol(TC), triglycerides(TG), LDL-cholesterol(LDL-CH) and HDL-cholesterol(HDL-CH) in normal rats at zero time. ( Al-Jamal and Alqadi,2011)

TC

TG

LDL-CH

HDL-CH

Normal Rats

Mean± SD (mg/dl)

92.3±3.3

88±4.42

60.2±5.5

51.3±2.2

Cholesterol(CH) is a type of fat, found in your blood. It is produced by your body and also comes from the foods you eat (animal products). Cholesterol is needed by your body to maintain the health of your cells. Too much cholesterol leads to coronary artery disease. Your blood cholesterol level is related to the foods you eat or to genetic conditions (passed down from other generations of family members). (as cited in Cleveland Clinic,2011).

High Density Lipoprotein (HDL) "Good cholesterol"is a lipoprotein (a combination of fat and protein) found in the blood. It is called "good" cholesterol because it removes excess cholesterol from the blood and takes it to the liver. A high HDL level is related to lower risk of heart and blood vessel disease. (as cited in Cleveland Clinic,2011).

Low Density Lipoprotein (LDL) "Bad cholesterol"is a lipoprotein (a combination of fat and protein) found in the blood. It is called "bad" cholesterol because it picks up cholesterol from the blood and takes it to the cells. A high LDL level is related to a higher risk of heart and blood vessel disease. (as cited in Cleveland Clinic,2011).

Triglycerides(TG) are a type of fat found in the blood. The blood level of this type of fat is most affected by the foods you eat (such as sugar, fat or alcohol) but can also be high due to being overweight, having thyroid or liver disease and genetic conditions. High levels of triglycerides are related to a higher risk of heart and blood vessel disease. (as cited in Cleveland Clinic,2011)

The role of the liver in lipid metabolism

As a key metabolic organ, the liver is central to the imbalance of high-caloric diets, and particularly dietary fat consumption. By interacting with the intestinal tract and adipose tissue, the liver plays a key role in various aspects of lipid metabolism. Increasing activation of transcription factors, such as carbohydrate responsive element binding protein (ChREBP), sterol response element binding protein-1c (SREBP-1c), or forkhead box 01 (Fox01), may contribute to fatty acid synthesis. ( Z Gastroenterol. 2007).

Eventually the accumulation of lipid droplets into the hepatocytes results in hepatic steatosis, which may develop as a consequence of multiple dysfunctions such as alterations in beta-oxidation, very low density lipoprotein secretion, and pathways involved in the synthesis of fatty acids. In addition an increased circulating pool of non-esterified fatty acid may also to be a major determinant in the pathogenesis fatty liver disease.( J Anim Physiol Anim Nutr (Berl). 2008).Satiety signals come from the stomach, which controls short-term eating. Cholecystokinin (CCK) is a peptide hormone and neurotransmitter involved in the feeling of satiety and proper function of the digestive tract.It is a group of similar proteins sharing rather specific hormonal activities in the digestive tract, and neurotransmitter functions in the central nervous system (CNS).The 7s/full/oby2006300a.html"satiety effect of CCK seems to be a physiological mechanism to avoid overeating and obesity. In animal studies, males are affected more than females. Bendleton.2009).

The aim of our experiment is to study the effects of Trapogopon Porrifolius intake on blood lipids and appetite for long and short terms using different methods.Therefore ,our research is done for about four weeks on sixty-four adult male Sprague-Dawley rats that were randomly distributed into 8 groups of 8 rats each and each group was given a different diet :

Standard chow diet

Standard chow diet + plant extract dose 1(50mg)

Standard chow diet + plant extract dose 2(100mg)

Standard chow diet + plant extract dose 3(250mg)

High-fat diet (10% saturated fat)

High-fat diet + plant extract dose 1

High-fat diet + plant extract dose 2

High-fat diet + plant extract dose 3

2.Materials and Methods

Plant extraction

Plant shoots were extracted from Tragopogon Porrifolius and boiled for 15mins. Three doses were prepared for use during the whole experiments, with dose 1 being the most dilute and dose 3 being the most concentrated.

Animals and diet

64 adult male Spargue -Dawley rats(Lebanese American University Stock) aged initially 8-10 weeks old were housed in temperature and humidity -controlled room under a 12:12 light / dark cycle(light on at 0800h).The animals were weight-matched and assigned to one of the 8 following dietary groups: Standers chow diet, Standard chow diet+ plant extract dose 1,Standard chow diet plant extract 2,Standard chow diet +plant extract dose 3 , High -fat diet(10% coconut oil),High fat diet +plant dose 1,High fat +plant extract dose 2 ,High fat diet +plant extract dose 3 (n=32).The diets were given ad libitum for 4 weeks and 8 groups had the same intial average body weight .All experiments were performed under an approved protocol according to the guidelines of the Guide for the Care and Use of Laboratory Animals (National Research Council of the United States 1985).Food intake and body weight gain were monitored during the whole experiment period.

Blood Lipid and Serum Cholecytokinin (CCK) analysis

Serum was separated in the collected blood by centrifugation at 2000g for 15 min .It was stored at -80c for subsequent analysis .Blood lipid analysis of the stool (total cholesterol ,HDL cholesterol, LDL cholesterol and triglycerides) were determined using the relevant Spinreact kits (Spinreact, Spain).Serum CCK levels were also determined by ELISA (Diagnostic System Laboratories ,Webster ,TX,USA) in order to quantify the stress of the animals .All serum samples were run in duplicate and analyzed within the same essay.

CCK levels in blood serum were measured using Enzyme Immunoassay (EIA). All serum samples were run in duplicate and analyzed within the same essay.

TG & CH in stools

Eight samples of stools were taken from one rat of each cage, incubated for 24hrs at 45 °C to determine water content in stools. After incubation, stools were crushed, mixed with hexane and incubated for 2hrs at 37 °C then centrifuged for 5mins at 5000 rpm. CH & TG kit were added to our samples and absorbance was detected using spectrophotometer. Absorbance is converted into mg/dl using the following formula:

Sample (A) Ã- 200 = mg/ dl Ã- 0.013 = m mol /1

Standard (A)

Standard (CH) = 0.318, standard (TG) =0.341.

Centrifugation

Centrifugation is a process that involves the sedimentation of mixtures with a centrifuge, used in industry and in laboratory settings. A centrifuge separates a heterogeneous mixture of solid and liquid by spinning it. (Harrison.2003)

Spectrophotometer

A spectrophotometer consists of two instruments, namely a spectrometer for producing light of any selected color (wavelength), and a photometer for measuring the intensity of light .(stoscheck.1990)

ELISA

The ELISA stands for enzyme-linked immunoassay.It is a fundamental tool of clinical immunology, and is used as an initial screen for HIV detection. Based on the principle of antibody-antibody interaction, this test allows for easy visualization of results. An HIV ELISA, sometimes called an HIV enzyme immunoassay (EIA) is the first and most basic test to determine if an individual is positive for a selected pathogen, such as HIV. The test is performed in a 8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which are about 1 cm high and 0.7 cm in diameter.( University of Arizona.1998)

Short-term satiety experiment

32 adult male Sprague-Dawley rats were fasted from food and water overnight, allocated into 4 groups of 8 rats each. Each group was receiving the following diet respectively, normal chow + normal water, normal chow + dose 1, normal chow + dose 2, normal chow + dose 3. A known large amount of food is given an hour later and the consumption of food is measured 1, 6 and 24 hours later. This study was repeated for three time.

3.Results

From the below histograms of the analysis of our stool samples, we detect that the amount of CH and TG decrease in the groups receiving the plant extract doses and mostly with the group having dose 3 of the plant.

Figure : Effect of different doses Tragopogon Porrifolius, taken with normal diet and high fat diet, on cholesterol levels in blood

NF: normal food, HFD: high fat diet

Figure : Effect of Tragopogon Porrifolius on triglycerides level in blood. (taken with normal and high fat diet).

NF: normal food, HFD: high fat diet

As we can notice while comparing the body weights of each rat as well as the food intake, the groups receiving the three different plant extract doses and mostly dose 3 had an insignificant increase in their body weights and food intake. The two other groups drinking normal water and diluted concentrations of the plant extract mainly dose 1 had a significant and a sharper increase in their body weights as well as their food intake. In fact the group with normal chow and water (not including the plant extract) were eating all the amount of food given to them.

Figure : Effect of different doses of Tragopogon Porrifolius on food intake (with normal chow

Figure : Effect of different doses of Tragopogon Porrifolius on food intake (High fat chow)

Figure : Effect of concentrated dose of Tragopogon Porrifolius on body weight. (Normal and high fat chow)

In the short-term satiety experiment results (done 3 times), we detect a significant decrease in the energy consumption mainly for the rats receiving the most concentrated plant extract dose (dose 3).

Table 2: Short term effect of Tragopogon Porrifolius on energy consumption

Average Energy (kJ) consumed per rat across the 3 experiments

Control

Dose 1

Dose 2

Dose 3

Average

SEM

Average

SEM

Average

SEM

Average

1-hr

70.1

20.6

38.8

22.4

37.8

17.1

26.0

6-hr

227.0

55.4

223.0

50.7

223.0

33.8

201.6

18-hr

434.6

4.9

395.0

29.1

431.4

12.2

381.4

24-hr

466.1

23.1

432.8

36.7

453.6

19.6

401.9

Values are mean ± SEM of energy consumption per rat across the 3 experiments

Evaluating the histograms below for the short term effect, we notice an important decrease of the food intake of the groups of rats receiving dose 3 of plant extraction compared to the control groups.

Figure : Short term effect of Tragopogon Porrifolius on food intake

ELISA immunoassay kit was performed to determine the CCK level in serum blood. However, due to technical and procedure errors, no relevant results were obtained.

4.Discussion

There are many errors committed during our experiments. These errors vary from limitations such as equipment constraints, limited availability of cages and limited amount of chow (for few days),to the inaccurate data taken and inaccurate timing. Indeed, the uncertainty and imprecision in the technical and procedure steps of ELISA gave us inappropriate results. But in general, we can say that our overall experiments were successful and that Tragopogon Porrifolius had a positive effect on blood lipids in both short and long-term satiety since we noted a drop off in body weight and food intake. In fact, oral administration of Tragopogon Porrifolius extract caused significant declines in the blood levels of TG & CH. Other experiments were done to reduce the food intake. In a research paper titled "Experimental anorexigenic effect of a membrane proteoglycan isolated from plants",Kidwai ,Upreti and other researchers (1993) have been able to isolate and characterize a proteoglycan from membranes of mung bean sprouts which reduced the food intake significantly when injected into murine model systems without any rebound. Due to its membrane origin, the glycoprotein causing satiety has been termed 'satiomem'.(Kidwai and Upreti,1993). In a randomized placebo-controlled study of mildly overweight, healthy women, the researchers hypothesized that oral supplementation with Satiereal (Inoreal Ltd, Plerin, France), a novel extract of saffron stigma, may reduce snacking and enhance satiety through its suggested mood-improving effect, and thus contribute to weight loss. (Gout,Bourges and Paineau-D.2010).

Our studies suggest that Tragopogon Porrifolius is accepted by the animal (Sprague-Dawley rat) body without any rebound or apparent unwanted effect. Therefore, Tragopogon Porrifolius with its regulatory control on hunger drive and satiety could provide a useful tool in the hands of physicians dealing with the problem of obesity. In addition, it can also be used in various nutritional research studies as a predictable parallel control group with controlled food intake depicting a true satiety condition. Finally, this beneficial plant can be conducted on humans.

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