Body Weight Reducing Activity In Rats Biology Essay

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6.1 INTRODUCTION

Obesity is one of the most reported health problems worldwide (Ball et al., 2009, Blackburn et al., 2009, Gletsu-Miller et al., 2009, Hatoum et al., 2009, Hernandez et al., 2009, Latzer et al., 2009, Mamun et al., 2009, Puhl and Heuer, 2009, Reinehr et al., 2009, Roehrig et al., 2009, Rutters et al., 2009, Stolley et al., 2009, Thande et al., 2009, Cho et al., 2009, Heinsbroek and van Dijk, 2008, MacEneaney et al., 2009, Zhao et al., 2009, Soriguer et al., 2009). Khat (Catha edulis) belongs to the suborder Rosidae and family Celastraceae. The main ingredients of khat leave include alkaloids, tannins and flavonoids (Feyissa and Kelly, 2008). The major alkaloids in fresh khat leaves are cathinone (0.95 mg/g), cathine (1.98 mg/g) and norephedrine (0.54 mg/g) (Dimba et al., 2004, Feyissa and Kelly, 2008, Geisshüsler and Brenneisen, 1987, Kalix, 1990, Widler et al., 1994). The major contents of alkaloids in adult or dried khat leaves are cathine and norephedrine at an approximate ratio of 4:1 (Feyissa and Kelly, 2008, Kalix, 1990, Schorno and Steinegger, 1979, Sporkert et al., 2003).

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Anorexia, a characteristic effect of khat has been used for centuries to alleviate the sensation of hunger (Feyissa and Kelly, 2008). However, detailed investigation into the anorectic effect of khat was only carried out in the last 30 years. Due to the relatively complex extraction method from the khat plant, chemically synthesized alkaloids were investigated (Halbach, 1972, Kalix, 1990). The synthesized alkaloid was reported to possess anorexic properties and body weight reducing activity in animals (Knoll, 1979, Zelger and Carlini, 1980, Goudie and Newton, 1985, Eisenberg et al., 1987, Nencini et al., 1988, Islam et al., 1990, Nencini et al., 1996, Wolgin and Munoz, 2006), when given intraventricularly (Knoll, 1979), intraperitoneally (Eisenberg et al., 1987, Foltin and Schuster, 1983, Goudie and Newton, 1985, Islam et al., 1990, Nencini et al., 1996, Nencini et al., 1988, Wolgin and Munoz, 2006, Woolverton and Johanson, 1984, Zelger and Carlini, 1980) or intravenously (Schuster and Johanson, 1979, Yanagita, 1979). At the same time the side effect of tolerance to the anorexic effects in rats (Schuster and Johanson, 1979, Foltin and Schuster, 1982, Wolgin and Munoz, 2006) was reported.

Other side effects associated with using of khat leaves were reported. These Including gastritis, insomnia, anorexia, constipation, headache, respiratory difficulties (Kennedy et al., 1983), reduction in placental blood flow and impairment in fetal growth in chronically catheterized late-pregnant guinea pigs (Jansson et al., 1988). Embryotoxic and teratogenic properties due to high dose of methanolic khat extract administered orally to rats during days 6 to 15 of gestation was also reported (Islam et al., 1994). This high dose of 125-500 mg/kg/day, which was many times more than the average normal dose, because the normal dose for an average habitual human user is approximately equivalent to 50 mg/kg of khat extract (Aziz et al., 2009). Moreover, the repeated oral administration of khat or the commercial S-(-)-cathinone was enhanced aggression in rats. These effects were produced by dose levels of S-(-)-cathinone-HCl at 1.5 mg/kg and fresh-khat-extract at 200 mg/kg (Banjaw et al., 2006). Al-Motarreb et al. (Al-Motarreb et al., 2002a, Al-Motarreb et al., 2002b) reported that chewing of fresh khat leaves resulted in acute myocardial infarction, while Kuczkowski (Kuczkowski, 2005) documented two cases of chest pain, sinus tachycardia and hypertension in two pregnant patients who had used fresh khat in familial gatherings. Mujlli et al. (Mujlli et al., 2005) reported that khat is a risk to blood pressure in acute cerebral infarction patients. However, these studies did not consider the adjustment of the confounding effects of smoking (since 80% of khat chewers are smokers), as such their findings might be due to either khat chewing or both khat chewing and smoking habits (Al-Hebshi and Skaug, 2005). Furthermore, recent review by Pennings et al. (Pennings et al., 2008) stated that based on a critical review (WHO, 2002), the WHO Expert Committee on Drug dependence recommended in 2006 that the potential for abuse and dependence of khat is low. The level of abuse and threat to public health is not significantly enough to warrant international control. In addition, epidemiological studies over the last 50 years (1945-2006) reviewed by Warfa et al. (Warfa et al., 2007) did not support a causal relationship between khat-chewing and mental illness.

In light of the results of all the above studies, it seems that khat alkaloid whether chemically synthesized or extracted from the leaves possesses anorexic and many other associated side effects. However, the severity of these side effects is influenced by the source of alkaloid used (natural or chemically synthesized), the dose level and the mode of administration. When pure alkaloid particularly cathinone were given parenterally (intraperitoneally, intraventricularly or intravenously), severe side effects are observed. However, moderate side effects are associated with ingestion of fresh khat leaves. In contrast, when chewing the fresh khat leaves in the traditional manner (a habitual user will chew the leaf slowly one by one, keep in the cheek pouch, swallow the juice and eject the residues at the end of the khat session for a period of 3-4 hr) results in a slow release of the alkaloid, as well as diminished side effects (Aziz et al., 2009, Cox and Rampes, 2003, Feyissa and Kelly, 2008, Toennes et al., 2003).

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The aim of the present study was to investigate the mode of administration and release rate of khat extract on body weight, anorexia, plasma cholesterol and triglycerides levels in rats.

The aim of the present study was to investigate the impact of chronic repeated administration and continuous administration of encapsulated khat extracts with emphasis the sustained release effect on the anorexic properties, body weight reducing activity, cholesterol and triglycerides levels in rats during two months of treatment.

6.2 MATERIALS AND METHODS

6.2.1 Materials

Khat extract microcapsules (KE235)

blank microcapsules without khat extract (KEblank) were also prepared following the similar procedure.

InjKE235

6.2.2 Animals

Sprague Dawley rats (5-10 months old, weighing between 200 to 285 g) were obtained from the Animal House at Universiti Sains Malaysia, Penang, Malaysia. The rats were housed in cages containing wood shavings as bedding, and fed with pellets containing high fat contents (30% butter) and water under a reversed light-dark cycle. The ambient temperature and relative humidity were 28 ± 1°C and 72 ± 2%, respectively. The animal studies were approved by the Animal Ethics Committee (AEC) of the School of Pharmaceutical Sciences, Universiti Sains Malaysia.

6.2.3 Effect of formulations on body weight food intake cholesterol and triglycerides

The rats were randomly divided into five groups of 12 rats each. Group A received 2 mL of 0.5% carboxymethylcellulose (CMC) as the control, group B received 2 mL of 0.5% CMC containing 20 mg/kg KE and group C received 2 mL of 0.5% CMC containing KE235 (equivalent to 20 mg/kg KE). Group D received 3 mL of empty microcapsules. Group E received 3 mL of InjKE235 (equivalent to 560 mg/kg KE).

During the first week (zero time), the rats were fed with known amount of food (F1). On day-6, the remaining food pellets were removed and weighed (F2). The amount of food intake by the rats was presented as the difference in weight of the food (F1-F2). The rats were then left fasted for at least 12 hr. On day-7, 1 mL of blood sample was collected from the tail using method reported by Weiss et al. (Weiss et al., 2000) and the rats were given pre-weighed amount of food. The body weight, blood pressure, heart beats, plasma cholesterol and triglyceride levels of the rats were measured and used as the baseline values.

Immediately after the collection of blood samples (pre-dose) on day-7, the rats in groups A, B and C were orally administered the different test formulations daily over a period of 8 weeks, using metal feeding needles. On the other hand, the rats in groups D and E were anesthetized before injected with the test samples at the subcutaneous dorsum once in 4 weeks using 22-gauge needles for 8 week. The same procedure as described earlier was repeated weekly until 8 weeks. A total number of 8 blood samples were collected from each rat.

6.2.4 Determination of cholesterol and triglycerides levels

The total cholesterol and triglycerides levels in the blood samples were determined using the AEROSET system and the ARCHITECT® c8000 system (Abbott Laboratories, Abbott Park, IL, USA), at the pathological laboratory, LAM WAH EE Hospital, Penang, Malaysia.

6.2.5 Statistical analysis

The results were treated statistically using SPSS software (Version 13, USA). One-way analysis of variance was employed. When there was a statistically significant difference, post-hoc Tukey Honestly Significant Difference (Tukey-HSD) test was applied. Pearson correlation test was used to correlate the release kinetic with BW, FI, Cho and Tri levels. A statistically significant difference was considered at p < 0.05.

6.3 RESULTS AND DISCUSSION

6.3.1 Body weight and food intake

Oral administration of KE or microcapsules KE235 reduced the body weight significantly in comparison with the control KEblank throughout the treatment period (p<0.05) (Table 1, Figure 1). KE induced a maximum reduction in body weight (9.13 ±1.20 %) by the end of the first treatment week. However, this reduction was rather transient and rebound gradually in the following weeks. At the end of the 4th treatment week, the body weight of the rats returned to original and continued to increase until the end of the study. A final increase of 10% was recorded for rats given KE. Microcapsules KE235 induced a maximum reduction in body weight (7.52 ±1.20 %) by the end of the third treatment week. Similarly, for rats given KE, their body weight rebound gradually and finally gained an increase of 7-8 %.

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The reduction in food intake in rats given KE and KE235 were more prominent than the reduction in body weight. KE induced a maximum reduction in food intake of 40.05 ±2.17% by the end of the first treatment week, whereas KE235 produced a maximum reduction of 29.84 ±1.67% by the end of the third treatment week. Although food intake in both cases rebound gradually in the following weeks, they were less than the pre-treatment period. At the end of the experiment, the reduction in food intake results was 1.14% and 13.77% for rats given KE and KE235, respectively.

In contrast, InjKE235 given subcutaneously showed an initial increase of 6% but then followed by a continuous reduction in body weight. A maximum of 18% reduction in body weight was attained at the end of 8 weeks. In addition, the anorexic effect of InjKE235 was also very significant (p<0.05), with a total reduction of 51% in food intake at the end of 8 weeks.

6.3.2 Cholesterol and triglycerides levels

The results showed that plasma cholesterol and triglycerides levels in rats given KE, KE235 and InjKE235 were significantly lower than the control groups (p<0.05) (Table 2, Figure 2). At the end of the first treatment week, KE induced 25% reduction in cholesterol and 30% reduction in triglyceride levels. However, plasma cholesterol and triglyceride levels rebound gradually. At the end of the experiment, the cholesterol and triglyceride levels of rats given KE increased by 24 and 16%, respectively. It is apparent that KE only exhibited a transient reduction in cholesterol and triglycerides levels. KE235 induced decrease in cholesterol (47%) and triglycerides (48%) levels at the end of the third treatment week, but rebound slightly in the following weeks. At the end of the experiment, KE235 maintained a reduction in the cholesterol and triglycerides levels of 12% and 9%, respectively. In contrast, InjKE235 when given subcutaneously showed a continuous and significant reduction in cholesterol and triglycerides levels (p<0.05). At the end of the study, more than 50% reduction in cholesterol and triglycerides levels were attained by InjKE235. The reduction in the total cholesterol and triglycerides levels was in the order of InjKE235 > KE235 > KE.

DISCUSSION

The recent literature review showed that animal study to investigate the anorexic properties of the entire khat extract was not available (Feyissa and Kelly, 2008). Therefore, one of the aims of this study was to address this shortfall. Although individual khat alkaloids have been shown to possess anorexic properties accompanied with cardiovascular side effects, most of these side effects were caused by the presence of cathinone. On the contrary, the khat alkaloids extracted from the dried khat leaves in our study were free of cathinone, as reported in previous work (Aziz et al., 2009) and was consistent with the findings of other researchers (Feyissa and Kelly, 2008, Kalix, 1990, Schorno and Steinegger, 1979, Sporkert et al., 2003). Furthermore, a low dose of khat extract (20 mg/kg) was selected in the present study to minimize the cardiovascular effects on the rats. In all the animal groups, ANOVA results did not demonstrate significant difference in their blood pressure or heart beats values except KE group at the first week which increases the BP significantly about 20% from the base line.

Daily oral administration of bolus khat-extract KE produced very fast release of the khat alkaloid as demonstrated by the t50% of KE of 0.18 hr and k1 of 5.43 hr-1. However, bolus KE administration only caused a transient decrease in the BW, FI, Cho and Tri levels. This may be attributed by the acute tolerance developed after oral ingestion of KE with immediate release of the khat alkaloids (Schechter, 1990, Eisenberg et al., 1987, Foltin and Schuster, 1983, Foltin et al., 1983, Nencini et al., 1988, Zelger and Carlini, 1980). In general, the mode of khat administration influences the absorption and the severity of its side effects (Toennes et al., 2003). Typically, khat leaves are ingested in two ways. The first involves chewing several leaves in the mouth and swallowing the juice slowly. The second entails slow mastication of each leave thoroughly during the 3-4 hours of khat session (Al-Hebshi and Skaug, 2005, Kalix, 1990). Comparing with the normal mode of khat administration, bolus administration of khat extract KE to the animals does not simulate closely the preferred khat chewing performed by khat users.

When khat-extract KE was formulated into KE235 microcapsules, the release of its alkaloids was found to be much sustained. In-vitro release of alkaloid from microcapsules showed that the t50% and k1 were prolonged to 1.58 hr and 0.53 hr-1, respectively. Study on the pharmacokinetics of khat chewing reported that the total amount of cathine absorbed (89%) was represented 84% buccal absorption with t-lag of 0.21 hr and 16% GIT absorption with t-lag of 1.46 hr. Also, the half-life of the distribution phase of 0.24 hr, terminal elimination half-life of 5.22 hr and mean residence time of 10 hr were observed (Toennes et al., 2003). Another study reported that cathine has a slow onset of action, with a serum half-life in humans of about 3 hours and it is excreted unchanged in the urine within about 24 hours (Cox and Rampes, 2003). In addition, it is well-known that gelatin microcapsules are useful to achieve the sustained release of many drugs (Chang et al., 2006, Lu et al., 2007, Pamujula et al., 2004, Tsuyoshi et al., 1987). These properties may explain why the slower release of khat alkaloid from the microcapsules facilitates better absorption into the systemic circulation to exert its effects. As the microcapsules move through the gastrointestinal tract, the gelatin coatings of the microcapsules are able to retain the particles longer in the upper gastrointestinal tract due to their mucoadhesive property (Bonferoni et al., 2004), leading to improved absorption profile (Wang et al., 2002). Our results showed that the daily oral administration of KE235 microcapsules exhibited a reduction in the body weight, food intake, cholesterol and triglycerides levels during the initial few weeks, but rebound slowly with time. Nevertheless, the total reduction in food intake, cholesterol and triglycerides levels of KE235 were more significant than KE indicating the effects of KE235 was a slowly developed phenomenon rather than an acute tolerance (Schechter, 1990).

The use of implants has been proposed for the sustained delivery of several therapeutic classes (Shively et al., 1995). One of the modalities most thoroughly investigated to prolong their duration of action is to embed them in a polymeric matrix and administer them subcutaneously (Benagiano et al., 2008). The 4 weekly subcutaneous implantation of InjKE235 produced prominently slower release of alkaloid from the microcapsules as shown by both t50% of 414 hr and k1 of about 0.0018 and 0.0022 hr-1 for in-vitro and ex-vivo respectively. In addition, InjKE235 also showed significantly greater reduction in the body weight, food intake, total cholesterol and triglycerides levels than KE and KE235. It is apparent that prolonged the release of alkaloid from InjKE235 has inhibited the development of the tolerance effect during the 8 weeks of the study.

Numerous researchers have reported some possible mechanisms of khat. The decreased of body weight due to increasing leptin was reported (Al-Dubai et al., 2006, Kaibara et al., 1998, Zelger et al., 1980). The decrease in plasma cholesterol and triglycerides concentration was attributed to the stimulating effect of khat on β-adrenergic receptors. Thus by activation of adenylyle cyclase, conversion of ATP to c-AMP and, consequently, increased c-AMP concentration that has stimulatory effect on lipolysis (Ahmed and El-Quirbi, 1993, Al-Dubai et al., 2006, Al-Habori and Al-Mamary, 2004, Tariq et al., 1989). It was reported that khat alkaloid enhancing the release of catecholamine from nerve terminals (Kalix, 1988) and subsequently, norepinephrine increasing lipolysis and increasing energy expenditure as well as decreasing appetite (Al-Dubai et al., 2006). However, khat was also found to enhance lipolysis in-vitro and in-vivo (Nencini, 1980).

In the present study, the formulated dosage forms of khat were administered orally through gastro intestinal tract (Yanagita) and subcutaneously through adipose tissue. Normally in GIT, appetite regulators namely ghrelin (produced by stomach), peptide YY3-36 (produced in intestine) and insulin (produced by pancreas), conveys information to the hypothalamus, and the responses are governed by the presence or absence of food. However, in fat tissue; leptin, adiponectin and resistin (produced by adipose tissue) are the regulators of food and energy balance which in response to the amount of fat stored in adipose tissue (Gale et al., 2004). Therefore the mechanism of action could be different in this study. Two mechanisms of action may possibly concern with the oral administration of khat extract. First, delayed gastric emptying of meal (Heymann et al., 1995) through the action of catecholamines which inhibit gastrointestinal motility (Al-Meshal et al., 1986). Second, the astringent characteristics of khat are also believed to delay intestinal absorption (Halbach, 1972). Yet, khat has no effect on ghrelin, YY3-36 (Murray et al., 2008) or, insulin (Saif-Ali et al., 2003) levels. Hence, the action of khat extract orally administered through GIT on anorexic properties might be more related to the astringent and delaying gastric emptying rate characteristics than the central effect. The difference in these characteristics degree between khat extract and khat extract microcapsules might be concerned with the degree of tolerance when administered repeatedly.

On the other hand, khat extract microcapsules administered subcutaneously might be concerned with the lipolysis effect through the stimulation of the β-adrenergic receptors as well as leptin hormone (Al-Dubai et al., 2006, Zelger et al., 1980). In addition, increase of peripheral norepinephrine would stimulate hormone sensitive lipase which hydrolyzes tissue triacylglycerol into free fatty acids and glycerol which are released into blood circulation. These in turn are transported to the liver where glycerol acts as gluconeogenic substrate and fatty acids as energy sources for glucose synthesis by the liver. Moreover, khat alkaloid has also been reported to significantly increase free fatty acids in rats and rabbit (Nencini, 1980).

It is well known that catecholamines play a central role in promoting lipolysis by white fat cells (Lafontan and Berlan, 1993, Morimoto et al., 1997, Okuda et al., 1994). Norepinephrine was induced lipolysis when examined in visceral and subcutaneous fat cells in rats due to the hormone-sensitive lipase activity(Morimoto et al., 1997). This effect is believed to be mediated by a β-adrenergic receptor adenylyle cyclase complex, which is located in the plasma membrane of fat cells and consists of at least three distinct components, these being a β-adrenergic receptor, a nucleotide regulatory protein and adenylyle cyclase (Lafontan and Berlan, 1993). The receptor-controlled increase in intracellular cyclic AMP concentrations promotes activation of cyclic AMP-dependent protein kinase A, which phosphorylates a serine residue on hormone-sensitive lipase and promotes its activation. The resulting phosphorylated hormone-sensitive lipase is believed to stimulate lipolysis in fat cells (Morimoto et al., 1997).

In addition, in this study, we noticed the development of pigmentation in some of the rats in the InjKE235 group, which may be an indication that khat might promotes release of α-melanocyte-stimulating hormone, a potent anorexigen at the central melanocortin-4 MC4R (Elias et al., 1999, Enriori et al., 2006).

β-adrenergic receptors

β1-receptor

In adipose tissue → Lipolysis (Hall, 2004).

β2-receptor

In GIT

Relaxation of smooth muscle and Contract sphincters

→ Decreases motility

→ Delay digestion

This explain the khat alkaloid effect reported

delayed gastric emptying of meal (Heymann et al., 1995) through the action of catecholamines which inhibit gastrointestinal motility (Al-Meshal et al., 1986).

the astringent characteristics of khat are also believed to delay intestinal absorption (Halbach, 1972).

In liver: Glycogenolysis and gluconeogenesis (Hall, 2004).

In adipose tissue → Lipolysis (Large et al., 1997).

In striated muscle

In skeletal muscle → Glycogenolysis and lactate release (Hall, 2004).

→ Tremor (Rang, 2003) (via protein kinase A mediated facilitation of presynaptic Ca2+ influx leading to acetylcholine release)

β3-receptor

In adipose tissue → Lipolysis (Ferrer-Lorente et al., 2005, Meyers et al., 1997).

Thermogenesis in skeletal muscle (Rang, 2003)

Khat → ↑catecholamines

Al-Meshal et al., 1986

Heymann et al., 1995

Halbach, 1972

Khat Alkaloid stimulates

β-adrenergic Receptors

Kalix 1988;

Tariq et al., 1989;

Ahmed & El-Quirbi 1993;

Al-Habori & Al-Mamary 2004;

Al-Dubai et al., 2006

β2

β1

β3

Hypothalamus

Energy expenditure

Appetite

Adipose Tissue (Subcutaneous)

Mechanism depending on the energy stored

Adipose Tissue

Leptin

Fat cells

β3

+ +

β2

+ +

β1

+ +

Lipolysis

Hall, 2004; Large et al., 1997; Ferrer-Lorente et al., 2005; Meyers et al., 1997

+ +

Muscles

β3

β2

+ +

Glycogenolysis

Lactate release Hall, 2004

Thermogenesis

Rang, 2003

Triglycerides

& Inhibits

Cholesterol synthesis

Gastro Intestinal Tract (Oral Administration)

The mechanism of action is depending on the presence of food

Astringent characteristics of khat are believed to delay intestinal absorption

Relaxation of smooth muscle and Contract sphincters

↓ Motility

↓ Digestion

Stomach

β2

+ +

Intestine

β2

+ +

Delayed gastric emptying rate

Delay action of Ghrelin and Insulin

Glycogenolysis gluconeogenesis

Liver

β2

+ +

Blood

↑ Glucose

PYY

Hall, 2004

Our argument

In the present study, khat alkaloids were administered continuously following two different dosing regiments. The first dosing regiment involved a once daily administration of 20 mg/kg body weight of KE or KE235 over a period of 60 days. The in-vitro release rate values of alkaloid from KE and KE235 were 50.38 and 5.09 mg/kg/hr respectively. Hence, the rate of absorption and metabolism by the liver of alkaloid of KE could be faster than KE235. On the other hand, as the rate of release of alkaloid of KE235 was relatively slower, it follows that the rate of absorption is expected to be slower. Therefore, the development of tolerance for KE could be faster than the KE235. Consequently, the daily repeated administration showed that the response effect was fading out after one week for KE (animal adapted after exposure for 6 times repeated administration of oral dose) and after three weeks for the KE235 (animal adapted after exposure for 18 times repeated administration of oral dose).

The second dosing regiment was subcutaneous injection of InjKE235 containing 480 mg/kg body weight of alkaloid once every four weeks over 8-week treatment period. The total amount of alkaloid released from in-vitro InjKE235 was about 85% (release rate of 0.89 mg/hr) or absorbed from the ex-vivo InjKE235 was 77% (release rate of 0.60 mg/hr). This way did not exhibit the first-pass metabolism. The exposure times for the repeated administration are very less (2 times only).

6.4 CONCLUSION

The reduction of food intake, body weight, cholesterol and triglycerides (as indicators of anti-obesity effect) was in the order of InjKE235 > Oral-KE235 > Oral-KE. The importance of the sustained release of khat alkaloids which, mimics the way of khat use by chewing exhibit a great potential in reducing food intake, body weight, cholesterol and triglycerides. In conclusion, the release rate of the khat alkaloid plays an important role in enhancing the anti-obesity effect, maintaining the therapeutic level and minimizing the associated side effects.

Table 1: The change in body weight and food intake (%), Mean ± SEM, N=12.

Formulation

Time of treatments (week)

1st

2nd

3rd

4th

5th

6th

7th

8th

Change in body weight (%)

A- Control-Oral

6.17±1.17

9.18±1.39

18.46±3.67

23.92±5.09

27.23±5.51

29.86±5.58

30.54±5.35

30.67±5.22

B- Control-Inj

4.63±0.99

6.90±1.13

14.03±3.26

18.14±4.53

21.48±4.52

23.38±4.56

24.82±4.35

26.21±7.21

C- Oral-KE

-9.13±1.20

-3.49±0.77

-1.59±0.49

1.94±2.46

4.60±2.92

7.79±2.72

9.30±2.61

10.34±4.13

D- Oral-KE235

-3.43±0.98

-4.58±0.70

-7.52±1.20

-2.15±2.49

3.75±2.73

6.86±2.70

8.00±2.52

7.31±2.93

E- Inj-KE235

5.61±1.15

3.53±1.71

0.41±2.05

-1.63±3.07

-6.78±2.47

-12.20±2.06

-17.96±2.06

-18.27±2.03

Statistical analysis

F

37.731

26.220

20.215

10.830

13.398

19.008

27.459

17.106

Sig.

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

Tukey-HSD

A&B

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

A&C

P<0.001

P<0.001

P<0.001

P<0.01

P<0.01

P<0.01

P<0.01

P<0.05

A&D

P<0.001

P<0.001

P<0.001

P<0.001

P<0.01

P<0.01

P<0.001

P<0.01

A&E

P>0.05

P<0.05

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

B&C

P<0.001

P<0.001

P<0.001

P<0.05

P<0.05

P<0.05

P<0.05

P>0.05

B&D

P<0.001

P<0.001

P<0.001

P<0.01

P<0.05

P<0.05

P<0.05

P<0.05

B&E

P>0.05

P>0.05

P<0.01

P<0.01

P<0.001

P<0.001

P<0.001

P<0.001

C&D

P<0.01

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

C&E

P<0.001

P<0.01

P>0.05

P>0.05

P>0.05

P<0.01

P<0.001

P<0.01

D&E

P<0.001

P<0.001

P>0.05

P>0.05

P>0.05

P<0.01

P<0.001

P<0.01

Change in food intake (%)

A- Control-Oral

10.35±2.01

23.89±1.94

27.84±2.75

30.36±1.12

32.66±2.05

34.57±1.55

35.29±1.56

34.79±1.56

B- Control-Inj

-6.87±3.52

-11.14±1.50

-2.77±3.93

4.50±5.25

6.63±4.40

9.26±4.84

12.26±4.33

10.70±4.69

C- Oral-KE

-40.05±2.17

-29.14±0.80

-21.49±1.38

-9.87±0.61

-4.17±1.04

-1.80±1.05

-0.15±1.30

-1.14±1.35

D- Oral-KE235

-16.28±2.28

-25.84±1.86

-29.84±1.67

-23.89±2.70

-16.91±2.65

-14.09±1.11

-13.27±3.00

-13.77±2.07

E- Inj-KE235

-8.22±3.23

-18.47±4.18

-27.85±2.84

-36.37±2.83

-42.31±2.82

-48.71±2.12

-50.99±2.06

-50.84±1.91

Statistical analysis

F

45.704

82.400

81.310

75.409

97.471

144.268

142.422

146.913

Sig.

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

Tukey-HSD

A&B

P<0.01

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

A&C

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

A&D

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

A&E

P<0.01

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

B&C

P<0.001

P<0.01

P<0.01

P<0.05

P>0.05

P>0.05

P<0.05

P<0.05

B&D

P>0.05

P<0.01

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

B&E

P>0.05

P>0.05

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

C&D

P<0.001

P>0.05

P>0.05

P<0.05

P<0.05

P<0.05

P<0.05

P<0.05

C&E

P<0.001

P<0.05

P>0.05

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

D&E

P>0.05

P>0.05

P>0.05

P>0.05

P<0.001

P<0.001

P<0.001

P<0.001

Table 2: The change in cholesterol and/or triglycerides levels (%), Mean ± SEM, N=12.

Formulation

Time of treatments (week)

Change in cholesterol level (%)

Change in triglycerides level (%)

1st

2nd

3rd

4th

5th

6th

7th

8th

A- Control-Oral

21.29±11.57

60.05±18.36

71.64±14.26

94.09±17.00

24.13±14.60

57.88±21.05

70.58±16.74

93.92±21.85

B- Control-Imp

7.00±24.68

40.58±14.01

71.06±14.17

99.50±19.08

16.92±25.09

35.29±17.41

68.20±14.74

93.26±19.50

C- Oral-KE

-24.79±5.49

-13.02±3.10

5.33±6.54

23.59±10.06

-29.86±8.77

-15.81±9.10

0.21±11.57

16.08±14.12

D- Oral-KE235

-30.55±3.26

-47.06±12.37

-27.89±3.08

-11.71±4.91

-33.45±3.46

-48.24±12.84

-28.55±3.63

-9.19±1.82

E- Inj-KE235

-9.83±3.92

-26.32±4.84

-37.43±5.68

-53.46±2.91

-8.89±4.54

-23.54±3.56

-34.47±3.78

-51.36±3.37

Statistical analysis

F

2.932

14.404

28.192

28.099

3.628

9.587

19.891

19.156

Sig.

P>0.05

P<0.001

P<0.001

P<0.001

P<0.05

P<0.01

P<0.001

P<0.001

Tukey-HSD

A&B

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

A&C

P>0.05

P<0.05

P<0.01

P<0.05

P>0.05

P<0.05

P<0.05

P<0.05

A&D

P>0.05

P<0.01

P<0.001

P<0.01

P>0.05

P<0.01

P<0.01

P<0.01

A&E

P>0.05

P<0.01

P<0.001

P<0.001

P>0.05

P<0.05

P<0.01

P<0.001

B&C

P>0.05

P>0.05

P<0.01

P<0.05

P>0.05

P>0.05

P<0.05

P<0.05

B&D

P>0.05

P<0.01

P<0.001

P<0.01

P>0.05

P<0.05

P<0.01

P<0.01

B&E

P>0.05

P<0.05

P<0.001

P<0.001

P>0.05

P>0.05

P<0.01

P<0.001

C&D

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

C&E

P>0.05

P>0.05

P>0.05

P<0.05

P>0.05

P>0.05

P>0.05

P>0.05

D&E

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

P>0.05

A

B

Figure 1: The change in body weight (A) and food intake (B). Mean ± SEM, N=12.

A

B

Figure 2: The change in cholesterol (A) and triglycerides levels (B). Mean ± SEM, N=12.