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Doi:10.1016/s0271-5317(03)00221-5Nutrition Research 24 (2004) 45–58 Efficacy of a novel, natural extract of (–)-hydroxycitric acid (HCA-SX) and a combination of HCA-SX, niacin- bound chromium and Gymnema sylvestre extract in weight management in human volunteers: A pilot study Harry G. Preussa,*, Debasis Bagchib, Manashi Bagchib, C.V. Sanyasi Raoc, S. Satyanarayanad, Dipak K. Deye aDept. of Physiology and Biophysics, Georgetown University Medical Center, Med-Dent Building, Room 103 SE, 3900 Reservoir Road NW, Washington, DC 20057, USA bDept. of Pharmacy Sciences, Creighton University Medical Center, 2500 California Plaza, Omaha, NE 68178, USA cDept of General Medicine, ASR Academy of Medical Sciences, Elluru, AP, India dDept of Pharmacy, Andhra University, Visakhapatnam, AP, India eDept. of Statistics, University of Connecticut, Storrs, CT 06269, USA Received 9 June 2003; received in revised form 18 September 2003; accepted 19 September 2003 In this pilot study, the efficacy of a novel, natural extract of a highly bioavailable, calcium- potassium salt of (–)-hydroxycitric acid (HCA-SX) alone and in combination with a niacin-boundchromium (NBC) and Gymnema sylvestra extract (GSE) was evaluated for weight loss in moderatelyobese subjects by monitoring changes in body weight, body mass index (BMI), appetite, lipid profiles,serum leptin and serotonin levels, and enhanced excretion of urinary fat metabolites. Garciniacambogia-derived (–)-hydroxycitric acid (HCA) has been shown to reduce appetite, inhibit fatsynthesis and decrease body weight without stimulating the central nervous system. NBC has shownthe ability to restore insulin function, metabolize fat, turn protein into muscle, and convert sugar intoenergy, which plays a role in appetite regulation and facilitates weight loss. Gymnema sylvestre is atraditional herb that helps to promote weight loss possibly through its ability to reduce cravings forsweets and control blood sugar levels. A randomized, double-blind, placebo-controlled human clinicalstudy was conducted in thirty obese subjects (ages 21-50, BMI⬎26 kg/m2) for eight weeks in Elluru,India. The subjects were randomly divided into three groups (10 subjects/group) and given HCA-SX4,667 mg (60% HCA providing 2,800 mg HCA/day) (Group A), a combination of HCA-SX 4,667 mg,NBC 4 mg (providing 400 g elemental Cr) and GSE 400 mg (providing 100 mg gymnemic acid)(Group B), or placebo (Group C) daily in 3 equally divided doses 30-60 min before each meal. This 0271-5317/04/$ – see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.nutres.2003.09.007 H.G. Preuss et al. / Nutrition Research 24 (2004) 45–58 HCA-SX dose was extrapolated from previously conducted in vitro and in vivo studies. In addition,subjects received 2,000 kcal diet/day and underwent a 30 min/day supervised walking program, 5days/week. At the end of 8 weeks, body weight and BMI decreased by 6.3%, respectively, in GroupA. Food intake was reduced by 4%. Total cholesterol, LDL and triglycerides levels were reduced by6.3%, 12.3% and 8.6%, respectively, while HDL and serotonin levels increased by 10.7% and 40%,respectively. Serum leptin levels were decreased by 36.6%, and the enhanced excretion of urinary fatmetabolites, including malondialdehyde (MDA), acetaldehyde (ACT), formaldehyde (FA) and ace-tone (ACON), increased by 125-258%. Under these same conditions, Group B reduced body weightand BMI by 7.8% and 7.9%, respectively. Food intake was reduced by 14.1%. Total cholesterol, LDLand triglyceride levels were reduced by 9.1%, 17.9% and 18.1%, respectively, while HDL andserotonin levels increased by 20.7% and 50%, respectively. Serum leptin levels decreased by 40.5%and enhanced excretion of urinary fat metabolites increased by 146-281%. Group C reduced bodyweight and BMI by only 1.6% and 1.7%, respectively, food intake was increased by 2.8%, and LDL,triglycerides and total cholesterol decreased by 0.8%, 0.2% and 0.8%, respectively. HDL werereduced by 4.1% while serum leptin levels were increased by 0.3%, and excretion of urinary fatmetabolites did not change in MDA, ACT and FA, and marginally increased in the case of ACON.
No adverse effects were observed. Results demonstrate that HCA-SX and, to a greater degree, thecombination of HCA-SX, NBC and GSE can serve as safe weight management supplements. 2004Elsevier Inc. All rights reserved.
Keywords: Obesity; Lipids; Urinary metabolites; Garcinia cambogia; Niacin-bound chromium; Gymnemasylvestre Current statistics demonstrate that more than half of U.S. adults are overweight (61%), defined as having a body mass index (BMI) greater than 25 kg/m2, while more than a quarter(26%) of U.S. adults are obese, having a BMI of greater than 30 kg/m2 Sixty threepercent of men and 55% of women are now overweight or obese in this country Providing an even worse outlook, national data indicate that 10.5-15.5% of children ages 6through 19 are severely overweight According to the World Health Organization, thereare over 300 million obese adults globally Low levels of physical activity and sedentarylifestyles have generally been implicated in the worldwide trend of weight gain Obesity, resulting from an imbalance between energy intake and expenditure, is the second leading cause of premature death in America. Other potential risks of obesity includecardiovascular diseases, diabetes, cancer and hormonal imbalances in women, leading tosterility Low caloric diets with and without exercise can help with temporary weight loss.
Weight loss drugs that suppress appetite, reduce food intake, increase energy expenditureand/or affect nutrient partitioning or metabolism have potential efficacy but are unfortunatelyfrequently accompanied by adverse side effects Therefore, supplementation with safeand natural products in addition to a healthy diet and exercise may be helpful.
* Corresponding author. Tel.: ⫹ 1-202-680-1441; fax: ⫹ 1-202-687-8788.
E-mail address: firstname.lastname@example.org (H.G. Preuss).
H.G. Preuss et al. / Nutrition Research 24 (2004) 45–58 (–)-Hydroxycitric acid (HCA) has been reported to cause weight loss in humans without stimulating the central nervous system HCA is derived from the fruit rinds of Garciniacambogia, which exhibits a distinctive sour taste and has been used for culinary purposes inSouthern Asia for centuries to make meals more "filling", and has been reported to reducefood intake in experimental animals, suggesting its role in the treatment of obesity HCA is a competitive inhibitor of ATP-citrate lyase, an extra-mitochondrial enzyme in-volved in the initial steps of de novo lipogenesis Consequently, HCA reduces thetransformation of citrate into acetyl coenzyme A, a step necessary for the formation of fattyacids in the liver. In addition, there is increased production of hepatic glycogen in thepresence of HCA, which may activate glucoreceptors leading to a sensation of fullness andreduced appetite Earlier successful animal trials suggest that the human doseof HCA typically recommended in dietary supplements and used in previous clinical trials(1,500 mg HCA/day) is sub-optimal. Several publications have reported the efficacy of HCAin weight management Acute oral, acute dermal, primary dermal, and primary eye irritation studies demonstrated the safety of HCA-SX The LD in rats was found to be greater than 5 gm/kg. HCA-SX bioavailability was found to be significantly higher in fasting individuals when consumed atleast 30-60 min prior to food consumption Chromium is important for energy production and plays a role in regulating appetite.
Administration of 600 g elemental chromium as NBC (ChromeMate) in two divided dosesdaily over a period of 2 months to African-American women with a moderate diet andexercise regimen influenced weight and fat loss and sparing of muscle and body compositionTheir blood chemistries revealed no significant adverse effects Another study atthe University of Texas found that young obese women consuming 400 g of elementalchromium as NBC per day with exercise experienced significant weight loss over an eightweek period. Insulin response to an oral glucose load was also lowered in the obese subjects,and no adverse effects were observed Preuss et al. conducted a long term study for12 months in Fischer F344/BN rats using a chronic dose of 400 g of elemental chromiumper day and no adverse effects were observed in body and organ weights, and bloodchemistries Gymnema helps to promote weight control by its ability to reduce the cravings for sweets and control blood sugar levels A peptide isolated from Gymnema, gurmarin, has alsobeen shown to block the sweet taste of glucose and sucrose in animal models Gurmarintemporarily binds to the sweet and bitter receptors on the tongue, thereby blocking the tastesensation and reducing sweet cravings Preuss et al. showed a significant loweringof cholesterol with Gymnema sylvestre ingestion in hypertensive rats fed a high sucrose diet,whereas the placebo group showed a significant increase in cholesterol levels. Gymnema isregarded as very safe and has been administered (400 mg/day) to patients with insulin-dependent diabetic mellitus (IDDM) for 10-12 months with no adverse side effects The effective dose of HCA-SX was determined by a previous ex vivo study on serotonin release from isolated rat brain cortex and in in vivo studies Based on thesestudies the human equivalency dose of HCA-SX used in the present study was calculated tobe 2,800 mg/day, which is significantly greater than the 1,500 mg/day that is normallyrecommended in dietary supplements H.G. Preuss et al. / Nutrition Research 24 (2004) 45–58 The present study was designed to examine the efficacy of optimal doses of HCA-SX alone and in combination with NBC and GSE given on an empty stomach in thirty humanvolunteers. Effects of these supplements were investigated on body weight, BMI (anindicator of obesity health risk), appetite (as determined by weighing the remaining food),lipid profiles, serum leptin levels (a biomarker of obesity regulatory gene), serotonin levels,and excretion of urinary fat metabolites (a biomarker of fat oxidation).
2. Subjects and methods
2.1. Subjects In this study conducted in Elluru, India, each subject was obese, ages 21-50 years, with a body mass index (BMI) ranging from 30.0 to 50.8 kg/m2 (BMI requirement was greaterthan 26⬎kg/m2). Additional inclusion criteria consisted of having a negative pregnancy test,possessing the ability to understand the risks/benefits of the protocol, willingness to partic-ipate in a 30 min supervised walking-exercise program (5 days a week), eat the vegetarianor non-vegetarian prescribed diets of approximately 2,000 kcal/day (17% protein, 25% fat,and 58% carbohydrate) divided into three meals, sign an informed consent form, completea standard health questionnaire, and participate in 3 clinic visits at 0, 4, and 8 weeks. Subjectswere excluded if they were pregnant or nursing, presently taking other weight loss medica-tions, had a history of thyroid disease, cardiovascular disease, or diabetes, suffered fromintractable obesity, had defined weight limits or had experienced any recent, unexplainedweight loss or gain. Subjects were required to fast overnight, and blood and urine sampleswere obtained at each clinic visit in the early morning to avoid diurinal variation. Anindividual diary was maintained for each subject.
Advertisements were placed in local newspapers and overweight subjects who responded and met the inclusion criteria during a screening were scheduled for a baseline visit. Theevaluation included a questionnaire, physical examination, electrocardiogram, and screeningblood studies. Subjects were then randomized into three groups (10 subjects/group) withequal probability through a random number generator. An Institutional Review Boardapproval IRB #01-001 was obtained from ASR Academy of Medicinal Sciences for thisstudy. All subjects gave written consent prior to participation.
2.2. Weight reduction protocol A detailed evaluation was performed at the beginning, week four, and week eight of treatment. Bodyweight, BMI, appetite, lipid profile, serum leptin and serotonin levels andexcretion of urinary fat metabolites were evaluated. The patients' diaries were checked on adaily basis.
Body weights of the subjects were measured using an Essae Digi (Model DS-410) digital weighing scale (Essae-Teraoka Pvt. Ltd., Bangalore, India). Height was measured using aBenson Track and Field height scale. BMI was calculated by body weight in kilogramsdivided by square of height in meters. Appetite reduction was estimated by weighing the H.G. Preuss et al. / Nutrition Research 24 (2004) 45–58 remaining food after each meal. Lipid profile, including high density lipoprotein (HDL), lowdensity lipoprotein (LDL), very low density lipoprotein (VLDL)W.S.William SeroyviaHPLC in conjunction with GCMS using a selective ion monitoring technique as described byShara et al. The following six key parameters were monitored in a randomized, double-blind, placebo- controlled study over a period of eight weeks: To assess whether optimal doses ofHCA-SX, and the combination of HCA-SX, NBC plus GSE (HCA-SX Formula) producesa greater reduction in body weight than placebo, To evaluate whether HCA-SX andHCA-SX Formula produces a greater reduction in BMI than placebo, To assess whetherHCA-SX and HCA-SX Formula has an inhibitory effect on appetite as compared to placebo,To assess whether HCA-SX and HCA-SX Formula produces a beneficial effect on lipidprofile, including LDL, HDL, triglycerides, VLDL, and total cholesterol, as compared toplacebo, To assess whether HCA-SX and HCA-SX Formula has an inhibitory effect onserum leptin and serotonin levels compared to placebo, and To evaluate whetherHCA-SX and HCA-SX Formula causes fat oxidation as estimated by enhanced excretion ofurinary fat metabolites, including malondialdehyde, acetaldehyde, formaldehyde, and ace-tone as compared to placebo.
Subjects were divided into three groups. Group A was given a daily dose of HCA-SX 4,667 mg (60% HCA providing 2,800 mg HCA per day), Group B was given a daily doseof a combination of HCA 4,667 mg, NBC 4 mg (400 g elemental chromium) plus GSE 400mg (100 mg gymnemic acid), and Group C was given a placebo (microcrystalline cellulose)in three equally divided doses 30-60 min before breakfast, lunch and dinner for eight weeks.
2.3. Study materials A natural, highly bioavailable, water-soluble, tasteless and odorless calcium-potassium salt of 60% HCA extract from Garcinia cambogia commercially known as Super CitriMax(HCA-SX), niacin-bound chromium supplement commercially known as ChromeMate (con-taining 10% elemental chromium) and a standardized extract of Gymnema sylvestre extractcommercially known as Gymnema (GSE, GYM-250) (containing 25% gymnemic acid) wereobtained from InterHealth Nutraceuticals, Inc., (Benicia, CA). Unless stated otherwise, allother chemicals and reagents were obtained from Sigma Chemical Co. (St. Louis, MO) andwere of analytical grade or the highest grade available.
2.4. Dose determination Earlier animal studies by Sullivan et al indicate that higher levels of HCA than those typically recommended in dietary supplements for human beings are required toproduce consistent and reliable results. Levels of HCA-SX used in our study were deter-mined by extrapolation of optional micromolar concentrations of HCA required to evokepeak levels of serotonin release in rat brain cortex study Considering a 5-fold fastermetabolism in rats compared to humans, the ex viso dose extrapolates to a human dose of2,800 mg of HCA per day, which approximates the human equivalent does used in the earlieranimal studies conducted by Sullivan et al. H.G. Preuss et al. / Nutrition Research 24 (2004) 45–58 Table 1Effects of placebo (Group C), HCA-SX alone (Group A) and HCA-SX formula (Group B) on body weight,BMI, and serum leptin levels in human subjects.
Placebo (Group C) Data are presented as group mean ⫾ SEM. Subjects were given placebo (Group C), HCA-SX alone (Group A) or HCA-SX formula (Group B) for 8 weeks. See Subjects and methods section for details. Values withnon-identical superscripts are significantly different (p ⬍ 0.05).
2.5. Data analysis Two-tailed Student's t test, with a level of 5% significance, was performed on all three groups for each variable to detect any significant changes. The data set that was analyzed hadeleven variables of interest, which are body weight, body mass index (BMI), low densitylipoproteins (LDL), high density lipoproteins (HDL), triglycerides, very low density lipopro-teins (VLDL), total cholesterol, serum leptin, serotonin, enhanced excretion of urinary fatmetabolites, and remaining food. In each group, longitudinal data was collected for 3 time points denoted by Initial (I), Middle (M) and Final (F) for the first 10 variables, and at 8 time points for the last variable,which is "remaining food".
To compare the differences at a 5% level of significance, we have differences for "I & M", "M & F" and "I & F" for the first 10 variables. For remaining food, since data was collectedat 8 time points, there are 28 possible paired differences. Basic summary statistics and testfor differences with respect to least square means, among the time points was conducted foreach of the variables for each group at each respective timepoint. P⬍0.05 was consideredstatistically significant.
The present clinical study on HCA reported that subjects taking higher, more optimal doses of a highly bioavailable form of HCA (HCA-SX) not only had a significant weightloss, but reduced food intake, increased fat oxidation, decreased LDL, triglycerides and totalcholesterol, increased HDL levels, and decreased BMI compared to placebo. The study alsodemonstrated some surprising new results: high doses of HCA-SX significantly loweredserum leptin levels and increased serotonin levels as determined by our previous in vitro and Table 2Effects of placebo (Group C), HCA-SX alone (Group A) and HCA-SX formula (Group B) on appetite in human subjects. Remaining food in grams on theplate as an index of appetite suppression.
82.22 ⫾ 35.22a 78.33 ⫾ 27.91a 101.11 ⫾ 35.92a 122.22 ⫾ 57.80a 80.00 ⫾ 32.49a 61.11 ⫾ 31.20a 22.22 ⫾ 16.90b 38.89 ⫾ 23.24a 236.50 ⫾ 51.87a 258.00 ⫾ 80.85a 237.50 ⫾ 40.46a 185.50 ⫾ 40.56a 279.00 ⫾ 84.61a 226.50 ⫾ 56.05a 342.00 ⫾ 60.34b 317.50 ⫾ 24.42b 218.50 ⫾ 26.44a 216.00 ⫾ 44.36a 252.50 ⫾ 38.69a 372.50 ⫾ 57.46b 344.00 ⫾ 79.20b 418.00 ⫾ 81.37b 388.00 ⫾ 49.12b 505.00 ⫾ 45.89b Data are presented as group mean ⫾ SEM. Subjects were given placebo (Group C), HCA-SX alone (Group A) or HCA-SX formula (Group B) for 8 weeks. See Subjects and methods section for details. Values with non-identical superscripts in each row are significantly different (p ⬍ 0.05).
H.G. Preuss et al. / Nutrition Research 24 (2004) 45–58 Table 3Effects of placebo (Group C), HCA-SX alone (Group A) and HCA-SX formula (Group B) on lipid profile inhuman subjects.
Total Cholesterol 140.00 ⫾ 27.99a 186.60 ⫾ 10.98a 138.00 ⫾ 27.54a 182.90 ⫾ 10.52b 140.00 ⫾ 26.46a 185.10 ⫾ 10.07a 153.50 ⫾ 31.90a 147.20 ⫾ 31.42a 140.30 ⫾ 31.32b Data presented as group mean ⫾ SEM. Subjects were given placebo (Group C). HCA-SX alone (Group A) or HCA-SX formula (Group B) for 8 weeks. See Subjects and methods section for details. Values with non-identicalsuperscripts are significantly different (p ⬍ 0.05).
in vivo animal studies The addition of NBC and GSE generally caused greatersignificant changes in all parameters measured.
demonstrates the changes in body weight, BMI, serum leptin and serotonin following supplementation of placebo (Group C), HCA-SX (Group A) and HCA-SX For-mula (Group B) over the period of eight weeks. There was a distinct change observed at theend of four weeks and eight weeks in both Group A and Group B. In Group C, approximately0.83 and 1.39 kg reduction in body weights were observed at the end of four and eight weeks,respectively. Under the same conditions, approximately 2.8 and 5.5 kg reduction in bodyweights were observed in Group A, and 3.6 and 6.8 kg reduction in body weights wereobserved in the Group B, respectively, at the end of four and eight weeks. Thus, at the endof eight weeks, Group C only showed a reduction of 1.7% BMI while there were a 6.3% and7.9% reduction in BMI observed in Group A and Group B, respectively.
Group C showed no change in serum leptin levels at the end of four weeks and a 0.3% increase in serum leptin levels at the end of eight weeks, while both Group A and Group Bexhibited a significant reduction. Approximately 17.8% and 36.6% reduction in serum leptinlevels were observed in Group A and 19.5% and 40.5% reduction in serum leptin levels wereobserved in Group B, respectively, at the end of four and eight weeks. In Group C, anincrease of approximately 8.6% and 21% was observed in serotonin levels at the end of fourand eight weeks, respectively. However, Group A demonstrated a 23% and 40% increase andGroup B demonstrated a 23% and 50% increase at the end of four and eight weeks,respectively.
demonstrates the amount of remaining food over the period of eight weeks for each group, which reflects a trend of appetite suppression in both Group A and Group B.
Group C exhibited a slight increase in food consumption of 2.8%. Approximately a 4% and14.1% reduction in appetite was observed in Group A and Group B at the end of eight weeks,respectively.
demonstrates the changes in lipid profiles, including LDL, HDL, triglycerides, H.G. Preuss et al. / Nutrition Research 24 (2004) 45–58 Table 4Effects of placebo (Group C), HCA-SX alone (Group A) and HCA-SX formula (Group B) on enhancedexcretion of urinary fat metabolites (nmoles/ml of urine) 4.011 ⫾ 0.0375a Data are presented as group mean ⫾ SEM. Subjects were given placebo (Group C), HCA-SX alone (Group A) or HCA-SX formula (Group B) for 8 weeks. See Subjects and methods section for details. Values withnon-identical superscripts are significantly different (p ⬍ 0.05).
VLDL and total cholesterol in Groups A, B and C. There was some reduction in LDL andtriglycerides in Group A, however, the changes that were observed in Group B were evenmore pronounced. Group B demonstrated a boost in HDL levels, while little effect wasobserved in Group A. The overall total cholesterol level decreased significantly in bothGroups A and B. Approximately 3.8% and 12.3% reduction in LDL levels were observed inGroup A, while under these same conditions approximately 8% and 17.9% reduction in LDLwere observed in Group B at the end of four and eight weeks, respectively. However, GroupC only showed a 1.6% and 0.8% decrease in LDL levels at the end of four and eight weeks,respectively. In Group A, approximately 0.3% and 10.7% increases in HDL levels wereobserved and 6.9% and 20.7% increases in HDL levels were observed in Group B, respec-tively, at the end of four and eight weeks. No significant changes were observed in GroupC. Approximately 4.1% and 8.6% reduction in triglyceride levels were observed in Group Aand 15.1% and 18.1% reduction in triglyceride levels were observed in Group B, respec-tively, at the end of four and eight weeks, respectively. No significant changes were observedin Group C. No significant changes were observed in the VLDL levels in any of the groups.
Approximately, 2.8% and 6.3% reduction in total cholesterol levels were observed in GroupA and 3.7% and 9.1% reduction in total cholesterol were observed in Group B at the end offour and eight weeks, respectively. No significant changes were observed in Group C.
demonstrates the enhanced excretion of urinary fat metabolites, including MDA, ACT, FA and ACON, which were quantified as biomarkers of fat oxidation. Approximately125-258% increases in total urinary fat metabolites in Group A and 146-281% increases intotal urinary fat metabolites in Group B were observed at the end of eight weeks, respec-tively, as compared to the control samples. In Group C, excretion of urinary fat metabolitesdid not change in MDA, ACT and FA, and marginally increased in the case of ACON at theend of eight weeks.
In Group A, MDA, ACT, FA and ACON increased by 160%, 110%, 140% and 110% at the end of four weeks, respectively, and 260%, 150%, 160% and 130% at the end of 8 weeks, H.G. Preuss et al. / Nutrition Research 24 (2004) 45–58 respectively. In Group B, MDA, ACT, FA and ACON increased by 190%, 120%, 140% and130% at the end of four weeks, respectively, and 280%, 150%, 190% and 150% at the endof eight weeks, respectively. In Group C, excretion of MDA, ACT and FA decreased at theend of four eight weeks of treatment, while excretion of ACON decreased at the end of fourweeks and increased at the end of eight weeks Thus, both HCA-SX alone and in combination with NBC and GSE demonstrated signif- icant reduction in body weight, BMI, LDL, triglycerides, total cholesterol and serum leptinas well as significant increases in HDL, serotonin levels and excretion of urinary fatmetabolites.
3.1. Adverse events and drop outs Twenty-nine of the initial thirty subjects completed the study. During the eight week study, adverse events were noted on a daily basis on each subject. There were no seriousadverse events noted in any of the groups in this study. It is important to emphasis that thenumber of patients reporting adverse events in the supplemented groups was not significantlydifferent from Group C.
One subject dropped out of this study on the 21st day for personal reasons, which was not a result of an adverse event caused by the treatment. No patient was removed or dropped outof the study as a result of an adverse event caused by the treatment.
Obesity, a chronic disequilibrium between food consumption and energy expenditure, continues to be a major health problem in developed and developing countries. Obesity andits related metabolic and cardiovascular complications continue to present an escalatingchallenge to contemporary medicine HCA, found in citrus fruits such as oranges and lemons, is an organic acid similar to citric acid but its properties are remarkably different from citric acid. HCA has been shown toreduce appetite, inhibit fat synthesis, and decrease body weight without stimulating thecentral nervous system Furthermore, HCA does not cause nervousness, rapidheart rate, high blood pressure, or insomnia, symptoms that are often associated with dietarystimulants such as ephedra (Ma-Huang), caffeine or phenylpropanolamine Thus, HCAmay prove to be a safe alternative to these popular diet aids In our previous studies and in the present study, we have demonstrated other important mechanisms including the appetite suppression by HCA and serotonin release byrat brain cortex in vitro, regulatory roles on the lowering of lipid profiles, and increased fatoxidation as demonstrated by enhanced excretion of urinary fat metabolites.
Studies by Ramos et al. (1995) demonstrated that 500 mg HCA (CitriMax) taken three times per day before meals for 8 weeks resulted in 215% greater weight loss in twentyoverweight adults than those taking a placebo. No adverse events were reported Ofadded importance, a significant reduction was also observed in cholesterol and triglyceridelevels. Mattes and Borman (2000) demonstrated that a daily dose of 1.2 gm HCA along with H.G. Preuss et al. / Nutrition Research 24 (2004) 45–58 a daily diet of 5,020 kJ (1,200 kcal) for 12 weeks resulted in a significant difference in weightloss (3.7 ⫾3.1 vs 2.4 ⫾2.9 kg) compared to placebo A 6-week randomized, placebo-controlled, single-blind, cross-over study was conducted by Westerterp-Plantenga and Ko-vacs (2002) in twelve males and twelve females using a daily dose of 900 mg HCA for twoweeks. Results demonstrated that HCA supplementation reduced 24 hr energy intake inhumans, while satiety was sustained In examining the "negative studies", Heymsfeld, et al. (1998) provided what became an accepted daily dose of 1.5 gm HCA along with a diet of 5,020 kJ/day or 1,200 kCal/day dietfor 12 weeks, and reported that no significant difference in weight loss was observed betweenthe placebo and treatment groups However, several problems with the study may beresponsible for the negative results. Heymsfeld, et al. (1998) quantified the HCA content, butdid not assess the bioavailability of the HCA sample used in their study Many HCAproducts are less than 50% soluble in water and poorly absorbed. Also, the low-calorie diet(5,020 kJ/day or 1,200 kcal/day) may have accounted for the substantial decreases in bodyweight of both treatment and placebo groups blunting the ability of HCA to show curbedappetite and reduced food intake.
Our present study used a highly bioavailable calcium-potassium salt of HCA and the optimal dose was determined based on the mechanistic observation of serotonin release in ratbrain cortex. Supplements were given on an empty stomach, since we had previouslydemonstrated that HCA-SX should be consumed at least 30-60 min before meals to enhancebioavailability. This more efficacious dose was extrapolated from ex vivo and in vivo studies.
Furthermore, our study was designed to better determine the effects of HCA on satiety. A 2,000 kcal/day or 8,372 kj/day was administered, and all unconsumed food was weighed asan approximation of appetite reduction. Supplementation with HCA-SX, and to a greaterdegree HCA-SX combined with NBC and GSE, significantly reduced appetite as determinedby increased amounts of remaining food.
Perceived small weight loss in both the HCA-SX (Group A) and HCA-SX formula (Group B) group is supported by the improvement in BMI which means sparing lean muscle andburning fat, as demonstrated by enhanced excretion of urinary fat metabolites.
Another possible mechanism of action may be HCA's ability to down-regulate the gene that influences obesity and body weight. Leptin is a 167 amino acid protein hormone encodedby the obesity regulatory gene. Synthesized and secreted by adipocytes (fat cells), leptin ispresent in the bloodstream in amounts related to the amount of fat in the body. Leptin actsprimarily on the brain, where it binds to receptors and activates signals that inhibit foodintake and increase energy expenditure When receptor-binding activity is dimin-ished, "leptin resistance" develops, i.e., plasma leptin levels increase and lose their ability toinhibit food intake and increase energy expenditure. Studies show that plasma leptin levelsare higher in overweight than in non-overweight individuals, and higher in women than inmen. Leptin has been shown to be able to modulate insulin secretion and action through thesereceptors Our findings in conjunction with earlier studies demonstrate that intracel-lular energy production is important for acute leptin secretion and that potassium and calciumflux may play roles in coupling intracellular energy production to leptin secretion We hypothesize that the calcium-potassium salt of HCA (HCA-SX) may play a role indown-regulating leptin, the obesity regulatory gene.
H.G. Preuss et al. / Nutrition Research 24 (2004) 45–58 Serotonin (5-HT) has been implicated in the control of eating behavior and body weight Our previous study demonstrated HCA-SX's ability to increase the availability ofserotonin in isolated rat brain cortex and serve as a mild serotonin receptor reuptake inhibitorIt is important to note that obese subjects generally show a low level of circulatingserotonin level, Our present study, demonstrated increased levels of circulating serotonin inboth supplemented groups as compared to the control subjects.
The current study also suggests that HCA has the ability to augment fat breakdown. This is based upon the enhanced excretion of urinary fat metabolites following usage, includingmalondialdehyde (MDA), acetaldehyde (ACT), formaldehyde (FA), and acetone (ACON),markers of oxidative fat degradation. The sources of these four lipid metabolites are notentirely clear, but the increases in these products are probably due to enhanced ␤-oxidationof fat. Dhanakoti and Draper demonstrated that urinary MDA excretion was enhancedfollowing enhanced oxidative stress. Furthermore, the fate of radiolabeled MDA adminis-tered to rats was found to be extensively metabolized to acetate and carbon dioxide. Basedon these observations, the urinary ACT identified in this study may arise from the breakdownof MDA, which is formed as a result of fat oxidation/lipid peroxidation. The enhancedformation of ACON in response to a consequence of enhanced ␤-oxidation is also wellknown Winters et al. reported that rat liver microsomes metabolized glycerolto FA. Glycerol is a product of the metabolism of triglycerides by adipose tissue and othertissues that possess the enzyme that activates glycerol, namely, glycerol kinase. Liver andbrown tissues are known to have high glycerol kinase levels Other possible sourcesof FA might include the breakdown of MDA to acetate or ACT and a one carbon fragmentand/or the cleavage of a one carbon fragment from acetoacetic acid with the formationof ACON. Under this situation, it should be pointed out that the triglyceride levels weresignificantly reduced in both HCA-SX and HCA-SX formula groups, accompanied bysignificant increases in urinary excretion of FA. These results suggest that these supplementsmay induce enhanced production of glycerol kinase in the biological system, however, thishas yet to be proven.
High levels of total cholesterol, LDL cholesterol and triglycerides, as well as low levels of HDL cholesterol, are all risk factors for cardiovascular diseases, diabetes and stroke. Thecurrent study shows that supplementation with HCA-SX, and to a greater degree HCA-SX,NBC plus GSE, significantly improves blood lipid profiles.
Taken together, these studies demonstrate that optimal doses of HCA-SX alone and in combination with NBC plus GSE given on an empty stomach are safe, bioavailable, andhighly efficacious diet aids that can help reduce excess, or maintain healthy body weight andBMI, and promote healthy blood lipid levels. The reduced serum leptin levels, decreasedappetite, reduced food intake, and increased fat oxidation may be, at least in part, responsiblefor this positive outcome and decrease the risk factors for obesity related degenerativediseases and mortality.
 Jequier E. Pathways to obesity. Int J Obes Relat Metab Disord 2002;260:S12–7.
H.G. Preuss et al. / Nutrition Research 24 (2004) 45–58  Cole TJ, Bellizzi MC, Flegal KM, Dietz WH. Establishing a standard definition for child overweight and obesity worldwide: International survey. Br Med J 2000;320:1240 –3.
 Roberts SB, McCrory MA, Saltzman E. The influence of dietary composition on energy intake and body weight. J Am Coll Nutr 2002;21:140S–5S.
 Ogden CL, Flegal KM, Carroll MD, Johnson CL. Prevalence and trends in overweight among US children and adolescents, 1999-2000. JAMA 2002;288:1728 –32.
 World Health Organization. Report: Controlling the global obesity epidemic. Available at www.who.int/ nut/obs.htm. Updated August 15, 2003.
 Popkin BM, Paeratakul S, Zhai F, Keyou G. A review of dietary and environmental correlates of obesity with emphasis on developing countries. Obes Res 1995;3:145S–53S.
 Sullivan AC, Triscari J, Cheng L. Appetite regulation by drugs and endogenous substances. Curr Concep Nutr 1983;12:139 – 67.
 Haller CA, Benowitz NL. Adverse cardiovascular and central nervous system events associated with dietary supplements containing ephedra alkaloids. N Engl J Med 2000;343:1833– 8.
 Clouatre, D, Rosenbaum, M. The diet and health benefits of HCA. A Keats Good Health Guide. pp. 9, 1994.
 Sergio W. A natural food, the malabar tamarind, may be effective in the treatment of obesity. Med Hypotheses 1988;27:39 – 40.
 Jena BS, Jayaprakasha GK, Singh RP, Sakariah KK. Chemistry and biochemistry of (–)-hydroxycitric acid from Garcinia. J Agric Food Chem 2002;50:10 –22.
 Lowenstein JM. Effect of (–)-hydroxycitrate on fatty acid synthesis by rat liver in vivo. J Biol Chem 1971;246:629 –32.
 Sullivan AC, Triscari J, Hamilton JG, Miller ON, Wheatley VR. Effect of (–)-hydroxycitrate upon the accumulation of lipid in the rat. I. lipogenesis. Lipids 1974;9:121– 8.
 Sullivan AC, Triscari J, Hamilton JG, Miller ON. Effect of (–)-hydroxycitrate upon the accumulation of lipid in the rat. II. appetite. Lipids 1974;9:129 –34.
 Triscari J, Sullivan AC. Anti-obesity activity of a novel lipid synthesis inhibitor. Int J Obes 1984;8:227–39.
 Ramos RR, Saenz JLS, Aguilar RJA. Extract of Garcinia cambogia in controlling obesity. Investigacion Medica Internacional 1995;22:97–100.
 Mattes DR, Bormann L. Effects of (–)-Hydroxycitric Acid on Appetitive Variables. Physiol Behav 2000;  Leonhardt M, Hrupka B, Langhans W. Effect of hydroxycitrate on food intake and body weight regain after a period of restrictive feeding in male rats. Physiol Behav 2001;74:191– 6.
 Westerterp-Plantenga MS, Kovacs EMR. The effect of (–)-hydroxycitrate on energy intake and satiety in overweight humans. Int J Obes 2002;26:870 –2.
 Shara, M, Ohia, SE, Yasmin, T, Zardetto-Smith, A, Kincaid, A, Bagchi, M, Chatterjee, A, Bagchi, D, Stohs, SJ. Dose- and time-dependent effects of a novel (–)-hydroxycitric acid extract on body weight, hepatic andtesticular lipid peroxidation, DNA fragmentation and histopathological data over a period of 90 days. MolCell Biochem (in press).
 Ohia SE, Opere CA, LeDay AM, Bagchi M, Bagchi D, Stohs SJ. Safety and mechanism of appetite suppression by a novel hydroxycitric acid extract (HCA-SX). Mol Cell Biochem 2002;238:89 –103.
 Loe YC, Bergeron N, Rodriguez N, Schwarz JM. Gas chromatography/mass spectrometry method to quantify blood hydroxycitrate concentration. Anal Biochem 2001;292:148 –54.
 Crawford V, Scheckenbach R, Preuss HG. Effects of niacin-bound chromium supplementation on body composition in overweight African-American women. Diab Obes Metab 1999;1:331–7.
 Grant KE, Chandler RM, Castle AL, Ivy JL. Chromium and exercise training: effect on obese women. Med Sci Sports Exer 1997;29:992–98.
 Preuss HG, Montamarry S, Echard B, Scheckenbach R, Bagchi D. Long-term effects of chromium, grape seed extract, and zinc on various metabolic parameters of rats. Mol Cell Biochem 2001;223:95–102.
 Prakash AO, Mathur S, Mathur R. Effect of feeding Gymnema sylvestre leaves on blood glucose in beryllium nitrate treated rats. J Ethnopharmacol 1986;18:143– 6.
H.G. Preuss et al. / Nutrition Research 24 (2004) 45–58  Ninomiya Y, Imoto T. Gurmarin inhibition of sweet taste responses in mice. Am J Physiol 1995;268: R1019 –25.
 Preuss HG, Jarrell ST, Scheckenbach R, Lieberman S, Anderson RA. Comparative effects of chromium, vanadium and Gymnema sylvestre on sugar-induced blood pressure elevations in SHR. J Am Coll Nutr1998;17:116 –23.
 Shanmugasundaram ERB, Rajeswari G, Baskaran K, Kumar BRR, Shanmugasundaram KR, Ahmath BK.
Use of Gymnema sylvestre leaf extract in the control of blood in insulin-dependent diabetes mellitus. JEthnopharmacol 1990;30:281–94.
 Ohia SE, Awe O, LeDay AM, Opere CA, Bagchi D. Effect of hydroxycitric acid on serotonin release from isolated rat brain cortex. Res Commun Mol Pathol Pharmacol 2001;109:210 – 6.
 Shara MA, Dickson PH, Bagchi D, Stohs SJ. Excretion of formaldehyde, malondialdehyde, acetaldehyde and acetone in the urine of rats in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin, paraquat, endrin andcarbon tetrachloride. J Chromatogr 1992;576:221–33.
 Committee for Proprietary Medicinal Products. Note for Guidance on Clinical Investigation of Drugs Used in Weight Control. London, England: The European Agency for the Evaluation of Medical Products; 1997.
 Montgomery DC. Design and analysis of experiments, 5th ed. New York: John Wiley & Sons, Inc., 2001.
 Littell RC, Stroup WW, Freund RJ. SAS for linear models, 4th ed. Cary, NC: SAS Institute Inc, 2002.
 CNN.com.Health. Report: Dietary supplement warning system lacking. Available at www.cnn.com. Ac- cessed February 25, 2003.
 Heymsfield SB, Allison DB, Vasselli JR, Pietrobelli A, Greenfield D, Nunez C. Garcinia cambogia (hydroxycitric acid) as a potential antiobesity agent: a randomized controlled trial. JAMA 1998;280:1596 –600.
 Dagogo-Jack S. Human leptin regulation and promise in pharmacotherapy. Curr Drug Targets 2001;2:181–  Adeyemi E, Abdulle A. A comparison of plasma leptin levels in obese and lean individuals in the United Arab emirates. Nutr Res 2000;20:157– 66.
 Lerario DDG, Ferreira SRG, Miranda WL, Chacra AR. Influence of dexamethasone and weight loss on the regulation of serum leptin levels in obese individuals. Braz J Med Biol Res 2001;34:479 – 87.
 Levy JR, Gyarmati J, Lesko JM, Adler RA, Stevens W. Dual regulation of leptin secretion: intracellular energy and calcium dependence of regulated pathway. Am J Physiol Endocrinol Metab 2000;278:E892–901.
 Leibowitz SF, Alexander JT. Hypothalamic serotonin in control of eating behavior, meal size, and body weight. Biol Psychiatry 1988;44:851– 64.
 Casper RC. Serotonin, a major player in the regulation of feeding and affect. Biol Psychiatry 1998;44:795–7.
 Dhanakoti SN, Draper HH. Response of urinary malondialdehyde to factors that stimulate lipid peroxidation in vivo. Lipids 1987;22:643– 6.
 Foster DW. Diabetes mellitus. Braunwald E, Isselbacher KJ, Petersdorf RG, Wilson JD, Martin JB, Fauci AS, editors. Harrison's Principles of Internal Medicine. 11th. New York, NY: McGraw-Hill, 1987.
 Winters DK, Clejan LA, Cederbaum AI. Oxidation of glycerol to formaldehyde by rat liver microsomes.
Biochem Biophy Res Commun 1988;153:612–7.
 Winters DK, Cederbaum AI. Oxidation of glycerol to formaldehyde by rat liver microsomes. Effects of cytochrome P-450 inducing agents. Biochem Pharmacol 1990;39:697–705.
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Lampbrush Chromosomes of the Chicken: Cytological Maps of Macrobivaients L.A. Chelysheva, I.V. Solovei, A.V. Rodionov, A.F. Yakovlev, E.R. Biological Research Institute. Leningrad University and the All-Union Research Institute of Farm Animal Breeding and Genetics, Leningrad The morphology of lampbrush chromosomes of the chicken has been studied. We identified six pairs of autosome bivalents and the sexual ZW-bivalent. The cytological map of the lampbrush macrobivalents have been constructed.